Nathan J Cherrington

Interests

No activities entered.

Courses

Scholarly Contributions

Chapters

• Hodgson, E., Cherrington, N. J., Philpot, R. M., & Rose, R. L. (1999). BIOCHEMICAL ASPECTS OF FLAVIN-CONTAINING MONOOXYGENASES (FMOs). In Molecular and Applied Aspects of Oxidative Drug Metabolizing Enzymes(pp 55–70). Springer, Boston, MA. doi:10.1007/978-1-4615-4855-3_4
  More info

  Xenobiotics are metabolized by many enzymes, including a number of isoforms of cytochrome P450 (P450), the flavin-containing monooxygenases (FMO), prostaglandin synthetase, alcohol and aldehyde dehydrogenases, molybdenum hydroxylases, esterases, as well as a number of transferases, particularly the glutathione S-transferases, the glucuronyl transferases and the sulfotransferases.1,2 Both activation and detoxication reac tions can be catalyzed by any of these enzymes but P450 is the most important with regard to activation of toxicants. While many of the reactions carried out by the FMO and P450 are similar there are differences between them. The most significant of these differences are that FMO does not, so far as is known, carry out oxidations at carbon atoms,3 prefering soft nucleophiles as substrates and carrying out oxidations at heteroatoms, such as nitro gen, sulfur, selenium and phosphorus, in organic molecules.

Journals/Publications

• Burgess, J. L., Cherrington, N. J., Jung, A. M., Beitel, S. C., Gutenkunst, S. L., Billheimer, D., Jahnke, S. A., Littau, S. R., White, M., & Hoppe-Jones, C. (2023). Excretion of polybrominated diphenyl ethers and AhR activation in breastmilk among firefighters. Toxicological Sciences. doi:10.1093/toxsci/kfad017
• Frost, K. L., Jilek, J. L., Sinari, S., Klein, R. R., Billheimer, D., Wright, S. H., & Cherrington, N. J. (2023). Renal Transporter Alterations in Patients with Chronic Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-Associated, Viral Hepatitis, and Alcohol-Viral Combination. Drug metabolism and disposition: the biological fate of chemicals, 51(2), 155-164.
  More info

  Alterations in hepatic transporters have been identified in precirrhotic chronic liver diseases (CLDs) that result in pharmacokinetic variations causing adverse drug reactions (ADRs). However, the effect of CLD on the expression of renal transporters is unknown despite the overwhelming evidence of kidney injury in CLD patients. This study determines the transcriptomic and proteomic expression profiles of renal drug transporters in patients with defined CLD etiology. Renal biopsies were obtained from patients with a history of CLD etiologies, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), viral hepatitis C (HCV), and combination ALD/HCV. A significant decrease in organic anion transporter (OAT)-3 was identified in NASH, ALD, HCV, and ALD/HCV (1.56 ± 1.10; 1.01 ± 0.46; 1.03 ± 0.43; 0.86 ± 0.57 pmol/mg protein) relative to control (2.77 ± 1.39 pmol/mg protein). Additionally, a decrease was shown for OAT4 in NASH (24.9 ± 5.69 pmol/mg protein) relative to control (43.8 ± 19.9 pmol/mg protein) and in urate transporter 1 (URAT1) for ALD and HCV (1.56 ± 0.15 and 1.65 ± 0.69 pmol/mg protein) relative to control (4.69 ± 4.59 pmol/mg protein). These decreases in organic anion transporter expression could result in increased and prolonged systemic exposure to drugs and possible toxicity. Renal transporter changes, in addition to hepatic transporter alterations, should be considered in dose adjustments for CLD patients for a more accurate disposition profile. It is important to consider a multiorgan approach to altered pharmacokinetics of drugs prescribed to CLD patients to prevent ADRs and improve patient outcomes. SIGNIFICANCE STATEMENT: Chronic liver diseases are known to elicit alterations in hepatic transporters that result in a disrupted pharmacokinetic profile for various drugs. However, it is unknown if there are alterations in renal transporters during chronic liver disease, despite strong indications of renal dysfunction associated with chronic liver disease. Identifying renal transporter expression changes in patients with chronic liver disease facilitates essential investigations on the multifaceted relationship between liver dysfunction and kidney physiology to offer dose adjustments and prevent adverse drug reactions.
• Frost, K. L., Jilek, J. L., Toth, E. L., Goedken, M. J., Wright, S. H., & Cherrington, N. J. (2023). Representative Rodent Models for Renal Transporter Alterations in Human Nonalcoholic Steatohepatitis. Drug metabolism and disposition: the biological fate of chemicals, 51(8), 970-981.
  More info

  Alterations in renal elimination processes of glomerular filtration and active tubular secretion by renal transporters can result in adverse drug reactions. Nonalcoholic steatohepatitis (NASH) alters hepatic transporter expression and xenobiotic elimination, but until recently, renal transporter alterations in NASH were unknown. This study investigates renal transporter changes in rodent models of NASH to identify a model that recapitulates human alterations. Quantitative protein expression by surrogate peptide liquid chromatography-coupled mass spectrometry (LC-MS/MS) on renal biopsies from NASH patients was used for concordance analysis with rodent models, including methionine/choline deficient (MCD), atherogenic (Athero), or control rats and MCD (), C57BL/6J fast-food thioacetamide (FFDTH), American lifestyle-induced obesity syndrome (ALIOS), or control mice. Demonstrating clinical similarity to NASH patients, FFDTH, and ALIOS showed decreases in glomerular filtration rate (GFR) by 76%, 28%, and 24%. Organic anion transporter 3 (OAT3) showed an upward trend in all models except the FFDTH (from 3.20 to 2.39 pmol/mg protein), making the latter the only model to represent human OAT3 changes. OAT5, a functional ortholog of human OAT4, significantly decreased in FFDTH, and ALIOS (from 4.59 to 0.45, 1.59, and 2.83 pmol/mg protein, respectively) but significantly increased for MCD (1.67 to 4.17 pmol/mg protein), suggesting that the mouse models are comparable to human for these specific transport processes. These data suggest that variations in rodent renal transporter expression are elicited by NASH, and the concordance analysis enables appropriate model selection for future pharmacokinetic studies based on transporter specificity. These models provide a valuable resource to extrapolate the consequences of human variability in renal drug elimination. SIGNIFICANCE STATEMENT: Rodent models of nonalcoholic steatohepatitis that recapitulate human renal transporter alterations are identified for future transporter-specific pharmacokinetic studies to facilitate the prevention of adverse drug reactions due to human variability.
• Hau, R. K., Wright, S. H., & Cherrington, N. J. (2023).

  Addressing the Clinical Importance of Equilibrative Nucleoside Transporters in Drug Discovery and Development

  . Clinical Pharmacology & Therapeutics, 114(4), 780-794. doi:10.1002/cpt.2984
• Hau, R. K., Wright, S. H., & Cherrington, N. J. (2023). Drug Transporters at the Human Blood-Testis Barrier. Drug metabolism and disposition: the biological fate of chemicals, 51(5), 560-571.
  More info

  Transporters are involved in the movement of many physiologically important molecules across cell membranes and have a substantial impact on the pharmacological and toxicological effect of xenobiotics. Many transporters have been studied in the context of disposition to, or toxicity in, organs such as the kidney and liver; however, transporters in the testes are increasingly gaining recognition for their role in drug transport across the blood-testis barrier (BTB). The BTB is an epithelial membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules that form intercellular junctional complexes to protect developing germ cells from the external environment. Consequently, many charged or large polar molecules cannot cross this barrier without assistance from a transporter. SCs express a variety of drug uptake and efflux transporters to control the flux of endogenous and exogenous molecules across the BTB. Recent studies have identified several transport pathways in SCs that allow certain drugs to circumvent the human BTB. These pathways may exist in other species, such as rodents and nonhuman primates; however, there is (1) a lack of information on their expression and/or localization in these species, and (2) conflicting reports on localization of some transporters that have been evaluated in rodents compared with humans. This review outlines the current knowledge on the expression and localization of pharmacologically relevant drug transporters in human testes and calls attention to the insufficient and contradictory understanding of testicular transporters in other species that are commonly used in drug disposition and toxicity studies. SIGNIFICANCE STATEMENT: While the expression, localization, and function of many xenobiotic transporters have been studied in organs such as the kidney and liver, the characterization of transporters in the testes is scarce. This review summarizes the expression and localization of common pharmacologically-relevant transporters in human testes that have significant implications for the development of drugs that can cross the blood-testis barrier. Potential expression differences between humans and rodents highlighted here suggest rodents may be inappropriate for some testicular disposition and toxicity studies.
• Hau, R. K., Wright, S. H., & Cherrington, N. J. (2023). In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier. Drug metabolism and disposition: the biological fate of chemicals, 51(9), 1157-1168.
  More info

  The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.
• Jung, A. M., Beitel, S. C., Gutenkunst, S. L., Billheimer, D., Jahnke, S. A., Littau, S. R., White, M., Hoppe-Jones, C., Cherrington, N., & Burgess, J. L. (2023). Excretion of polybrominated diphenyl ethers and AhR activation in breastmilk among firefighters. Toxicological sciences : an official journal of the Society of Toxicology, 192(2), 223-32.
  More info

  Excretion of toxicants accumulated from firefighter exposures through breastmilk represents a potential hazard. We investigated if firefighting exposures could increase the concentration of polybrominated diphenyl ethers (PBDEs) and aryl hydrocarbon receptor (AhR) activation in excreted breastmilk. Firefighters and non-firefighters collected breastmilk samples prior to any firefighting responses (baseline) and at 2, 8, 24, 48, and 72 hours after a structural fire (firefighters only). Five PBDE analytes (BDEs 15, 28, 47, 99, and 153) detected in at least 90% of samples were summed for analyses. The AhR in vitro DR CALUX® bioassay assessed the mixture of dioxin-like compounds and toxicity from breastmilk extracts. Baseline PBDEs and AhR response were compared between firefighters and non-firefighters. Separate linear mixed models assessed changes in sum of PBDEs and AhR response among firefighters over time and effect modification by interior or exterior response was assessed. Baseline PBDE concentrations and AhR responses did not differ between the 21 firefighters and 10 non-firefighters. There were no significant changes in sum of PBDEs or AhR response among firefighters over time post-fire, and no variation by interior or exterior response. Plots of sum of PBDEs and AhR response over time demonstrated individual variation but no consistent pattern. Currently, our novel study results do not support forgoing breastfeeding after a fire exposure. However, given study limitations and the potential hazard of accumulated toxicants from firefighter exposures excreted via breastfeeding, future studies should consider additional contaminants and measures of toxicity by which firefighting may impact maternal and child health.
• Marie, S., Frost, K. L., Hau, R. K., Martinez-Guerrero, L., Izu, J. M., Myers, C. M., Wright, S. H., & Cherrington, N. J. (2023). Predicting disruptions to drug pharmacokinetics and the risk of adverse drug reactions in non-alcoholic steatohepatitis patients. Acta pharmaceutica Sinica. B, 13(1), 1-28.
  More info

  The liver plays a central role in the pharmacokinetics of drugs through drug metabolizing enzymes and transporters. Non-alcoholic steatohepatitis (NASH) causes disease-specific alterations to the absorption, distribution, metabolism, and excretion (ADME) processes, including a decrease in protein expression of basolateral uptake transporters, an increase in efflux transporters, and modifications to enzyme activity. This can result in increased drug exposure and adverse drug reactions (ADRs). Our goal was to predict drugs that pose increased risks for ADRs in NASH patients. Bibliographic research identified 71 drugs with reported ADRs in patients with liver disease, mainly non-alcoholic fatty liver disease (NAFLD), 54 of which are known substrates of transporters and/or metabolizing enzymes. Since NASH is the progressive form of NAFLD but is most frequently undiagnosed, we identified other drugs at risk based on NASH-specific alterations to ADME processes. Here, we present another list of 71 drugs at risk of pharmacokinetic disruption in NASH, based on their transport and/or metabolism processes. It encompasses drugs from various pharmacological classes for which ADRs may occur when used in NASH patients, especially when eliminated through multiple pathways altered by the disease. Therefore, these results may inform clinicians regarding the selection of drugs for use in NASH patients.
• Vignaux, P. A., Lane, T. R., Puhl, A. C., Hau, R. K., Wright, S. H., Cherrington, N. J., & Ekins, S. (2023). Transporter Inhibition Profile for the Antivirals Tilorone, Quinacrine and Pyronaridine. ACS omega, 8(13), 12532-12537.
  More info

  Pyronaridine, tilorone and quinacrine are cationic molecules that have activity against Ebola, SARS-CoV-2 and other viruses. All three molecules have also demonstrated activity against Ebola in mice, while pyronaridine showed efficacy against SARS-CoV-2 in mice. We have recently tested these molecules and other antivirals against human organic cation transporters (OCTs) and apical multidrug and toxin extruders (MATEs). Quinacrine was found to be an inhibitor of OCT2, while tilorone and pyronaridine were less potent, and these displayed variability depending on the substrate used. To assess whether any of these three molecules have other potential interactions with additional transporters, we have now screened them at 10 μM against various human efflux and uptake transporters including P-gp, OATP1B3, OAT1, OAT3, MRP1, MRP2, MRP3, BCRP, as well as confirmational testing against OCT1, OCT2, MATE1 and MATE2K. Interestingly, in this study tilorone appears to be a more potent inhibitor of OCT1 and OCT2 than pyronaridine or quinacrine. However, both pyronaridine and quinacrine appear to be more potent inhibitors of MATE1 and MATE2K. None of the three compounds inhibited MRP1, MRP2, MRP3, OAT1, OAT3, P-gp or OATP1B3. Similarly, we previously showed that tilorone and pyronaridine do not inhibit OATP1B1 and have confirmed that quinacrine behaves similarly. In total, these observations suggest that the three compounds only appear to interact with OCTs and MATEs to differing extents, suggesting they may be involved in fewer clinically relevant drug-transporter interactions involving pharmaceutical substrates of the other major transporters tested.
• Cherrington, N. J., Hau, R. K., & Wright, S. H. (2022). PF ‐07321332 (Nirmatrelvir) does not interact with human ENT1 or ENT2 : Implications for COVID ‐19 patients. Clinical and Translational Science, 15(7), 1599-1605. doi:10.1111/cts.13292
• Cherrington, N. J., Wright, S. H., Billheimer, D., Klein, R. R., Sinari, S., Yu, A., & Frost, K. L. (2022). Renal Transporter Alterations in Patients with Chronic Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-associated, Viral Hepatitis, and Alcohol-Viral Combination. Drug Metabolism and Disposition. doi:10.1124/dmd.122.001038
  More info

  Alterations in hepatic transporters have been identified in pre-cirrhotic chronic liver diseases (CLD) that result in pharmacokinetic variations causing adverse drug reactions (ADRs). However, the effect of CLD on the expression of renal transporters is unknown despite the overwhelming evidence of kidney injury in CLD patients. This study determines the transcriptomic and proteomic expression profiles of renal drug transporters in patients with defined CLD etiology. Renal biopsies were obtained from patients with a history of CLD etiologies, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), viral hepatitis C (HCV), and combination ALD/HCV. A significant decrease in organic anion transporter 3 (OAT3) was identified in NASH, ALD, HCV, and ALD/HCV (1.56{plus minus} 1.10; 1.01{plus minus} 0.46; 1.03{plus minus} 0.43; 0.86{plus minus} 0.57 pmol/mg protein) relative to control (2.77{plus minus} 1.39 pmol/mg protein). Additionally, a decrease was shown for OAT4 in NASH (24.9{plus minus} 5.69 pmol/mg protein) relative to control (43.8{plus minus} 19.9 pmol/mg protein) and in urate transporter 1 (URAT1) for ALD and HCV (1.56{plus minus} 0.15 and 1.65{plus minus} 0.69 pmol/mg protein) relative to control (4.69{plus minus} 4.59 pmol/mg protein). These decreases in organic anion transporter expression could result in increased and prolonged systemic exposure to drugs and possible toxicity. Renal transporter changes, in addition to hepatic transporter alterations should be considered in dose adjustments for CLD patients for a more accurate disposition profile. It is important to consider a multi-organ approach to altered pharmacokinetics of drugs prescribed to CLD patients to prevent ADRs and improve patient outcomes. Significance Statement Chronic liver diseases are known to elicit alterations in hepatic transporters that result in a disrupted pharmacokinetic profile for various drugs. However, it is unknown if there are alterations in renal transporters during chronic liver disease, despite strong indications of renal dysfunction associated with chronic liver disease. Identifying renal transporter expression changes in patients with chronic liver disease facilitates essential investigations on the multifaceted relationship between liver dysfunction and kidney physiology to offer dose adjustments and prevent adverse drug reactions.
• Frost, K. L., Jilek, J. L., Thompson, A. D., Klein, R. R., Sinari, S., Torabzedehkorasani, E., Billheimer, D. D., Schnellmann, R. G., & Cherrington, N. J. (2022). Increased Renal Expression of Complement Components in Patients With Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-Associated, Viral Hepatitis, and Alcohol-Viral Combination. Toxicological sciences : an official journal of the Society of Toxicology, 189(1), 62-72.
  More info

  Inflammatory liver diseases, including nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), hepatitis C virus (HCV), and ALD/HCV, account for nearly 2 million deaths annually. Despite increasing evidence that liver dysfunction impacts renal physiology, there is limited supportive clinical information, due to limited diagnosis of liver disease, complexity in liver disease etiology, and inadequacy of renal function tests. Human kidney biopsies with liver and renal pathology were obtained from patients with nonalcoholic fatty liver disease (NAFLD), NASH, ALD, HCV, and ALD/HCV (n = 5-7). Each liver disease showed renal pathology with at least 50% interstitial nephritis, 50% interstitial fibrosis, and renal dysfunction by estimated glomerular filtration rate (NAFLD 36.7 ± 21.4; NASH 32.7 ± 15.0; ALD 16.0 ± 11.0; HCV 27.6 ± 11.5; ALD/HCV 21.0 ± 11.2 ml/min/1.73 m2). Transcriptomic analysis identified 55 genes with expression changes in a conserved direction in response to liver disease. Considering association with immune regulation, protein levels of alpha-2-macroglobulin, clusterin, complement C1q C chain (C1QC), CD163, and joining chain of multimeric IgA and IgM (JCHAIN) were further quantified by LC-MS/MS. C1QC demonstrated an increase in NASH, ALD, HCV, and ALD/HCV (42.9 ± 16.6; 38.8 ± 18.4; 39.0 ± 13.5; 40.1 ± 20.1 pmol/mg protein) relative to control (19.2 ± 10.4 pmol/mg protein; p ≤ 0.08). Renal expression changes identified in inflammatory liver diseases with interstitial pathology suggest the pathogenesis of liver associated renal dysfunction. This unique cohort overcomes diagnostic discrepancies and sample availability to provide insight for mechanistic investigations on the impact of liver dysfunction on renal physiology.
• Hau, R. K., Tash, J. S., Georg, G. I., Wright, S. H., & Cherrington, N. J. (2022). Physiological Characterization of the Transporter-Mediated Uptake of the Reversible Male Contraceptive H2-Gamendazole Across the Blood-Testis Barrier. The Journal of pharmacology and experimental therapeutics, 382(3), 299-312.
  More info

  The blood-testis barrier (BTB) is formed by a tight network of Sertoli cells (SCs) to limit the movement of reproductive toxicants from the blood into the male genital tract. Transporters expressed at the basal membranes of SCs also influence the disposition of drugs across the BTB. The reversible, nonhormonal contraceptive, H2-gamendazole (H2-GMZ), is an indazole carboxylic acid analog that accumulates over 10 times more in the testes compared with other organs. However, the mechanism(s) by which H2-GMZ circumvents the BTB are unknown. This study describes the physiologic characteristics of the carrier-mediated process(es) that permit H2-GMZ and other analogs to penetrate SCs. Uptake studies were performed using an immortalized human SC line (hT-SerC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Uptake of H2-GMZ and four analogs followed Michaelis-Menten transport kinetics (one analog exhibited poor penetration). H2-GMZ uptake was strongly inhibited by indomethacin, diclofenac, MK-571, and several analogs. Moreover, H2-GMZ uptake was stimulated by an acidic extracellular pH, reduced at basic pHs, and independent of extracellular Na, K, or Cl levels, which are intrinsic characteristics of OATP-mediated transport. Therefore, the characteristics of H2-GMZ transport suggest that one or more OATPs may be involved. However, endogenous transporter expression in wild-type Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human embryonic kidney-293 (HEK-293) cells limited the utility of heterologous transporter expression to identify a specific OATP transporter. Altogether, characterization of the transporters involved in the flux of H2-GMZ provides insight into the selectivity of drug disposition across the human BTB to understand and overcome the pharmacokinetic and pharmacodynamic difficulties presented by this barrier. SIGNIFICANCE STATEMENT: Despite major advancements in female contraceptives, male alternatives, including vasectomy, condom usage, and physical withdrawal, are antiquated and the widespread availability of nonhormonal, reversible chemical contraceptives is nonexistent. Indazole carboxylic acid analogs such as H2-GMZ are promising new reversible, antispermatogenic drugs that are highly effective in rodents. This study characterizes the carrier-mediated processes that permit H2-GMZ and other drugs to enter Sertoli cells and the observations made here will guide the development of drugs that effectively circumvent the BTB.
• Jilek, J. L., Frost, K. L., Marie, S., Myers, C. M., Goedken, M., Wright, S. H., & Cherrington, N. J. (2022). Attenuated Ochratoxin A Transporter Expression in a Mouse Model of Nonalcoholic Steatohepatitis Protects against Proximal Convoluted Tubule Toxicity. Drug metabolism and disposition: the biological fate of chemicals, 50(10), 1389-1395.
  More info

  Ochratoxin A (OTA) is an abundant mycotoxin, yet the toxicological impact of its disposition is not well studied. OTA is an organic anion transporter (OAT) substrate primarily excreted in urine despite a long half-life and extensive protein binding. Altered renal transporter expression during disease, including nonalcoholic steatohepatitis (NASH), may influence response to OTA exposure, but the impact of NASH on OTA toxicokinetics, tissue distribution, and associated nephrotoxicity is unknown. By inducing NASH in fast food-dieted/thioacetamide-exposed mice, we evaluated the effect of NASH on a bolus OTA exposure (12.5 mg/kg by mouth) after 3 days. NASH mice presented with less gross toxicity (44% less body weight loss), and kidney and liver weights of NASH mice were 11% and 24% higher, respectively, than healthy mice. Organ and body weight changes coincided with reduced renal proximal tubule cells vacuolation, degeneration, and necrosis, though no OTA-induced hepatic lesions were found. OTA systemic exposure in NASH mice increased modestly from 5.65 ± 1.10 to 7.95 ± 0.61 mg*h/ml per kg BW, and renal excretion increased robustly from 5.55% ± 0.37% to 13.11% ± 3.10%, relative to healthy mice. Total urinary excretion of OTA increased from 24.41 ± 1.74 to 40.07 ± 9.19 g in NASH mice, and kidney-bound OTA decreased by ∼30%. Renal OAT isoform expression (OAT1-5) in NASH mice decreased by ∼50% with reduced OTA uptake by proximal convoluted cells. These data suggest that NASH-induced OAT transporter reductions attenuate renal secretion and reabsorption of OTA, increasing OTA urinary excretion and reducing renal exposure, thereby reducing nephrotoxicity in NASH. SIGNIFICANCE STATEMENT: These data suggest a disease-mediated transporter mechanism of altered tissue-specific toxicity after mycotoxin exposure, despite minimal systemic changes to ochratoxin A (OTA) concentrations. Further studies are warranted to evaluate the clinical relevance of this functional model and the potential effect of human nonalcoholic steatohepatitis on OTA and other organic anion substrate toxicity.
• Kovalchuk, N., Jilek, J. L., Van Winkle, L. S., Cherrington, N. J., & Ding, X. (2022). Role of Lung P450 Oxidoreductase in Paraquat-Induced Collagen Deposition in the Lung. Antioxidants (Basel, Switzerland), 11(2).
  More info

  Paraquat (PQ) is an agrochemical known to cause pulmonary fibrosis. PQ-induced collagen deposition in the lung is thought to require enzymatic formation of PQ radicals, but the specific enzymes responsible for this bioactivation event in vivo have not been identified. We tested the hypothesis that lung P450 oxidoreductase (POR or CPR) is important in PQ-induced lung fibrosis in mice. A lung--null mouse model was utilized, which undergoes doxycycline-induced, Cre recombinase-mediated deletion of the gene specifically in airway Club cells and alveolar type 2 cells in the lung. The lungs of lung--null mice and their wild-type littermates were collected on day 15 after a single intraperitoneal injection of saline (control) or PQ (20 mg/kg). Lung tissue sections were stained with picrosirius red for detection of collagen fibrils. Fibrotic lung areas were found to be significantly smaller (1.6-fold for males and 1.4-fold for females) in PQ-treated lung--null mice than in sex- and treatment-matched wild-type mice. The levels of collagen in lung tissue homogenate were also lower (1.4-2.3-fold; < 0.05) in PQ-treated lung--null mice compared to PQ-treated wild-type mice. In contrast, plasma PQ toxicokinetic profiles were not different between sex-matched wild-type and lung--null mice. Taken together, these results indicate that lung POR plays an important role in PQ-induced pulmonary fibrosis.
• Tripp, D. K., Marie, S., & Cherrington, N. J. (2022). Strategies to Diagnose Nonalcoholic Steatohepatitis: A Novel Approach to Take Advantage of Pharmacokinetic Alterations.. Drug metabolism and disposition: the biological fate of chemicals, 50(4), 492-499. doi:10.1124/dmd.121.000413
  More info

  Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is diagnosed by a liver biopsy. Because of the invasiveness of a biopsy, the majority of patients with NASH are undiagnosed. Additionally, the prevalence of NAFLD and NASH creates the need for a simple screening method to differentiate patients with NAFLD versus NASH. Noninvasive strategies for diagnosing NAFLD versus NASH have been developed, typically relying on imaging techniques and endogenous biomarker panels. However, each technique has limitations, and none can accurately predict the associated functional impairment of drug metabolism and disposition. The function of several drug-metabolizing enzymes and drug transporters has been described in NASH that impacts drug pharmacokinetics. The aim of this review is to give an overview of the existing noninvasive strategies to diagnose NASH and to propose a novel strategy based on altered pharmacokinetics using an exogenous biomarker whose disposition and elimination pathways are directly impacted by disease progression. Altered disposition of safe and relatively inert exogenous compounds may provide the sensitivity and specificity needed to differentiate patients with NAFLD and NASH to facilitate a direct indication of hepatic impairment on drug metabolism and prevent subsequent adverse drug reactions. SIGNIFICANCE STATEMENT: This review provides an overview of the main noninvasive techniques (imaging and panels of biomarkers) used to diagnose NAFLD and NASH along with a biopsy. Pharmacokinetic changes have been identified in NASH, and this review proposes a new approach to predict NASH and the related risk of adverse drug reactions based on the assessment of drug elimination disruption using exogenous biomarkers.
• Wright, S. H., Klein, R. R., Hau, R. K., & Cherrington, N. J. (2022). Localization of Xenobiotic Transporters Expressed at the Human Blood-Testis Barrier.. Drug metabolism and disposition: the biological fate of chemicals, DMD-AR-2021-000748. doi:10.1124/dmd.121.000748
  More info

  The blood-testis barrier (BTB) is formed by basal tight junctions between adjacent Sertoli cells (SCs) of the seminiferous tubules and acts as a physical barrier to protect developing germ cells in the adluminal compartment from reproductive toxicants. Xenobiotics, including antivirals, male contraceptives, and cancer chemotherapeutics, are known to cross the BTB, although the mechanisms that permit barrier circumvention are generally unknown. This study used immunohistological staining of human testicular tissue to determine the site of expression for xenobiotic transporters that facilitate transport across the BTB. OAT1, OAT2, and OCTN1 primarily localized to the basal membrane of SCs, whereas OCTN2, MRP3, MRP6, and MRP7 localized to SC basal membranes and peritubular myoid cells (PMCs) surrounding the seminiferous tubules. CNT2 localized to Leydig cells (LCs), PMCs, and SC apicolateral membranes. OCT1, OCT2, and OCT3 mostly localized to PMCs and LCs, although there was minor staining in developing germ cells for OCT3. OATP1A2, OATP1B1, OATP1B3, OATP2A1, OATP2B1 and OATP3A1-v2 localized to SC basal membranes with diffuse staining for some transporters. Notably, OATP1C1 and OATP4A1 primarily localized to LCs. Positive staining for MATE1 was only observed throughout the adluminal compartment. Definitive staining for CNT1, OAT3, MATE2, and OATP6A1 was not observed. The location of these transporters is consistent with their involvement in the movement of xenobiotics across the BTB. Altogether, the localization of these transporters provides insight into the mechanisms of drug disposition across the BTB and will be useful in developing tools to overcome the pharmacokinetic and pharmacodynamic difficulties presented by the BTB. Significance Statement Although the total mRNA and protein expression of drug transporters in the testes has been explored, the localization of many transporters at the blood-testis barrier (BTB) has not been determined. This study applied immunohistological staining in human testicular tissues to identify the cellular localization of drug transporters in the testes. The observations made in this study have implications for the development of drugs that can effectively use transporters expressed at the basal membranes of Sertoli cells to bypass the BTB.
• Zorn, K. M., Wright, S. H., Miller, S. R., Mcgrath, M. E., Ekins, S., & Cherrington, N. J. (2022). Response to Comments on "Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites".. Molecular pharmacology, 101(2), 121-122. doi:10.1124/molpharm.121.000448
• Cherrington, N. J., Galligan, J. J., Miller, S. R., Jilek, J. L., McGrath, M. E., Hau, R. K., Jennings, E. Q., & Wright, S. H. (2021). Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters. Pharmacology Research & Perspectives, 9(4). doi:10.1002/prp2.831
• Cherrington, N., Frost, K., & Jilek, J. (2021). Human Renal Xenobiotic Transporter Expression is Altered in Progression of Non‐Alcoholic Fatty Liver Disease as revealed by Quantitative Targeted Proteomics. The FASEB Journal, 35(S1). doi:10.1096/fasebj.2021.35.s1.02733
• Jilek, J. L., Frost, K. L., Jacobus, K. A., He, W., Toth, E. L., Goedken, M., & Cherrington, N. J. (2021). Altered cisplatin pharmacokinetics during nonalcoholic steatohepatitis contributes to reduced nephrotoxicity. Acta pharmaceutica Sinica. B, 11(12), 3869-3878.
  More info

  Disease-mediated alterations to drug disposition constitute a significant source of adverse drug reactions. Cisplatin (CDDP) elicits nephrotoxicity due to exposure in proximal tubule cells during renal secretion. Alterations to renal drug transporter expression have been discovered during nonalcoholic steatohepatitis (NASH), however, associated changes to substrate toxicity is unknown. To test this, a methionine- and choline-deficient diet-induced rat model was used to evaluate NASH-associated changes to CDDP pharmacokinetics, transporter expression, and toxicity. NASH rats administered CDDP (6 mg/kg, i.p.) displayed 20% less nephrotoxicity than healthy rats. Likewise, CDDP renal clearance decreased in NASH rats from 7.39 to 3.83 mL/min, renal secretion decreased from 6.23 to 2.80 mL/min, and renal CDDP accumulation decreased by 15%, relative to healthy rats. Renal copper transporter-1 expression decreased, and organic cation transporter-2 and ATPase copper transporting protein-7b increased slightly, reducing CDDP secretion. Hepatic CDDP accumulation increased 250% in NASH rats relative to healthy rats. Hepatic organic cation transporter-1 induction and multidrug and toxin extrusion protein-1 and multidrug resistance-associated protein-4 reduction may contribute to hepatic CDDP sequestration in NASH rats, although no drug-related toxicity was observed. These data provide a link between NASH-induced hepatic and renal transporter expression changes and CDDP renal clearance, which may alter nephrotoxicity.
• Miller, S. R., Hau, R. K., Cherrington, N. J., Miller, S. R., Hau, R. K., & Cherrington, N. J. (2021). Implications of Species Differences in Function and Localization of Transporters at the Blood-Testis Barrier.. Toxicological sciences : an official journal of the Society of Toxicology, 181(1), 1-2. doi:10.1093/toxsci/kfab024
• Wright, S. H., Myers, C. M., Marie, S., Jilek, J. L., Goedken, M., Frost, K. L., & Cherrington, N. J. (2021). Attenuated Ochratoxin A Transporter Expression in a Mouse Model of Nonalcoholic Steatohepatitis Protects against Proximal Convoluted Tubule Toxicity.. Drug metabolism and disposition: the biological fate of chemicals, DMD-MR-2021-000451. doi:10.1124/dmd.121.000451
  More info

  Ochratoxin A (OTA) is an abundant mycotoxin, yet the toxicological impact of its disposition is not well studied. OTA is an organic anion transporter (OAT) substrate primarily excreted in urine despite a long half-life and extensive protein binding. Altered renal transporter expression during disease, including nonalcoholic steatohepatitis (NASH), may influence response to OTA exposure, but the impact of NASH on OTA toxicokinetics, tissue distribution, and associated nephrotoxicity are unknown. By inducing NASH in fast food-dieted/thioacetamide-exposed mice, we evaluated the effect of NASH on a bolus OTA exposure (12.5 mg/kg p.o.) after 3 days. NASH mice presented with less gross toxicity (44% less bodyweight loss) and kidney and liver weights of NASH mice were 11% and 24%, respectively, higher than healthy mice. Organ and body weight changes coincided with reduced renal proximal tubule cells vacuolation, degeneration and necrosis though no OTA-induced hepatic lesions were found. OTA systemic exposure in NASH mice increased modestly from 5.65 {plus minus} 1.10 to 7.95 {plus minus} 0.61 mg*h/mL/kg BW, renal excretion increased robustly from 5.55 {plus minus} 0.37% to 13.11 {plus minus} 3.10%, relative to healthy mice. Total urinary excretion of OTA increased from 24.41 {plus minus} 1.74 to 40.07 {plus minus} 9.19 μg in NASH mice and kidney-bound OTA decreased ~30%. Renal OAT isoform expression (OAT1-5) in NASH mice decreasd by ~50% with reduced OTA uptake by proximal convoluted cells. These data suggest that NASH-induced OAT transporter reductions attenuate renal secretion and reabsorption of OTA, increasing OTA urinary excretion and reducing renal exposure, thereby reducing nephrotoxicity in NASH. Significance Statement These data suggest a disease-mediated transporter mechanism of altered tissue-specific toxicity following mycotoxin exposure, despite minimal systemic changes to OTA concentrations. Further studies are warranted to evaluate the clinical relevance of this functional model and the potential effect of human NASH on OTA and other organic anion substrate toxicity.
• Zorn, K. M., Galligan, J. J., Zhang, X., Zorn, K. M., Zhang, X., Wright, S. H., Miller, S. R., Wright, S. H., Jilek, J. L., Miller, S. R., Jennings, E. Q., Jilek, J. L., Hau, R. K., Jennings, E. Q., Hau, R. K., Galligan, J. J., Foil, D. H., Foil, D. H., Ekins, S., , Ekins, S., et al. (2021). Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.. Molecular pharmacology, 99(2), 147-162. doi:10.1124/molpharm.120.000169
  More info

  Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.
• Zorn, K. M., Wright, S. H., Miller, S. R., Lane, T. R., Ekins, S., Cherrington, N. J., Zorn, K. M., Wright, S. H., Miller, S. R., Lane, T. R., Ekins, S., & Cherrington, N. J. (2021). Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1.. Drug metabolism and disposition: the biological fate of chemicals, 49(7), 479-489. doi:10.1124/dmd.121.000423
  More info

  Equilibrativenucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors using Kt and IC50 data curated from the literature. Substrate pharmacophores for ENT1 and ENT2 are distinct, with partial overlap of hydrogen bond donors, whereas the inhibitor pharmacophores predominantly feature hydrogen bond acceptors. Mizoribine and ribavirin mapped to the ENT1 substrate pharmacophore and proved to be substrates of the ENTs. The presence of the ENT-specific inhibitor 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) decreased mizoribine accumulation in ENT1 and ENT2 cells (ENT1, ∼70% decrease, P = 0.0046; ENT2, ∼50% decrease, P = 0.0012). NBMPR also decreased ribavirin accumulation in ENT1 and ENT2 cells (ENT1: ∼50% decrease, P = 0.0498; ENT2: ∼30% decrease, P = 0.0125). Darunavir mapped to the ENT1 inhibitor pharmacophore and NBMPR did not significantly influence darunavir accumulation in either ENT1 or ENT2 cells (ENT1: P = 0.28; ENT2: P = 0.53), indicating that darunavir's interaction with the ENTs is limited to inhibition. These computational and in vitro models can inform compound selection in the drug discovery and development process, thereby reducing time and expense of identification and optimization of ENT-interacting compounds. SIGNIFICANCE STATEMENT: This study developed computational models of human equilibrative nucleoside transporters (ENTs) to predict drug interactions and validated these models with two compounds in vitro. Identification and prediction of ENT1 and ENT2 substrates allows for the determination of drugs that can penetrate tissues expressing these transporters.
• Zorn, K. M., Wright, S. H., Miller, S. R., Mcgrath, M. E., Ekins, S., & Cherrington, N. J. (2021). Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites.. Molecular pharmacology, 100(6), 548-557. doi:10.1124/molpharm.121.000333
  More info

  Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs, remdesivir, molnupiravir, and molnupiravir's active metabolite, β-d-N4-hydroxycytidine (EIDD-1931), and four non-nucleoside molecules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H]uridine uptake (ENT1 IC50: 39 μM; ENT2 IC50: 77 μM), followed by EIDD-1931 (ENT1 IC50: 259 μM; ENT2 IC50: 467 μM), whereas molnupiravir was a modest inhibitor (ENT1 IC50: 701 μM; ENT2 IC50: 851 μM). Other proposed antivirals failed to inhibit ENT-mediated [3H]uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) by 30% in ENT1 cells (P = 0.0248) and 27% in ENT2 cells (P = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (P = 0.0463) and by 64% in ENT2 cells (P = 0.0132), which supported computational predictions that both are ENT substrates that may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. SIGNIFICANCE STATEMENT: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.
• Frost, K., Toth, E., Jilek, J. L., Jacobus, K., He, W., Goedken, M., & Cherrington, N. J. (2020). Altered Cisplatin Pharmacokinetics during Nonalcoholic Steatohepatitis Contributes to Reduced Nephrotoxicity. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.05732
• Galligan, J. J., Zhang, X. R., Wright, S. H., Miller, S. R., Jennings, E. Q., Hau, R. K., Galligan, J. J., & Cherrington, N. J. (2020). Modeling Blood-Testis Barrier Characteristics with Novel CRISPR/Cas9 Functional ENT1 and ENT2 Knockout HeLa Cell Lines. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.08972
• Morales, M. N., Wright, S. H., Miller, S. R., Jilek, J. L., Hau, R. K., & Cherrington, N. J. (2020). Nucleoside Reverse Transcriptase Inhibitor Interaction with Human Equilibrative Nucleoside Transporters 1 and 2.. Drug metabolism and disposition: the biological fate of chemicals, 48(7), 603-612. doi:10.1124/dmd.120.090720
  More info

  Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 μM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 μM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.
• Toth, E. L., Csanaky, I. L., Clarke, J. D., & Cherrington, N. J. (2020). Interaction of Oatp1b2 expression and nonalcoholic steatohepatitis on pravastatin plasma clearance.. Biochemical pharmacology, 174, 113780. doi:10.1016/j.bcp.2019.113780
  More info

  The downregulation of hepatic uptake transporters, including those of the OATP family, are a well known consequence of nonalcoholic steatohepatitis (NASH). Prior studies have shown that the combination of NASH and Oatp1b2 knockout synergistically reduces the clearance of pravastatin (PRAV) in the methionine and choline deficient (MCD) mouse model of NASH, and the current study therefore aimed to determine the impact of NASH and genetic heterozygosity of Oatp1b2 on PRAV clearance, modeling the overlap between the 24% of the human population who are heterozygous for non-functioning OATP1B1, and the ~15% with NASH, potentially placing these people at higher risk of statin-induced myopathy. Therefore, male C57BL/6 wild-type (WT), Oatp1b2+/- (HET), and Oatp1b2-/- (KO) mice were fed either a control (methionine and choline sufficient) or methionine and choline-deficient (MCD) diet to induce NASH. After six weeks of feeding, pravastatin was administered via the carotid artery. Blood and bile samples were collected throughout 90 min after PRAV administration. The concentration of PRAV in plasma, bile, liver, kidney, and muscle was determined by liquid chromatography-tandem mass spectrometry. MCD diet did not alter the plasma AUC values of PRAV in either WT or HET mice. However, the MCD diet increased plasma AUC by 4.4-fold in KO mice. MCD diet and nonfunctional Oatp1b2 synergistically increased not only plasma AUC but also the extrahepatic tissue concentration of pravastatin, whereas the partially decreased function of Oatp1b2 and NASH together were insufficient in significantly altering PRAV pharmacokinetics. These data suggest that a single copy of fully functional OATP1B1 in NASH patients may be sufficient to avoid the increase of pravastatin toxicity.
• Wright, S. H., Hau, R. K., & Cherrington, N. J. (2020). Expression and Immunolocalization of Xenobiotic Transporters at the Human Blood‐Testis Barrier. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.08992
• Wright, S. H., Miller, S. R., Hau, R. K., & Cherrington, N. J. (2020). Generation of a hTERT-Immortalized Human Sertoli Cell Model to Study Transporter Dynamics at the Blood-Testis Barrier.. Pharmaceutics, 12(11), 1005. doi:10.3390/pharmaceutics12111005
  More info

  The blood-testis barrier (BTB) formed by adjacent Sertoli cells (SCs) limits the entry of many chemicals into seminiferous tubules. Differences in rodent and human substrate-transporter selectivity or kinetics can misrepresent conclusions drawn using rodent in vitro models. Therefore, human in vitro models are preferable when studying transporter dynamics at the BTB. This study describes a hTERT-immortalized human SC line (hT-SerC) with significantly increased replication capacity and minor phenotypic alterations compared to primary human SCs. Notably, hT-SerCs retained similar morphology and minimal changes to mRNA expression of several common SC genes, including AR and FSHR. The mRNA expression of most xenobiotic transporters was within the 2-fold difference threshold in RT-qPCR analysis with some exceptions (OAT3, OCT3, OCTN1, OATP3A1, OATP4A1, ENT1, and ENT2). Functional analysis of the equilibrative nucleoside transporters (ENTs) revealed that primary human SCs and hT-SerCs predominantly express ENT1 with minimal ENT2 expression at the plasma membrane. ENT1-mediated uptake of [3H] uridine was linear over 10 min and inhibited by NBMPR with an IC50 value of 1.35 ± 0.37 nM. These results demonstrate that hT-SerCs can functionally model elements of transport across the human BTB, potentially leading to identification of other transport pathways for xenobiotics, and will guide drug discovery efforts in developing effective BTB-permeable compounds.
• Wright, S. H., Miller, S. R., Hau, R. K., & Cherrington, N. J. (2020). Generation of a hTERT-Immortalized Human Sertoli Cell Model to Study Transporter Dynamics at the Blood-Testis Barrier.. Pharmaceutics, 12(11). doi:10.3390/pharmaceutics12111005
  More info

  The blood-testis barrier (BTB) formed by adjacent Sertoli cells (SCs) limits the entry of many chemicals into seminiferous tubules. Differences in rodent and human substrate-transporter selectivity or kinetics can misrepresent conclusions drawn using rodent in vitro models. Therefore, human in vitro models are preferable when studying transporter dynamics at the BTB. This study describes a hTERT-immortalized human SC line (hT-SerC) with significantly increased replication capacity and minor phenotypic alterations compared to primary human SCs. Notably, hT-SerCs retained similar morphology and minimal changes to mRNA expression of several common SC genes, including AR and FSHR. The mRNA expression of most xenobiotic transporters was within the 2-fold difference threshold in RT-qPCR analysis with some exceptions (OAT3, OCT3, OCTN1, OATP3A1, OATP4A1, ENT1, and ENT2). Functional analysis of the equilibrative nucleoside transporters (ENTs) revealed that primary human SCs and hT-SerCs predominantly express ENT1 with minimal ENT2 expression at the plasma membrane. ENT1-mediated uptake of [3H] uridine was linear over 10 min and inhibited by NBMPR with an IC50 value of 1.35 ± 0.37 nM. These results demonstrate that hT-SerCs can functionally model elements of transport across the human BTB, potentially leading to identification of other transport pathways for xenobiotics, and will guide drug discovery efforts in developing effective BTB-permeable compounds.
• Clarke, J. D., Dzierlenga, A., Arman, T., Toth, E., Li, H., Lynch, K. D., Lynch, D. D., Goedken, M., Paine, M. F., & Cherrington, N. J. (2019). Nonalcoholic fatty liver disease alters microcystin-LR toxicokinetics and acute toxicity. Toxicon, 162, 1-8. doi:10.1016/j.toxicon.2019.03.002
• Lake, A. D., N, H. R., Leamon, C. P., Low, P. S., & Cherrington, N. J. (2019). Folate receptor-beta expression as a diagnostic target in human & rodent nonalcoholic steatohepatitis. Toxicology and Applied Pharmacology, 368, 49-54. doi:10.1016/j.taap.2019.02.009
• Toth, E. L., Clarke, J. D., Csanaky, I. L., & Cherrington, N. J. (2019). Interaction of Oatp1b2 Expression and Nonalcoholic Steatohepatitis on Pravastatin Plasma Clearance. Biochemical Pharmacology. doi:10.1016/j.bcp.2019.113780. [Epub ahead of print]
• Cherrington, N. J., & Dzierlenga, A. L. (2018). Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis. Journal of Biochemical and Molecular Toxicology, 32(3), e22035. doi:10.1002/jbt.22035
• Cherrington, N. J., Toth, E., & Li, H. (2018). Alcohol Metabolism in the Progression of Human Nonalcoholic Steatohepatitis. Toxicological Sciences, 164(2), 428-438. doi:10.1093/toxsci/kfy106
• Dzierlenga, A. L., & Cherrington, N. J. (2018). Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis. Journal of biochemical and molecular toxicology.
  More info

  Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance-associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.
• Li, H., Toth, E., & Cherrington, N. J. (2018). Alcohol Metabolism in the Progression of Human Nonalcoholic Steatohepatitis. Toxicological sciences : an official journal of the Society of Toxicology, 164(2), 428-438.
  More info

  Alcohol metabolism is a well-characterized biological process that is dominated by the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) families. Nonalcoholic steatohepatitis (NASH) is the advanced inflammatory stage of nonalcoholic fatty liver disease (NAFLD) and is known to alter the metabolism and disposition of numerous drugs. The purpose of this study was to investigate the alterations in alcohol metabolism processes in response to human NASH progression. Expression and function of ADHs, ALDHs, and catalase were examined in normal, steatosis, NASH (fatty) and NASH (not fatty) human liver samples. ALDH4A1 mRNA was significantly decreased in both NASH groups, while no significant changes were observed in the mRNA levels of other alcohol-related enzymes. The protein levels of ADH1A, ADH1B, and ADH4 were each decreased in the NASH groups, which was consistent with a decreased overall ADH activity. The protein level of ALDH2 was significantly increased in both NASH groups, while ALDH1A1 and ALDH1B1 were only decreased in NASH (fatty) samples. ALDH activity represented by oxidation of acetaldehyde was decreased in the NASH (fatty) group. The protein level of catalase was decreased in both NASH groups, though activity was unchanged. Furthermore, the significant accumulation of 4-hydroxynonenal protein adduct in NASH indicated significant oxidative stress and a potential reduction in ALDH activity. Collectively, ADH and ALDH expression and function are profoundly altered in the progression of NASH, which may have a notable impact on ADH- and ALDH-associated cellular metabolism processes and lead to significant alterations in drug metabolism mediated by these enzymes.
• Li, H., Toth, E., & Cherrington, N. J. (2018). Asking the Right Questions With Animal Models: Methionine- and Choline-Deficient Model in Predicting Adverse Drug Reactions in Human NASH. Toxicological sciences : an official journal of the Society of Toxicology, 161(1), 23-33.
  More info

  In the past few decades, great conceptual and technological advances have been made in the field of toxicology, but animal model-based research still remains one of the most widely used and readily available tools for furthering our current knowledge. However, animal models are not perfect in predicting all systemic toxicity in humans. Extrapolating animal data to accurately predict human toxicities remains a challenge, and researchers are obligated to question the appropriateness of their chosen animal model. This paper provides an assessment of the utility of the methionine- and choline-deficient (MCD) diet fed animal model in reflecting human nonalcoholic steatohepatitis (NASH) and the potential risks of adverse drug reactions and toxicities that are associated with the disease. As a commonly used NASH model, the MCD model fails to exhibit most metabolic abnormalities in a similar manner to the human disease. The MCD model, on the other hand, closely resembles human NASH histology and reflects signatures of drug transporter alterations in humans. Due to the nature of the MCD model, it should be avoided in studies of NASH pathogenesis, metabolic parameter evaluation, and biomarker identification. But it can be used to accurately predict altered drug disposition due to NASH-associated transporter alterations.
• Miller, S. R., & Cherrington, N. J. (2018). Transepithelial transport across the blood–testis barrier. Reproduction (Cambridge, England).
  More info

  The blood–testis barrier protects developing germ cells by limiting the entry of xenobiotics into the adluminal compartment. There is strong evidence that the male genital tract can serve as a sanctuary site, an area of the body where tumors or viruses are able to survive treatments because most drugs are unable to reach therapeutic concentrations. Recent work has classified the expression and localization of endogenous transporters in the male genital tract as well as the discovery of a transepithelial transport pathway as the molecular mechanism by which nucleoside analogs may be able to circumvent the blood–testis barrier. Designing drug therapies that utilize transepithelial transport pathways may improve drug disposition to this sanctuary site. Strategies that improve disposition into the male genital tract could reduce the rate of testicular relapse, decrease viral load in semen, and improve therapeutic strategies for male fertility.
• Toth, E. L., Li, H., Dzierlenga, A. L., Clarke, J. D., Vildhede, A., Goedken, M., & Cherrington, N. J. (2018). Gene-by-Environment Interaction of Bcrp and Methionine- and Choline-Deficient Diet-Induced Nonalcoholic Steatohepatitis Alters SN-38 Disposition. Drug metabolism and disposition: the biological fate of chemicals, 46(11), 1478-1486.
  More info

  Disease progression to nonalcoholic steatohepatitis (NASH) has profound effects on the expression and function of drug-metabolizing enzymes and transporters, which provide a mechanistic basis for variable drug response. Breast cancer resistance protein (BCRP), a biliary efflux transporter, exhibits increased liver mRNA expression in NASH patients and preclinical NASH models, but the impact on function is unknown. It was shown that the transport capacity of multidrug resistance protein 2 (MRP2) is decreased in NASH. SN-38, the active irinotecan metabolite, is reported to be a substrate for Bcrp, whereas SN-38 glucuronide (SN-38G) is a Mrp2 substrate. The purpose of this study was to determine the function of Bcrp in NASH through alterations in the disposition of SN-38 and SN-38G in a knockout (Bcrp KO) and methionine- and choline-deficient (MCD) model of NASH. Sprague Dawley [wild-type (WT)] rats and Bcrp rats were fed either a methionine- and choline-sufficient (control) or MCD diet for 8 weeks to induce NASH. SN-38 (10 mg/kg) was administered i.v., and blood and bile were collected for quantification by liquid chromatography-tandem mass spectrometry. In Bcrp rats on the MCD diet, biliary efflux of SN-38 decreased to 31.9%, and efflux of SN-38G decreased to 38.7% of control, but WT-MCD and KO-Control were unaffected. These data indicate that Bcrp is not solely responsible for SN-38 biliary efflux, but rather implicate a combined role for BCRP and MRP2. Furthermore, the disposition of SN-38 and SN-38G is altered by Bcrp and NASH in a gene-by-environment interaction and may result in variable drug response to irinotecan therapy in polymorphic patients.
• Cherrington, N. J., Clarke, J. D., Novak, P., Lake, A. D., & Hardwick, R. N. (2017). Impaired N-linked glycosylation of uptake and efflux transporters in human non-alcoholic fatty liver disease. Liver International, 37(7), 1074-1081. doi:10.1111/liv.13362
• Cherrington, N. J., Klein, D. M., Harding, M. C., & Crowther, M. K. (2017). Localization of nucleoside transporters in rat epididymis: Klein et al.. Journal of Biochemical and Molecular Toxicology, 31(8), e21911. doi:10.1002/jbt.21911
• Clarke, J. D., Novak, P., Lake, A. D., Hardwick, R. N., & Cherrington, N. J. (2017). Impaired N-linked glycosylation of uptake and efflux transporters in human non-alcoholic fatty liver disease. Liver international : official journal of the International Association for the Study of the Liver.
  More info

  N-linked glycosylation of proteins is critical for proper protein folding and trafficking to the plasma membrane. Drug transporters are one class of proteins that have reduced function when glycosylation is impaired. N-linked glycosylation of plasma proteins has also been investigated as a biomarker for several liver diseases, including non-alcoholic fatty liver disease (NAFLD). The purpose of this study was to assess the transcriptomic expression of genes involved in protein processing and glycosylation, and to determine the glycosylation status of key drug transporters during human NAFLD progression.
• Han, J., Dzierlenga, A. L., Lu, Z., Billheimer, D. D., Torabzadeh, E., Lake, A. D., Li, H., Novak, P., Shipkova, P., Aranibar, N., Robertson, D., Reily, M. D., Lehman-McKeeman, L. D., & Cherrington, N. J. (2017). Metabolomic profiling distinction of human nonalcoholic fatty liver disease progression from a common rat model. Obesity (Silver Spring, Md.), 25(6), 1069-1076.
  More info

  Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD).
• Klein, D. M., Harding, M. C., Crowther, M. K., & Cherrington, N. J. (2017). Localization of nucleoside transporters in rat epididymis. Journal of biochemical and molecular toxicology, 31(8).
  More info

  The epididymis relies on transporters for the secretion of nucleosides and influence the disposition of nucleoside analogs (NSA). Since these compounds can cross the blood-testis barrier (BTB), it is important to understand if the epididymis reabsorbs NSA drugs. The purpose of this study is to determine the localization of nucleoside transporters expressed within rat epididymis to demonstrate the potential of epididymal reabsorption. Using immunohistochemistry, we determined that equilibrative nucleoside transporter 1 (ENT1) is localized to the basolateral membrane of epithelial cells, ENT2 is expressed in the nucleus of the epithelium and CNT2 is expressed by basal cells. The expression pattern for these transporters suggests that nucleosides are able to access the epithelial cells of the epididymal duct via the blood, but not from the lumen. We did not find any evidence for a transepithelial reabsorption pathway indicating the NSA drugs that cross the BTB remain within the epididymis.
• Lee, S., Kim, S., Hwang, S., Cherrington, N. J., & Ryu, D. Y. (2017). Dysregulated expression of proteins associated with ER stress, autophagy and apoptosis in tissues from nonalcoholic fatty liver disease. Oncotarget, 8(38), 63370-63381.
  More info

  Nonalcoholic fatty liver disease (NAFLD) is categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) and has emerged as a risk factor for more critical clinical conditions. However, the underlying mechanisms of NAFLD pathogenesis are not fully understood. In this study, expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis and autophagy were analyzed in normal, NAFL and NASH human livers by western blotting. Levels of some ER stress-transducing transcription factors, including cleaved activating transcription factor 6, were higher in NASH than in the normal tissues. However, the expression of a majority of the ER chaperones and foldases analyzed, including glucose-regulated protein 78 and ER protein 44, was lower in NASH than in the normal tissues. Levels of apoptosis markers, such as cleaved poly (ADP-ribose) polymerase, were also lower in NASH tissues, in which expression of some B-cell lymphoma-2 family proteins was up- or down-regulated compared to the normal tissues. The level of the autophagy substrate p62 was not different in NASH and normal tissues, although some autophagy regulators were up- or down-regulated in the NASH tissues compared to the normal tissues. Levels of most of the proteins analyzed in NAFL tissues were either similar to those in one of the other two types, NASH and normal, or were somewhere in between. Together, these findings suggest that regulation of certain important tissues processes involved in protein quality control and cell survival were broadly compromised in the NAFLD tissues.
• Li, H., Canet, M. J., Clarke, J. D., Billheimer, D., Xanthakos, S. A., Lavine, J. E., Erickson, R. P., & Cherrington, N. J. (2017). Pediatric Cytochrome P450 Activity Alterations in Nonalcoholic Steatohepatitis. Drug metabolism and disposition: the biological fate of chemicals, 45(12), 1317-1325.
  More info

  Variable drug responses depend on individual variation in the activity of drug-metabolizing enzymes, including cytochrome P450 enzymes (CYP). As the most common chronic liver disease in children and adults, nonalcoholic steatohepatitis (NASH) has been identified as a source of significant interindividual variation in hepatic drug metabolism. Compared with adults, children present age-related differences in pharmacokinetics and pharmacodynamics. The purpose of this study was to determine the impact of fatty liver disease severity on the activity of a variety of CYP enzymes in children and adolescents. Healthy and nonalcoholic fatty liver disease pediatric subjects aged 12-21 years inclusive received an oral cocktail of four probe drugs: caffeine (CYP1A2, 100 mg), omeprazole (CYP2C19, 20 mg), losartan (CYP2C9, 25 mg), and midazolam (CYP3A4, 2 mg). Venous blood and urine were collected before administration and 1, 2, 4, and 6 hours after administration. Concentrations of the parent drugs and CYP-specific metabolites were quantified in plasma and urine using liquid chromatography with tandem mass spectrometry. In plasma, the decreased metabolic area under the curve (AUC) ratio, defined as the metabolite AUC to parent AUC, of omeprazole indicated significant decreases of CYP2C19 (P = 0.002) enzymatic activities in NASH adolescents, while the urine analyses did not show significant differences and were highly variable. A comparison between the present in vivo pediatric studies and a previous ex vivo study in adults indicates distinct differences in the activities of CYP1A2 and CYP2C9. These data demonstrate that pediatric NASH presents an altered pattern of CYP activity and NASH should be considered as a confounder of drug metabolism for certain CYP enzymes. These differences could lead to future investigations that may reveal unexpected variable drug responses that should be considered in pediatric dosage recommendations.
• Li, H., Clarke, J. D., Dzierlenga, A. L., Bear, J., Goedken, M. J., & Cherrington, N. J. (2017). In vivo cytochrome P450 activity alterations in diabetic nonalcoholic steatohepatitis mice. Journal of biochemical and molecular toxicology, 31(2).
  More info

  Nonalcoholic steatohepatitis (NASH) has been identified as a source of significant interindividual variation in drug metabolism. A previous ex vivo study demonstrated significant changes in hepatic Cytochrome P450 (CYP) activity in human NASH. This study evaluated the in vivo activities of multiple CYP isoforms simultaneously in prominent diabetic NASH mouse models. The pharmacokinetics of CYP selective substrates: caffeine, losartan, and omeprazole changed significantly in a diabetic NASH mouse model, indicating attenuation of the activity of Cyp1a2 and Cyp2c29, respectively. Decreased mRNA expression of Cyp1a2 and Cyp2c29, as well as an overall decrease in CYP protein expression, was found in the diabetic NASH mice. Overall, these data suggest that the diabetic NASH model only partially recapitulates the human ex vivo CYP alteration pattern. Therefore, in vivo determination of the effects of NASH on CYP activity should be conducted in human, and more appropriate models are required for future drug metabolism studies in NASH.
• Yu, A. M., Ingelman-Sundberg, M., Cherrington, N. J., Aleksunes, L. M., Zanger, U. M., Xie, W., Jeong, H., Morgan, E. T., Turnbaugh, P. J., Klaassen, C. D., Bhatt, A. P., Redinbo, M. R., Hao, P., Waxman, D. J., Wang, L., & Zhong, X. B. (2017). Regulation of drug metabolism and toxicity by multiple factors of genetics, epigenetics, lncRNAs, gut microbiota, and diseases: a meeting report of the 21st International Symposium on Microsomes and Drug Oxidations (MDO). Acta pharmaceutica Sinica. B, 7(2), 241-248.
  More info

  Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2-6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research.
• Cheng, Q., Aleksunes, L. M., Manautou, J. E., Cherrington, N. J., Scheffer, G. L., Yamasaki, H., & Slitt, A. L. (2016). Drug-metabolizing enzyme and transporter expression in a mouse model of diabetes and obesity. Molecular pharmaceutics, 5(1), 77-91.
  More info

  Obesity and type II diabetes pose a serious human health risk. Obese or diabetic patients usually take prescription drugs that require hepatic and renal metabolism and transport, and these patients sometimes display different pharmacokinetics of these drugs. Therefore, mRNA and protein expression of drug-metabolizing enzymes (DMEs) and transporters was measured in livers and kidneys of adult wild-type and ob/ob mice, which model obesity and diabetes. mRNA expression of numerous DMEs increased by at least 2-fold in livers of male ob/ob mice, including Cyp4a14, Cyp2b10, NAD(P)H:quinone oxidoreductase 1 (Nqo1), and sulfotransferase 2a1/2. In general, expression of uptake transporters was decreased in livers of ob/ob mice, namely organic anion-transporting polypeptides (Oatps) and sodium/taurocholate cotransporting polypeptide (Ntcp). In particular, Oatp1a1 mRNA and protein expression in livers of ob/ob mice was diminished to
• Cheng, Q., Taguchi, K., Aleksunes, L. M., Manautou, J. E., Cherrington, N. J., Yamamoto, M., & Slitt, A. L. (2016). Constitutive activation of nuclear factor-E2-related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte-specific deletion of Kelch-like ECH-associated protein 1. Journal of biochemical and molecular toxicology, 25(5), 320-9.
  More info

  Chemicals that activate nuclear factor-E2-related factor 2 (Nrf2) often increase multidrug-resistance-associated protein (Mrp) expression in liver. Hepatocyte-specific deletion of Kelch-like ECH-associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte-specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild-type and hepatocyte-specific Keap1-null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate-cysteine ligase catalytic subunit expression were increased in livers of Keap1-null mice. Organic anion-transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild-type mice. By contrast, Mrp 1-5 mRNA and protein levels were increased in Keap1-null mouse livers, with Mrp4 expression being more than 15-fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions.
• Cherrington, N. J., Li, H., Clarke, J. D., Dzierlenga, A. L., Bear, J., & Goedken, M. J. (2016). In vivo cytochrome P450 activity alterations in diabetic nonalcoholic steatohepatitis mice. Journal of Biochemical and Molecular Toxicology, 31(2), e21840. doi:10.1002/jbt.21840
• Ditzel, E. J., Li, H., Foy, C. E., Perrera, A. B., Parker, P., Renquist, B. J., Cherrington, N. J., & Camenisch, T. D. (2016). Altered Hepatic Transport by Fetal Arsenite Exposure in Diet-Induced Fatty Liver Disease. Journal of biochemical and molecular toxicology, 30(7), 321-30.
  More info

  Non-alcoholic fatty liver disease can result in changes to drug metabolism and disposition potentiating adverse drug reactions. Furthermore, arsenite exposure during development compounds the severity of diet-induced fatty liver disease. This study examines the effects of arsenite potentiated diet-induced fatty liver disease on hepatic transport in male mice. Changes were detected for Mrp2/3/4 hepatic transporter gene expression as well as for Oatp1a4/2b1/1b2. Plasma concentrations of Mrp and Oatp substrates were increased in arsenic exposure groups compared with diet-only controls. In addition, murine embryonic hepatocytes and adult primary hepatocytes show significantly altered transporter expression after exposure to arsenite alone: a previously unreported phenomenon. These data indicate that developmental exposure to arsenite leads to changes in hepatic transport which could increase the risk for ADRs during fatty liver disease.
• Donepudi, A. C., Cheng, Q., Lu, Z. J., Cherrington, N. J., & Slitt, A. L. (2016). Hepatic Transporter Expression in Metabolic Syndrome: Phenotype, Serum Metabolic Hormones, and Transcription Factor Expression. Drug metabolism and disposition: the biological fate of chemicals, 44(4), 518-26.
  More info

  Metabolic syndrome is a multifactorial disease associated with obesity, insulin resistance, diabetes, and the alteration of multiple metabolic hormones. Obesity rates have been rising worldwide, which increases our need to understand how this population will respond to drugs and exposure to other chemicals. The purpose of this study was to determine in lean and obese mice the ontogeny of clinical biomarkers such as serum hormone and blood glucose levels as well as the physiologic markers that correlate with nuclear receptor- and transporter-related pathways. Livers from male and female wild-type (WT) (C57BL/6) and ob/ob mice littermates were collected before, during, and after the onset of obesity. Serum hormone and mRNA levels were analyzed. Physiologic changes and gene expression during maturation and progression to obesity were performed and correlation analysis was performed using canonical correlations. Significant ontogenic changes in both WT and ob/ob mice were observed and these ontogenic changes differ in ob/ob mice with the development of obesity. In males and females, the ontogenic pattern of the expression of genes such as Abcc3, 4, Abcg2, Cyp2b10, and 4a14 started to differ from week 3, and became significant at weeks 4 and 8 in ob/ob mice compared with WT mice. In obese males, serum resistin, glucagon, and glucose levels correlated with the expression of most hepatic ATP-binding cassette (Abc) transporters, whereas in obese females, serum glucagon-like peptide 1 levels were correlated with most hepatic uptake transporters and P450 enzymes. Overall, the correlation between physiologic changes and gene expression indicate that metabolism-related hormones may play a role in regulating the genes involved in drug metabolism and transport.
• Dzierlenga, A. L., Clarke, J. D., & Cherrington, N. J. (2016). Nonalcoholic Steatohepatitis Modulates Membrane Protein Retrieval and Insertion Processes. Drug metabolism and disposition: the biological fate of chemicals, 44(11), 1799-1807.
  More info

  Interindividual variability in drug response in nonalcoholic steatohepatitis (NASH) can be mediated by altered regulation of drug metabolizing enzymes and transporters. Among these is the mislocalization of multidrug resistance-associated protein (MRP2)/Mrp2 away from the canalicular membrane, which results in decreased transport of MRP2/Mrp2 substrates. The exact mechanism of this mislocalization is unknown, although increased activation of membrane retrieval processes may be one possibility. The current study measures the activation status of various mediators implicated in the active membrane retrieval or insertion of membrane proteins to identify which processes may be important in rodent methionine and choline deficient diet-induced NASH. The mediators currently known to be associated with transporter mislocalization are stimulated by oxidative stressors and choleretic stimuli, which play a role in the pathogenesis of NASH. The activation of protein kinases PKA, PKCα, PKCδ, and PKCε and substrates radixin, myristoylated alanine-rich C-kinase substrate, and Rab11 were measured by comparing the expression, phosphorylation, and membrane translocation between control and NASH. Many of the mediators exhibited altered activation in NASH rats. Consistent with membrane retrieval of Mrp2, NASH rats exhibited a decreased phosphorylation of radixin and increased membrane localization of PKCδ and PKCε, thought to be mediators of radixin dephosphorylation. Altered activation of PKCδ, PKA, and PKCα may impair the Rab11-mediated active insertion of Mrp2. Overall, these data suggest alterations in membrane retrieval and insertion processes that may contribute to altered localization of membrane proteins in NASH.
• Dzierlenga, A. L., Clarke, J. D., Klein, D. M., Anumol, T., Snyder, S. A., Li, H., & Cherrington, N. J. (2016). Biliary Elimination of Pemetrexed Is Dependent on Mrp2 in Rats: Potential Mechanism of Variable Response in Nonalcoholic Steatohepatitis. The Journal of pharmacology and experimental therapeutics, 358(2), 246-53.
  More info

  Hepatic multidrug resistance-associated protein 2 (MRP2) provides the biliary elimination pathway for many xenobiotics. Disruption of this pathway contributes to retention of these compounds and may ultimately lead to adverse drug reactions. MRP2 mislocalization from the canalicular membrane has been observed in nonalcoholic steatohepatitis (NASH), the late stage of nonalcoholic fatty liver disease, which is characterized by fat accumulation, oxidative stress, inflammation, and fibrosis. MRP2/Mrp2 mislocalization is observed in both human NASH and the rodent methionine and choline-deficient (MCD) diet model, but the extent to which it impacts overall transport capacity of MRP2 is unknown. Pemetrexed is an antifolate chemotherapeutic indicated for non-small cell lung cancer, yet its hepatobiliary elimination pathway has yet to be determined. The purpose of this study was to quantify the loss of Mrp2 function in NASH using an obligate Mrp2 transport substrate. To determine whether pemetrexed is an obligate Mrp2 substrate, its cumulative biliary elimination was compared between wild-type and Mrp2(-/-) rats. No pemetrexed was detected in the bile of Mrp2(-/-) rats, indicating pemetrexed is completely reliant on Mrp2 function for biliary elimination. Comparing the biliary elimination of pemetrexed between MCD and control animals identified a transporter-dependent decrease in biliary excretion of 60% in NASH. This study identifies Mrp2 as the exclusive biliary elimination mechanism for pemetrexed, making it a useful in vivo probe substrate for Mrp2 function, and quantifying the loss of function in NASH. This mechanistic feature may provide useful insight into the impact of NASH on interindividual variability in response to pemetrexed.
• Klein, D. M., & Cherrington, N. J. (2016). Organic and inorganic transporters of the testis: A review. Spermatogenesis, 4(2), e979653.
  More info

  Transporters have a huge impact on the toxicology and pharmacological effects of xenobiotics in addition to being implicated in several diseases. While these important proteins have been well studied in organs such as the kidney or liver, characterization of transporters in the testis is still in the early stages. Knowledge of transporter function may greatly advance the field's understanding of the physiological and toxicological processes that occur in the testis. Several foundational studies involving both organic and inorganic transporters have been critical in furthering our understanding of how the testis interacts with endogenous and xenobiotic compounds. This review provides an overview of how transporters function, their clinical significance, and highlights what is known for many of the important transporters in the testis.
• Laho, T., Clarke, J. D., Dzierlenga, A. L., Li, H., Klein, D. M., Goedken, M., Micuda, S., & Cherrington, N. J. (2016). Effect of nonalcoholic steatohepatitis on renal filtration and secretion of adefovir. Biochemical pharmacology, 115, 144-51.
  More info

  Adefovir, an acyclic nucleotide reverse transcriptase inhibitor used to treat hepatitis B viral infection, is primarily eliminated renally through cooperation of glomerular filtration with active tubular transport. Nonalcoholic steatohepatitis is a variable in drug disposition, yet the impact on renal transport processes has yet to be fully understood. The goal of this study was to determine the effect of nonalcoholic steatohepatitis on the pharmacokinetics of adefovir in rats given a control or methionine and choline deficient diet to induce nonalcoholic steatohepatitis.
• Lake, A. D., Chaput, A. L., Novak, P., Cherrington, N. J., & Smith, C. L. (2016). Transcription factor binding site enrichment analysis predicts drivers of altered gene expression in nonalcoholic steatohepatitis. Biochemical pharmacology, 122, 62-71.
  More info

  The molecular mechanisms behind the transition from simple steatosis to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD) are not clearly understood. This hinders development of effective therapies for treatment and prevention of NASH. In this study expression profiling data from normal, steatosis, and NASH human livers were used to predict transcription factors that are misregulated as mechanistic features of NAFLD progression. Previously-published human NAFLD gene expression profiling data from normal, steatosis, and NASH livers were subjected to transcription factor binding site enrichment analysis. Selected transcription factors that bind enriched transcription factor binding sites were analyzed for changes in expression. Distinct transcription factor binding sites were enriched in genes significantly up- or down-regulated in NASH livers. Those enriched in up-regulated genes were bound by transcription factors such as FOXA, CEBP, and HNF1 family members, while those enriched in down-regulated genes were bound by nuclear receptors involved in xenobiotic sensing and lipid metabolism. Levels of mRNA and protein for selected transcription factors were significantly changed during disease progression. The study indicates that NAFLD progression involves changes in activity or expression of transcription factors that regulate genes involved in hepatic processes known to be altered in NASH. Transcription factors such as PPAR receptors, FoxA family members, and HNF4A might be targeted therapeutically to prevent NAFLD progression.
• Canet, M. J., Hardwick, R. N., Lake, A. D., Dzierlenga, A. L., Clarke, J. D., Goedken, M. J., & Cherrington, N. J. (2015). Renal xenobiotic transporter expression is altered in multiple experimental models of nonalcoholic steatohepatitis. Drug metabolism and disposition: the biological fate of chemicals, 43(2), 266-72.
  More info

  Nonalcoholic fatty liver disease is the most common chronic liver disease, which can progress to nonalcoholic steatohepatitis (NASH). Previous investigations demonstrated alterations in the expression and activity of hepatic drug transporters in NASH. Moreover, studies using rodent models of cholestasis suggest that compensatory changes in kidney transporter expression occur to facilitate renal excretion during states of hepatic stress; however, little information is currently known regarding extrahepatic regulation of drug transporters in NASH. The purpose of the current study was to investigate the possibility of renal drug transporter regulation in NASH across multiple experimental rodent models. Both rat and mouse NASH models were used in this investigation and include: the methionine and choline-deficient (MCD) diet, atherogenic diet, fa/fa rat, ob/ob and db/db mice. Histologic and pathologic evaluations confirmed that the MCD and atherogenic rats as well as the ob/ob and db/db mice all developed NASH. In contrast, the fa/fa rats did not develop NASH but did develop extensive renal injury compared with the other models. Renal mRNA and protein analyses of xenobiotic transporters suggest that compensatory changes occur in NASH to favor increased xenobiotic secretion. Specifically, both apical efflux and basolateral uptake transporters are induced, whereas apical uptake transporter expression is repressed. These results suggest that NASH may alter the expression and potentially function of renal drug transporters, thereby impacting drug elimination mechanisms in the kidney.
• Canet, M. J., Merrell, M. D., Harder, B. G., Maher, J. M., Wu, T., Lickteig, A. J., Jackson, J. P., Zhang, D. D., Yamamoto, M., & Cherrington, N. J. (2015). Identification of a functional antioxidant response element within the eighth intron of the human ABCC3 gene. Drug metabolism and disposition: the biological fate of chemicals, 43(1), 93-9.
  More info

  The ATP-binding cassette (ABC) family of transporters, including ABCC3, is a large family of efflux pumps that plays a pivotal role in the elimination of xenobiotics from the body. ABCC3 has been reported to be induced during hepatic stress conditions and through the progression of some forms of cancer. Several lines of evidence have implicated the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in this induction. However, although rodent models have been investigated, a functional antioxidant response element (ARE) in the human ABCC3 gene has not been identified. The purpose of this study was to identify and characterize the ARE(s) responsible for mediating the Nrf2-dependent induction of the human ABCC3 gene. A high-throughput chromatin immunoprecipitation-sequencing analysis performed in A549 cells revealed a specific interaction between Nrf2 and the eighth intron of the human ABCC3 gene rather than the more prototypical flanking region of the gene. Subsequent in silico analysis of the intron identified two putative ARE elements that contained the core consensus ARE sequence commonly found in several Nrf2-responsive genes. Functional characterization of these two AREs using luciferase-reporter constructs with ARE mutant constructs revealed that one of these putative AREs is functionally active. Finally, DNA pull-down assays confirmed specific binding of these intronic AREs by Nrf2 in vitro. Our findings identify a functional Nrf2 response element within the eighth intron of the ABCC3 gene, which may provide mechanistic insight into the induction of ABCC3 during antioxidant response stimuli.
• Canet, M. J., Merrell, M. D., Hardwick, R. N., Bataille, A. M., Campion, S. N., Ferreira, D. W., Xanthakos, S. A., Manautou, J. E., Hesham A-Kader, H., Erickson, R. P., & Cherrington, N. J. (2015). Altered regulation of hepatic efflux transporters disrupts acetaminophen disposition in pediatric nonalcoholic steatohepatitis. Drug metabolism and disposition: the biological fate of chemicals, 43(6), 829-35.
  More info

  Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation.
• Clarke, J. D., & Cherrington, N. J. (2015). Nonalcoholic steatohepatitis in precision medicine: Unraveling the factors that contribute to individual variability. Pharmacology & therapeutics, 151, 99-106.
  More info

  There are numerous factors in individual variability that make the development and implementation of precision medicine a challenge in the clinic. One of the main goals of precision medicine is to identify the correct dose for each individual in order to maximize therapeutic effect and minimize the occurrence of adverse drug reactions. Many promising advances have been made in identifying and understanding how factors such as genetic polymorphisms can influence drug pharmacokinetics (PK) and contribute to variable drug response (VDR), but it is clear that there remain many unidentified variables. Underlying liver diseases such as nonalcoholic steatohepatitis (NASH) alter absorption, distribution, metabolism, and excretion (ADME) processes and must be considered in the implementation of precision medicine. There is still a profound need for clinical investigation into how NASH-associated changes in ADME mediators, such as metabolism enzymes and transporters, affect the pharmacokinetics of individual drugs known to rely on these pathways for elimination. This review summarizes the key PK factors in individual variability and VDR and highlights NASH as an essential underlying factor that must be considered as the development of precision medicine advances. A multifactorial approach to precision medicine that considers the combination of two or more risk factors (e.g. genetics and NASH) will be required in our effort to provide a new era of benefit for patients.
• Clarke, J. D., Dzierlenga, A. L., Nelson, N. R., Li, H., Werts, S., Goedken, M. J., & Cherrington, N. J. (2015). Mechanism of Altered Metformin Distribution in Nonalcoholic Steatohepatitis. Diabetes, 64(9), 3305-13.
  More info

  Metformin is an antihyperglycemic drug that is widely prescribed for type 2 diabetes mellitus and is currently being investigated for the treatment of nonalcoholic steatohepatitis (NASH). NASH is known to alter hepatic membrane transporter expression and drug disposition similarly in humans and rodent models of NASH. Metformin is almost exclusively eliminated through the kidney primarily through active secretion mediated by Oct1, Oct2, and Mate1. The purpose of this study was to determine how NASH affects kidney transporter expression and metformin pharmacokinetics. A single oral dose of [(14)C]metformin was administered to C57BL/6J (wild type [WT]) and diabetic ob/ob mice fed either a control diet or a methionine- and choline-deficient (MCD) diet. Metformin plasma concentrations were slightly increased in the WT/MCD and ob/control groups, whereas plasma concentrations were 4.8-fold higher in ob/MCD mice compared with WT/control. The MCD diet significantly increased plasma half-life and mean residence time and correspondingly decreased oral clearance in both genotypes. These changes in disposition were caused by ob/ob- and MCD diet-specific decreases in the kidney mRNA expression of Oct2 and Mate1, whereas Oct1 mRNA expression was only decreased in ob/MCD mice. These results indicate that the diabetic ob/ob genotype and the MCD disease model alter kidney transporter expression and alter the pharmacokinetics of metformin, potentially increasing the risk of drug toxicity.
• Dzierlenga, A. L., Clarke, J. D., Hargraves, T. L., Ainslie, G. R., Vanderah, T. W., Paine, M. F., & Cherrington, N. J. (2015). Mechanistic basis of altered morphine disposition in nonalcoholic steatohepatitis. The Journal of pharmacology and experimental therapeutics, 352(3), 462-70.
  More info

  Morphine is metabolized in humans to morphine-3-glucuronide (M3G) and the pharmacologically active morphine-6-glucuronide (M6G). The hepatobiliary disposition of both metabolites relies upon multidrug resistance-associated proteins Mrp3 and Mrp2, located on the sinusoidal and canalicular membrane, respectively. Nonalcoholic steatohepatitis (NASH), the severe stage of nonalcoholic fatty liver disease, alters xenobiotic metabolizing enzyme and transporter function. The purpose of this study was to determine whether NASH contributes to the large interindividual variability and postoperative adverse events associated with morphine therapy. Male Sprague-Dawley rats were fed a control diet or a methionine- and choline-deficient diet to induce NASH. Radiolabeled morphine (2.5 mg/kg, 30 µCi/kg) was administered intravenously, and plasma and bile (0-150 or 0-240 minutes), liver and kidney, and cumulative urine were analyzed for morphine and M3G. The antinociceptive response to M6G (5 mg/kg) was assessed (0-12 hours) after direct intraperitoneal administration since rats do not produce M6G. NASH caused a net decrease in morphine concentrations in the bile and plasma and a net increase in the M3G/morphine plasma area under the concentration-time curve ratio, consistent with upregulation of UDP-glucuronosyltransferase Ugt2b1. Despite increased systemic exposure to M3G, NASH resulted in decreased biliary excretion and hepatic accumulation of M3G. This shift toward systemic retention is consistent with the mislocalization of canalicular Mrp2 and increased expression of sinusoidal Mrp3 in NASH and may correlate to increased antinociception by M6G. Increased metabolism and altered transporter regulation in NASH provide a mechanistic basis for interindividual variability in morphine disposition that may lead to opioid-related toxicity.
• Lake, A. D., Novak, P., Shipkova, P., Aranibar, N., Robertson, D. G., Reily, M. D., Lehman-McKeeman, L. D., Vaillancourt, R. R., & Cherrington, N. J. (2015). Branched chain amino acid metabolism profiles in progressive human nonalcoholic fatty liver disease. Amino acids, 47(3), 603-15.
  More info

  Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127% of normal), isoleucine (139%), and valine (147%) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.
• Camenisch, T. D., Sanchez-soria, P., Quach, S., Hardwick, R. N., Cherrington, N. J., Camenisch, T. D., & Broka, D. (2014). Fetal exposure to arsenic results in hyperglycemia, hypercholesterolemia, and nonalcoholic fatty liver disease in adult mice. Journal of Toxicology and Health, 1(1), 1. doi:10.7243/2056-3779-1-1
  More info

  Abstract Background: Exposure to arsenic is a major concern in the United States and worldwide, since this metalloid has been associated with a number of ailments, including cardiovascular and metabolic diseases.
• Canet, M. J., & Cherrington, N. J. (2014). Drug disposition alterations in liver disease: extrahepatic effects in cholestasis and nonalcoholic steatohepatitis. Expert opinion on drug metabolism & toxicology, 10(9), 1209-19.
  More info

  The pharmacokinetics (PK) of drugs and xenobiotics, namely pharmaceuticals, is influenced by a host of factors that include genetics, physiological factors and environmental stressors. The importance of disease on the disposition of xenobiotics has been increasingly recognized among medical professionals for alterations in key enzymes and membrane transporters that influence drug disposition and contribute to the development of adverse drug reactions.
• Canet, M. J., Hardwick, R. N., Lake, A. D., Dzierlenga, A. L., Clarke, J. D., & Cherrington, N. J. (2014). Modeling human nonalcoholic steatohepatitis-associated changes in drug transporter expression using experimental rodent models. Drug metabolism and disposition: the biological fate of chemicals, 42(4), 586-95.
  More info

  Nonalcoholic fatty liver disease is a prevalent form of chronic liver disease that can progress to the more advanced stage of nonalcoholic steatohepatitis (NASH). NASH has been shown to alter drug transporter regulation and may have implications in the development of adverse drug reactions. Several experimental rodent models have been proposed for the study of NASH, but no single model fully recapitulates all aspects of the human disease. The purpose of the current study was to determine which experimental NASH model best reflects the known alterations in human drug transporter expression to enable more accurate drug disposition predictions in NASH. Both rat and mouse NASH models were used in this investigation and include the methionine and choline deficient (MCD) diet model, atherogenic diet model, ob/ob and db/db mice, and fa/fa rats. Pathologic scoring evaluations demonstrated that MCD and atherogenic rats, as well as ob/ob and db/db mice, developed NASH. Liver mRNA and protein expression analyses of drug transporters showed that in general, efflux transporters were induced and uptake transporters were repressed in the rat MCD and the mouse ob/ob and db/db models. Lastly, concordance analyses suggest that both the mouse and rat MCD models as well as mouse ob/ob and db/db NASH models show the most similarity to human transporter mRNA and protein expression. These results suggest that the MCD rat and mouse model, as well as the ob/ob and db/db mouse models, may be useful for predicting altered disposition of drugs with similar kinetics across humans and rodents.
• Clarke, J. D., Hardwick, R. N., Lake, A. D., Canet, M. J., & Cherrington, N. J. (2014). Experimental nonalcoholic steatohepatitis increases exposure to simvastatin hydroxy acid by decreasing hepatic organic anion transporting polypeptide expression. Journal of Pharmacology and Experimental Therapeutics, 348(3), 452-458.
  More info

  PMID: 24403518;Abstract: Simvastatin (SIM)-induced myopathy is a dose-dependent adverse drug reaction (ADR) that has been reported to occur in 18.2% of patients receiving a 40- to 80-mg dose. The pharmacokinetics of SIM hydroxy acid (SIMA), the bioactive form of SIM, and the occurrence of SIM-induced myopathy are linked to the function of the organic anion transporting polypeptide (Oatp) hepatic uptake transporters. Genetic polymorphisms in SLCO1B1, the gene for human hepatic OATP1B1, cause decreased elimination of SIMA and increased risk of developing myopathy. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease, and is known to alter drug transporter expression and drug disposition. The purpose of this studywas to assess themetabolism and disposition of SIM in a diet-induced rodent model of NASH. Rats were fed a methionine- and choline-deficient diet for 8 weeks to induce NASH and SIM was administered intravenously. Dietinduced NASH caused increased plasma retention and decreased biliary excretion of SIMA due to decreased protein expression of multiple hepatic Oatps. SIM exhibited increased volume of distribution in NASH as evidenced by increased muscle, decreased plasma, and no change in biliary concentrations. Although Cyp3a and Cyp2c11 proteins were decreased in NASH, no alterations in SIMmetabolism were observed. These data, in conjunction with our previous data showing that human NASH causes a coordinated downregulation of hepatic uptake transporters, suggest that NASHmediated transporter regulation may play a role in altered SIMA disposition and the occurrence of myopathy. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.
• Clarke, J. D., Hardwick, R. N., Lake, A. D., Canet, M. J., & Cherrington, N. J. (2014). Experimental nonalcoholic steatohepatitis increases exposure to simvastatin hydroxy acid by decreasing hepatic organic anion transporting polypeptide expression. The Journal of pharmacology and experimental therapeutics, 348(3), 452-8.
  More info

  Simvastatin (SIM)-induced myopathy is a dose-dependent adverse drug reaction (ADR) that has been reported to occur in 18.2% of patients receiving a 40- to 80-mg dose. The pharmacokinetics of SIM hydroxy acid (SIMA), the bioactive form of SIM, and the occurrence of SIM-induced myopathy are linked to the function of the organic anion transporting polypeptide (Oatp) hepatic uptake transporters. Genetic polymorphisms in SLCO1B1, the gene for human hepatic OATP1B1, cause decreased elimination of SIMA and increased risk of developing myopathy. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease, and is known to alter drug transporter expression and drug disposition. The purpose of this study was to assess the metabolism and disposition of SIM in a diet-induced rodent model of NASH. Rats were fed a methionine- and choline-deficient diet for 8 weeks to induce NASH and SIM was administered intravenously. Diet-induced NASH caused increased plasma retention and decreased biliary excretion of SIMA due to decreased protein expression of multiple hepatic Oatps. SIM exhibited increased volume of distribution in NASH as evidenced by increased muscle, decreased plasma, and no change in biliary concentrations. Although Cyp3a and Cyp2c11 proteins were decreased in NASH, no alterations in SIM metabolism were observed. These data, in conjunction with our previous data showing that human NASH causes a coordinated downregulation of hepatic uptake transporters, suggest that NASH-mediated transporter regulation may play a role in altered SIMA disposition and the occurrence of myopathy.
• Clarke, J. D., Hardwick, R. N., Lake, A. D., Lickteig, A. J., Goedken, M. J., Klaassen, C. D., & Cherrington, N. J. (2014). Synergistic interaction between genetics and disease on pravastatin disposition. Journal of hepatology, 61(1), 139-47.
  More info

  A genome wide association study and multiple pharmacogenetic studies have implicated the hepatic uptake transporter organic anion transporting polypeptide-1B1 (OATP1B1) in the pharmacokinetics and musculoskeletal toxicity of statin drugs. Other OATP uptake transporters can participate in the transport of pravastatin, partially compensating for the loss of OATP1B1 in patients carrying the polymorphism. Non-alcoholic steatohepatitis (NASH) in humans and in a diet-induced rodent model alter the expression of multiple OATP transporters.
• Clarke, J. D., Novak, P., Lake, A. D., Shipkova, P., Aranibar, N., Robertson, D., Severson, P. L., Reily, M. D., Futscher, B. W., Lehman-Mckeeman, L. D., & Cherrington, N. J. (2014). Characterization of hepatocellular carcinoma related genes and metabolites in human nonalcoholic fatty liver disease. Digestive Diseases and Sciences, 59(2), 365-374.
  More info

  PMID: 24048683;Abstract: Background: The worldwide prevalences of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are estimated to range from 30 to 40 % and 5-17 %, respectively. Hepatocellular carcinoma (HCC) is primarily caused by hepatitis B infection, but retrospective data suggest that 4-29 % of NASH cases will progress to HCC. Currently the connection between NASH and HCC is unclear. Aims: The purpose of this study was to identify changes in expression of HCC-related genes and metabolite profiles in NAFLD progression. Methods: Transcriptomic and metabolomic datasets from human liver tissue representing NAFLD progression (normal, steatosis, NASH) were utilized and compared to published data for HCC. Results: Genes involved in Wnt signaling were downregulated in NASH but have been reported to be upregulated in HCC. Extracellular matrix/angiogenesis genes were upregulated in NASH, similar to reports in HCC. Iron homeostasis is known to be perturbed in HCC and we observed downregulation of genes in this pathway. In the metabolomics analysis of hepatic NAFLD samples, several changes were opposite to what has been reported in plasma of HCC patients (lysine, phenylalanine, citrulline, creatine, creatinine, glycodeoxycholic acid, inosine, and alpha-ketoglutarate). In contrast, multiple acyl-lyso-phosphatidylcholine metabolites were downregulated in NASH livers, consistent with observations in HCC patient plasma. Conclusions: These data indicate an overlap in the pathogenesis of NAFLD and HCC where several classes of HCC related genes and metabolites are altered in NAFLD. Importantly, Wnt signaling and several metabolites are different, thus implicating these genes and metabolites as mediators in the transition from NASH to HCC. © 2013 Springer Science+Business Media New York.
• Clarke, J. D., Sharapova, T., Lake, A. D., Blomme, E., Maher, J., & Cherrington, N. J. (2014). Circulating microRNA 122 in the methionine and choline-deficient mouse model of non-alcoholic steatohepatitis. Journal of applied toxicology : JAT, 34(6), 726-32.
  More info

  Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) and is a major cause of liver cirrhosis and hepatic failure. The methionine choline-deficient diet (MCD) is a frequently used hepatotoxicity animal model of NASH that induces hepatic transaminase (ALT, AST) elevations and hepatobiliary histological changes similar to those observed in human NASH. Liver-specific microRNA-122 (miR-122) has been shown as a key regulator of cholesterol and fatty acid metabolism in adult liver, and has recently been proposed as a sensitive and specific circulating biomarker of hepatic injury. The purpose of this study was to assess miR-122 serum levels in mice receiving an MCD diet for 0, 3, 7, 14, 28 and 56 days and compare the performance vs. routine clinical chemistry when benchmarked against the histopathological liver findings. MiR-122 levels were quantified in serum using RT-qPCR. Both miR-122 and ALT/AST levels were significantly elevated in serum at all timepoints. MiR-122 levels increased on average by 40-fold after 3 days of initiating the MCD diet, whereas ALT and AST changes were 4.8- and 3.3-fold, respectively. In general, miR-122 levels remained elevated across all time points, whereas the ALT/AST increases were less robust but correlated with the progressive severity of NASH as assessed by histopathology. In conclusion, serum levels of miR-122 can potentially be used as a sensitive biomarker for the early detection of hepatotoxicity and can aid in monitoring the extent of NAFLD-associated liver injury in mouse efficacy models.
• Hardwick, R. N., Clarke, J. D., Lake, A. D., Canet, M. J., Anumol, T., Street, S. M., Merrell, M. D., Goedken, M. J., Snyder, S. A., & Cherrington, N. J. (2014). Increased susceptibility to methotrexate-induced toxicity in nonalcoholic steatohepatitis. Toxicological sciences : an official journal of the Society of Toxicology, 142(1), 45-55.
  More info

  Hepatic drug metabolizing enzymes and transporters play a crucial role in determining the fate of drugs, and alterations in liver function can place individuals at greater risk for adverse drug reactions (ADRs). We have shown that nonalcoholic steatohepatitis (NASH) leads to changes in the expression and localization of enzymes and transporters responsible for the disposition of numerous drugs. The purpose of this study was to determine the effect of NASH on methotrexate (MTX) disposition and the resulting toxicity profile. Sprague Dawley rats were fed either a control or methionine-choline-deficient diet for 8 weeks to induce NASH, then administered a single ip vehicle, 10, 40, or 100 mg/kg MTX injection followed by blood, urine, and feces collection over 96 h with terminal tissue collection. At the onset of dosing, Abcc1-4, Abcb1, and Abcg2 were elevated in NASH livers, whereas Abcc2 and Abcb1 were not properly localized to the membrane, similar to that previously observed in human NASH. NASH rodents receiving 40-100 mg/kg MTX exhibited hepatocellular damage followed by initiation of repair, whereas damage was absent in controls. NASH rodents receiving 100 mg/kg MTX exhibited slightly greater renal toxicity, indicating multiple organ toxicity, despite the majority of the dose being excreted by 6 h. Intestinal toxicity in NASH however, was strikingly less severe than controls, and coincided with reduced fecal MTX excretion. Because MTX-induced gastrointestinal toxicity limits the dose escalation necessary for cancer remission, these data suggest a greater risk for life-threatening MTX-induced hepatic and renal toxicity in NASH in the absence of overt gastrointestinal toxicity.
• Klein, D. M., & Cherrington, N. J. (2014). Organic and inorganic transporters of the testis: A review.. Spermatogenesis, 4(2), e979653. doi:10.4161/21565562.2014.979653
  More info

  Transporters have a huge impact on the toxicology and pharmacological effects of xenobiotics in addition to being implicated in several diseases. While these important proteins have been well studied in organs such as the kidney or liver, characterization of transporters in the testis is still in the early stages. Knowledge of transporter function may greatly advance the field's understanding of the physiological and toxicological processes that occur in the testis. Several foundational studies involving both organic and inorganic transporters have been critical in furthering our understanding of how the testis interacts with endogenous and xenobiotic compounds. This review provides an overview of how transporters function, their clinical significance, and highlights what is known for many of the important transporters in the testis.
• Klein, D. M., Wright, S. H., & Cherrington, N. J. (2014). Localization of multidrug resistance-associated proteins along the blood-testis barrier in rat, macaque, and human testis. Drug Metabolism and Disposition, 42(1), 89-93.
  More info

  Abstract: The blood-testis barrier (BTB) prevents the entry of many drugs into seminiferous tubules, which can be beneficial for therapy not intended for the testis but may decrease drug efficacy for medications requiring entry to the testis. Previous data have shown that some of the transporters in the multidrug resistance-associated protein (MRP) family (ABCC) are expressed in the testis. By determining the subcellular localization of these transporters, their physiologic function and effect on drug disposition may be better predicted. Using immunohistochemistry (IHC), we determined the site of expression of the MRP transporters expressed in the testis, namely, MRP1, MRP4, MRP5, and MRP8, from immature and mature rats, rhesus macaques, and adult humans. We determined that in all species MRP1 was restricted to the basolateral membrane of Sertoli cells, MRP5 is located in Leydig cells, and MRP8 is located in round spermatids, whereas MRP4 showed speciesspecific localization. MRP4 is expressed on the basolateral membrane of Sertoli cells in human and nonhuman primates, but on the apical membrane of Sertoli cells in immature and mature rats, representing a potential caution when using rat models as a means for studying drug disposition across the BTB. These data suggest that MRP1 may limit drug disposition into seminiferous tubules, as may MRP4 in human and nonhuman primates but not in rats. These data also suggest that MRP5 and MRP8 may not have a major impact on the penetration of drugs across the BTB. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Klein, D. M., Wright, S. H., & Cherrington, N. J. (2014). Xenobiotic transporter expression along the male genital tract. Reproductive toxicology (Elmsford, N.Y.), 47, 1-8.
  More info

  The male genital tract plays an important role in protecting sperm by forming a distinct compartment separate from the body which limits exposure to potentially toxic substrates. Transporters along this tract can influence the distribution of xenobiotics into the male genital tract through efflux back into the blood or facilitating the accumulation of toxicants. The aim of this study was to quantitatively determine the constitutive mRNA expression of 30 xenobiotic transporters in caput and cauda regions of the epididymis, vas deferens, prostate, and seminal vesicles from adult Sprague-Dawley rats. The epididymis was found to express at least moderate levels of 18 transporters, vas deferens 15, seminal vesicles 23, and prostate 18. Constitutive expression of these xenobiotic transporters in the male genital tract may provide insight into the xenobiotics that can potentially be transported into these tissues and may provide the molecular mechanism for site specific toxicity of select agents.
• Kyriakides, M., Hardwick, R. N., Jin, Z., Goedken, M. J., Holmes, E., Cherrington, N. J., & Coen, M. (2014). Systems level metabolic phenotype of methotrexate administration in the context of non-alcoholic steatohepatitis in the rat. Toxicological sciences : an official journal of the Society of Toxicology, 142(1), 105-16.
  More info

  Adverse drug reactions (ADRs) represent a significant clinical challenge with respect to patient morbidity and mortality. We investigated the hepatotoxicity and systems level metabolic phenotype of methotrexate (MTX) in the context of a prevalent liver disease; non-alcoholic steatohepatitis (NASH). A nuclear magnetic resonance spectroscopic-based metabonomic approach was employed to analyze the metabolic consequences of MTX (0, 10, 40, and 100 mg/kg) in the urine and liver of healthy rats (control diet) and in a model of NASH (methionine-choline deficient diet). Histopathological analysis confirmed baseline (0 mg/kg) liver necrosis, liver inflammation, and lipid accumulation in the NASH model. Administration of MTX (40 and 100 mg/kg) led to liver necrosis in the control cohort, whereas the NASH cohort also displayed biliary hyperplasia and liver fibrosis (100 mg/kg), providing evidence of the synergistic effect of MTX and NASH. The complementary hepatic and urinary metabolic phenotypes of the NASH model, at baseline, revealed perturbation of multiple metabolites associated with oxidative and energetic stress, and folate homeostasis. Administration of MTX in both diet cohorts showed dose-dependent metabolic consequences affecting gut microbial, energy, nucleobase, nucleoside, and folate metabolism. Furthermore, a unique panel of metabolic changes reflective of the synergistic effect of MTX and NASH was identified, including the elevation of hepatic phenylalanine, urocanate, acetate, and both urinary and hepatic formiminoglutamic acid. This systems level metabonomic analysis of the hepatotoxicity of MTX in the context of NASH provided novel mechanistic insight of potential wider clinical relevance for further understanding the role of liver pathology as a risk factor for ADRs.
• Lake, A. D., Novak, P., Hardwick, R. N., Flores-Keown, B., Zhao, F., Klimecki, W. T., & Cherrington, N. J. (2014). The Adaptive endoplasmic reticulum stress response to lipotoxicity in progressive human nonalcoholic fatty liver disease. Toxicological Sciences, 137(1), 26-35.
  More info

  Abstract: Nonalcoholic fatty liver disease (NAFLD) may progress from simple steatosis to severe, nonalcoholic steatohepatitis (NASH) in 7%-14% of the U.S. population through a second "hit" in the form of increased oxidative stress and inflammation. Endoplasmic reticulum (ER) stress signaling and the unfolded protein response (UPR) are triggered when high levels of lipids and misfolded proteins alter ER homeostasis creating a lipotoxic environment within NAFLD livers. The objective of this study was to determine the coordinate regulation of ER stress-associated genes in the progressive stages of human NAFLD. Human liver samples categorized as normal, steatosis, NASH (Fatty), and NASH (Not Fatty) were analyzed by individual Affymetrix GeneChip Human 1.0 ST microarrays, immunoblots, and immunohistochemistry. A gene set enrichment analysis was performed on autophagy, apoptosis, lipogenesis, and ER stress/UPR gene categories. An enrichment of downregulated genes in the ER stress-associated lipogenesis and ER stress/UPR gene categories was observed in NASH. Conversely, an enrichment of upregulated ER stress-associated genes for autophagy and apoptosis gene categories was observed in NASH. Protein expression of the adaptive liver response protein STC2 and the transcription factor X-box binding protein 1 spliced (XBP-1s) were significantly elevated among NASH samples, whereas other downstream ER stress proteins including CHOP, ATF4, and phosphorylated JNK and eIF2a were not significantly changed in disease progression. Increased nuclear accumulation of total XBP-1 protein was observed in steatosis and NASH livers. The findings reveal the presence of a coordinated, adaptive transcriptional response to hepatic ER stress in human NAFLD. © The Author 2013. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
• Merrell, M. D., Nyagode, B. A., Clarke, J. D., Cherrington, N. J., & Morgan, E. T. (2014). Selective and cytokine-dependent regulation of hepatic transporters and bile acid homeostasis during infectious colitis in mice. Drug metabolism and disposition: the biological fate of chemicals, 42(4), 596-602.
  More info

  Various disease models have been shown to alter hepatic drug-metabolizing enzyme (DME) and transporter expression and to induce cholestasis through altered enzyme and transporter expression. Previously, we detailed the regulation of hepatic DMEs during infectious colitis caused by Citrobacter rodentium infection. We hypothesized that this infection would also modulate hepatic drug transporter expression and key genes of bile acid (BA) synthesis and transport. Mice lacking Toll-like receptor 4 (TLR4), interleukin-6 (IL-6), or interferon-gamma (IFNγ) and appropriate wild-type animals were orally infected with C. rodentium and sacrificed 7 days later. In two wild-type strains, drug transporter mRNA expression was significantly decreased by infection for Slc22a4, Slco1a1, Slco1a4, Slco2b1, and Abcc6, whereas the downregulation of Abcc2, Abcc3, and Abcc4 were strain-dependent. In contrast, mRNA expressions of Slco3a1 and Abcb1b were increased in a strain-dependent manner. Expression of Abcb11, Slc10a1, the two major hepatic BA transporters, and Cyp7a1, the rate-limiting enzyme of BA synthesis, was also significantly decreased in infected animals. None of the above effects were caused by bacterial lipopolysaccharide, since they still occurred in the absence of functional TLR4. The downregulation of Slc22a4 and Cyp7a1 was absent in IFNγ-null mice, and the downregulation of Slco1a1 was abrogated in IL-6-null mice, indicating in vivo roles for these cytokines in transporter regulation. These data indicate that C. rodentium infection modulates hepatic drug processing through alteration of transporter expression as well as DMEs. Furthermore, this infection downregulates important genes of BA synthesis and transport and may increase the risk for cholestasis.
• Reilly, B. G., Pham, T., Kronenfeld, J., Futch, L., Fraijo, S., Crowther, M. K., Cherrington, N. J., Cherrington, E., Adamson, B., Reilly, B. G., Pham, T., Kronenfeld, J., Futch, L., Fraijo, S., Crowther, M. K., Cherrington, N. J., Cherrington, E., & Adamson, B. (2014). P-glycoprotein - an ABC Transporter: Toxin Removal to Chemotherapy Interference (LB109). The FASEB Journal, 28(S1). doi:10.1096/fasebj.28.1_supplement.lb109
  More info

  Cancer claimed 7.6 million lives worldwide in 2013. Despite improvements in chemotherapy, most relapsed cancers become drug resistant due to an ATP-binding cassette transporter, P-glycoprotein (P-gp). P-gp is a gatekeeper in multiple tissues, controlling exposure to drugs and toxins by pumping them out of the cell. This homodimeric protein of 1,280 amino acids is located on the apical surface and extrudes lipid soluble molecules. Pharmaceutical companies must determine if their drugs are P-gp substrates before proceeding to clinical trials, as the amount of drug entering the body may vary widely due to P-gp function. P-gp may reduce the amount of drugs reaching circulation by transporting drugs back into the digestive tract lumen. Many chemicals act as modulators of P-gp’s function; for example, the flavonoid naringin in grapefruit juice interacts with P-gp and may significantly increase the uptake of some statin drugs. The active sites and conformational changes of P-gp have not been elucidated, as it ha...
• Bodeman, C. E., Dzierlenga, A. L., Tally, C. M., Mulligan, R. M., Lake, A. D., Cherrington, N. J., & McKarns, S. C. (2013). Differential regulation of hepatic organic cation transporter 1, organic anion-transporting polypeptide 1a4, bile-salt export pump, and multidrug resistance-associated protein 2 transporter expression in lymphocyte-deficient mice associates with interleukin-6 production. Journal of Pharmacology and Experimental Therapeutics, 347(1), 136-144.
  More info

  PMID: 23929842;PMCID: PMC3781416;Abstract: Cholestasis results from interrupted bile flow and is associated with immune-mediated liver diseases. It is unclear how inflammation contributes to cholestasis. The aim of this study was to determine whether T and B cells contribute to hepatic transporter expression under basal and inflammatory conditions. C57BL/6J wild-type mice or strains lacking T, B, or both T and B cells were exposed to lipopolysaccharide (LPS) or saline, and livers were collected 16 hours later. Branched DNA signal amplification was used to assess mRNA levels of organic anion-transporting polypeptides (Oatp) 1a1, 1a4, and 1b2; organic cation transporter (Oct) 1; canalicular bile-salt export pump (Bsep); multidrug resistance-associated proteins (Mrp) 2 and 3; and sodium-taurocholate cotransporting polypeptide (Ntcp). Real-time polymerase chain reaction analysis was used to correlate changes of transporter expression with interleukin-1b (IL-1b), IL-6, IL-17A, IL-17F, tumor necrosis factor-α (TNF-α), and interferon-γ expression in the liver. LPS treatment inhibited Bsep and Oct1 mRNA expression, and this was abrogated with a loss of T cells, but not B cells. In addition, the absence of T cells increased Mrp2 mRNA expression, whereas B cell deficiency attenuated Oatp1a4 mRNA in LPS-treated mice. Oatp1a1, Oatp1b2, Ntcp, and Mrp3 were largely unaffected by T or B cell deficiency. Lymphocyte deficiency altered basal and inflammatory IL-6, but not TNF-α or IL-1b, mRNA expression. Taken together, these data implicate lymphocytes as regulators of basal and inflammatory hepatic transporter expression and suggest that IL-6 signaling may play a critical role. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Bodeman, C. E., Dzierlenga, A. L., Tally, C. M., Mulligan, R. M., Lake, A. D., Cherrington, N. J., & McKarns, S. C. (2013). Differential regulation of hepatic organic cation transporter 1, organic anion-transporting polypeptide 1a4, bile-salt export pump, and multidrug resistance-associated protein 2 transporter expression in lymphocyte-deficient mice associates with interleukin-6 production. The Journal of pharmacology and experimental therapeutics, 347(1), 136-44.
  More info

  Cholestasis results from interrupted bile flow and is associated with immune-mediated liver diseases. It is unclear how inflammation contributes to cholestasis. The aim of this study was to determine whether T and B cells contribute to hepatic transporter expression under basal and inflammatory conditions. C57BL/6J wild-type mice or strains lacking T, B, or both T and B cells were exposed to lipopolysaccharide (LPS) or saline, and livers were collected 16 hours later. Branched DNA signal amplification was used to assess mRNA levels of organic anion-transporting polypeptides (Oatp) 1a1, 1a4, and 1b2; organic cation transporter (Oct) 1; canalicular bile-salt export pump (Bsep); multidrug resistance-associated proteins (Mrp) 2 and 3; and sodium-taurocholate cotransporting polypeptide (Ntcp). Real-time polymerase chain reaction analysis was used to correlate changes of transporter expression with interleukin-1b (IL-1b), IL-6, IL-17A, IL-17F, tumor necrosis factor-α (TNF-α), and interferon-γ expression in the liver. LPS treatment inhibited Bsep and Oct1 mRNA expression, and this was abrogated with a loss of T cells, but not B cells. In addition, the absence of T cells increased Mrp2 mRNA expression, whereas B cell deficiency attenuated Oatp1a4 mRNA in LPS-treated mice. Oatp1a1, Oatp1b2, Ntcp, and Mrp3 were largely unaffected by T or B cell deficiency. Lymphocyte deficiency altered basal and inflammatory IL-6, but not TNF-α or IL-1b, mRNA expression. Taken together, these data implicate lymphocytes as regulators of basal and inflammatory hepatic transporter expression and suggest that IL-6 signaling may play a critical role.
• Cherrington, N. J., Clarke, J. D., Sharapova, T., Lake, A. D., Blomme, E., & Maher, J. (2013). Circulating microRNA 122 in the methionine and choline-deficient mouse model of non-alcoholic steatohepatitis: miRNA 122 and NASH. Journal of Applied Toxicology, 34(6), 726-732. doi:10.1002/jat.2960
• Cherrington, N. J., Estrada, T. E., Frisk, H. A., Canet, M. J., Hardwick, R. N., Dvorak, B., Lux, K., & Halpern, M. D. (2013). The hepatic bile acid transporters Ntcp and Mrp2 are downregulated in experimental necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 304(1), G48-56.
  More info

  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants and is characterized by an extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. We have previously shown that, during the development of experimental NEC, the liver plays an important role in regulating inflammation in the ileum, and accumulation of ileal bile acids (BA) along with dysregulation of ileal BA transporters contributes to ileal damage. Given these findings, we speculated that hepatic BA transporters would also be altered in experimental NEC. Using both rat and mouse models of NEC, levels of Cyp7a1, Cyp27a1, and the hepatic BA transporters Bsep, Ntcp, Oatp2, Oatp4, Mrp2, and Mrp3 were investigated. In addition, levels of hepatic BA transporters were also determined when the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-18, which are both elevated in NEC, are neutralized during disease development. Ntcp and Mrp2 were decreased in NEC, but elevated ileal BA levels were not responsible for these reductions. However, neutralization of TNF-α normalized Ntcp, whereas removal of IL-18 normalized Mrp2 levels. These data show that the hepatic transporters Ntcp and Mrp2 are downregulated, whereas Cyp27a1 is increased in rodent models of NEC. Furthermore, increased levels of TNF-α and IL-18 in experimental NEC may play a role in the regulation of Ntcp and Mrp2, respectively. These data suggest the gut-liver axis should be considered when therapeutic modalities for NEC are developed.
• Cherrington, N. J., Estrada, T. E., Frisk, H. A., Canet, M. J., Hardwick, R. N., Dvorak, B., Lux, K., & Halpern, M. D. (2013). The hepatic bile acid transporters ntcp and Mrp2 are downregulated in experimental necrotizing enterocolitis. American Journal of Physiology - Gastrointestinal and Liver Physiology, 304(1), G48-G56.
  More info

  PMID: 23125159;PMCID: PMC3543632;Abstract: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants and is characterized by an extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. We have previously shown that, during the development of experimental NEC, the liver plays an important role in regulating inflammation in the ileum, and accumulation of ileal bile acids (BA) along with dysregulation of ileal BA transporters contributes to ileal damage. Given these findings, we speculated that hepatic BA transporters would also be altered in experimental NEC. Using both rat and mouse models of NEC, levels of Cyp7a1, Cyp27a1, and the hepatic BA transporters Bsep, Ntcp, Oatp2, Oatp4, Mrp2, and Mrp3 were investigated. In addition, levels of hepatic BA transporters were also determined when the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-18, which are both elevated in NEC, are neutralized during disease development. Ntcp and Mrp2 were decreased in NEC, but elevated ileal BA levels were not responsible for these reductions. However, neutralization of TNF-α normalized Ntcp, whereas removal of IL-18 normalized Mrp2 levels. These data show that the hepatic transporters Ntcp and Mrp2 are downregulated, whereas Cyp27a1 is increased in rodent models of NEC. Furthermore, increased levels of TNF-α and IL-18 in experimental NEC may play a role in the regulation of Ntcp and Mrp2, respectively. These data suggest the gut-liver axis should be considered when therapeutic modalities for NEC are developed. © 2013 the American Physiological Society.
• Cherrington, N., Augustine, L. M., Fisher, C. D., Lickteig, A. J., Aleksunes, L. M., Slitt, A. L., & Cherrington, N. J. (0). Gender divergent expression of Nqo1 in Sprague Dawley and August Copenhagen x Irish rats. Journal of biochemical and molecular toxicology, 22(2).
  More info

  In the mammalian liver, there is an abundance of enzymes that function to enable the safe and efficient elimination of potentially harmful xenobiotics that are encountered through environmental exposure. A variety of factors, including gender and genetic polymorphisms, contribute to the variation between an individual system's detoxification capacity and thus its ability to protect itself against oxidative stress, cellular damage, cell death, etc. NAD(P)H:quinone oxidoreducatase 1 (Nqo1) is an antioxidant enzyme that plays a major role in reducing reactive electrophiles, thereby protecting cells from free-radical damage and oxidative stress. The goal of this study was to determine the gender-specific expression and inducibility of Nqo1 in the Sprague Dawley (SD) and August Copenhagen x Irish (ACI) rat strains, two strains that are commonly used in drug metabolism and drug-induced enzyme induction, toxicity, and carcinogenesis studies. Nqo1 mRNA, protein, and activity levels were determined through 96 h in SD and ACI males and females following treatment with known Nqo1 inducers oltipraz and butylated hydroxyanisole. In the SD strain, gender dimorphic expression of Nqo1 was observed with female mRNA, protein, and activity levels being significantly higher than in males. In contrast, there were minimal differences in Nqo1 mRNA, protein, and activity levels between ACI males and females. The gender dimorphic expression of Nqo1 in the SD rats was maintained through the course of induction, with female-induced levels greater than male-induced levels indicating that SD females may have a greater capacity to protect against oxidative stress and thus a decreased susceptibility to carcinogens.
• Cherrington, N., Clarke, J. D., Novak, P., Lake, A. D., Shipkova, P., Aranibar, N., Robertson, D., Severson, P. L., Reily, M. D., Futscher, B. W., Lehman-McKeeman, L. D., & Cherrington, N. J. (2013). Characterization of Hepatocellular Carcinoma Related Genes and Metabolites in Human Nonalcoholic Fatty Liver Disease. Digestive diseases and sciences.
  More info

  The worldwide prevalences of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are estimated to range from 30 to 40 % and 5-17 %, respectively. Hepatocellular carcinoma (HCC) is primarily caused by hepatitis B infection, but retrospective data suggest that 4-29 % of NASH cases will progress to HCC. Currently the connection between NASH and HCC is unclear.
• Cherrington, N., Hardwick, R. N., Ferreira, D. W., More, V. R., Lake, A. D., Lu, Z., Manautou, J. E., Slitt, A. L., & Cherrington, N. J. (2013). Altered UDP-glucuronosyltransferase and sulfotransferase expression and function during progressive stages of human nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 41(3).
  More info

  The UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent major phase II drug-metabolizing enzymes that are also responsible for maintaining cellular homeostasis by metabolism of several endogenous molecules. Perturbations in the expression or function of these enzymes can lead to metabolic disorders and improper management of xenobiotics and endobiotics. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Because the liver plays a central role in the metabolism of xenobiotics, the purpose of the current study was to determine the effect of human NAFLD progression on the expression and function of UGTs and SULTs in normal, steatosis, NASH (fatty), and NASH (not fatty/cirrhosis) samples. We identified upregulation of UGT1A9, 2B10, and 3A1 and SULT1C4 mRNA in both stages of NASH, whereas UGT2A3, 2B15, and 2B28 and SULT1A1, 2B1, and 4A1 as well as 3'-phosphoadenosine-5'-phosphosulfate synthase 1 were increased in NASH (not fatty/cirrhosis) only. UGT1A9 and 1A6 and SULT1A1 and 2A1 protein levels were decreased in NASH; however, SULT1C4 was increased. Measurement of the glucuronidation and sulfonation of acetaminophen (APAP) revealed no alterations in glucuronidation; however, SULT activity was increased in steatosis compared with normal samples, but then decreased in NASH compared with steatosis. In conclusion, the expression of specific UGT and SULT isoforms appears to be differentially regulated, whereas sulfonation of APAP is disrupted during progression of NAFLD.
• Cherrington, N., Klein, D. M., Evans, K. K., Hardwick, R. N., Dantzler, W. H., Wright, S. H., & Cherrington, N. J. (2013). Basolateral uptake of nucleosides by Sertoli cells is mediated primarily by equilibrative nucleoside transporter 1. The Journal of pharmacology and experimental therapeutics, 346(1).
  More info

  The blood-testis barrier (BTB) prevents the entry of many xenobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiologic nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S-[(4-nitrophenyl)methyl]-6-thioinosine), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir disoproxil fumarate, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport.
• Cherrington, N., Klein, D. M., Wright, S. H., & Cherrington, N. J. (2013). Localization of Multidrug Resistance-Associated Proteins Along the Blood-testis Barrier in Rat, Macaque, and Human Testis. Drug metabolism and disposition: the biological fate of chemicals.
  More info

  The blood-testis barrier (BTB) prevents the entry of many drugs into seminiferous tubules thereby decreasing their efficacy. Previous data has shown that some of the transporters in the multidrug resistance-associated protein (MRP) family (ABCC) are expressed in the testis. By determining the subcellular localization of these transporters, their physiological function and effect on drug disposition may be better predicted. Using immunohistochemistry (IHC), we determined the site of expression of the MRP transporters expressed in testis, namely MRP1, MRP4, MRP5 and MRP8, in testis from immature and mature rats, rhesus macaques, and adult humans. We determined that in all species MRP1 was restricted to the basolateral membrane of Sertoli cells, MRP5 is located in Leydig cells, and MRP8 is located in round spermatids, whereas MRP4 showed species specific localization. MRP4 is expressed on the basolateral membrane of Sertoli cells in human and non-human primates, but on the apical membrane of Sertoli cells in immature and mature rats, representing a potential caution when using rat models as a means for studying drug disposition across the BTB. These data suggest that MRP1 acts as an obstacle in drug disposition into seminiferous tubules, as does MRP4 in human and non-human primates but not in rats. These data also suggest that MRP5 and MRP8 do not have a major impact on the penetration of drugs across the BTB.
• Cherrington, N., Lake, A. D., Novak, P., Hardwick, R. N., Flores-Keown, B., Zhao, F., Klimecki, W. T., & Cherrington, N. J. (2013). The Adaptive Endoplasmic Reticulum Stress Response to Lipotoxicity in Progressive Human Nonalcoholic Fatty Liver Disease. Toxicological sciences : an official journal of the Society of Toxicology.
  More info

  Nonalcoholic fatty liver disease (NAFLD) may progress from simple steatosis to severe, nonalcoholic steatohepatitis (NASH) in 7-14% of the U.S. population through a 2(nd) 'hit' in the form of increased oxidative stress and inflammation. ER stress signaling and the unfolded protein response (UPR) are triggered when high levels of lipids and misfolded proteins alter ER homeostasis creating a lipotoxic environment within NAFLD livers. The objective of this study was to determine the coordinate regulation of ER stress-associated genes in the progressive stages of human NAFLD. Human liver samples categorized as normal, steatosis, NASH (Fatty) and NASH (Not Fatty) were analyzed by individual Affymetrix GeneChip Human 1.0ST microarrays, immunoblots, and immunohistochemistry. A gene set enrichment analysis was performed on autophagy, apoptosis, lipogenesis and ER stress/UPR gene categories. An enrichment of downregulated genes in the ER stress associated-lipogenesis and ER stress/UPR gene categories was observed in NASH. Conversely, an enrichment of upregulated ER stress-associated genes for autophagy and apoptosis gene categories was observed in NASH. Protein expression of the adaptive liver response protein STC2 and the transcription factor XBP-1s were significantly elevated among NASH samples while other downstream ER stress proteins including CHOP, ATF4 and phosphorylated JNK and eIF2α were not significantly changed in disease progression. Increased nuclear accumulation of total XBP-1 protein was observed in steatosis and NASH livers. The findings reveal the presence of a coordinated, adaptive transcriptional response to hepatic ER stress in human NAFLD.
• Cherrington, N., Lake, A. D., Novak, P., Shipkova, P., Aranibar, N., Robertson, D., Reily, M. D., Lu, Z., Lehman-McKeeman, L. D., & Cherrington, N. J. (2013). Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease. Toxicology and applied pharmacology, 268(2).
  More info

  Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the 'classical' (neutral) and 'alternative' (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH.
• Cherrington, N., Merrell, M. D., Augustine, L. M., Slitt, A. L., & Cherrington, N. J. (0). Induction of drug metabolism enzymes and transporters by oltipraz in rats. Journal of biochemical and molecular toxicology, 22(2).
  More info

  Coordinate regulation of Phase-I and -II enzymes with xenobiotic transporters has been shown after treatment with microsomal enzyme inducers. The chemopreventive agent oltipraz (OPZ) induces Phase-I and -II drug-metabolizing enzymes such as CYP2B and NQO1. The purpose of this study was to examine the regulation of drug-metabolizing enzymes and transporters in response to OPZ treatment and to investigate a potential role for constitutive androstane receptor (CAR) in OPZ-mediated induction. Sprague-Dawley rats treated with OPZ exhibited increased mRNA and protein levels of both Nqo1 and Cyp2b1/2 by 24 h. To examine whether OPZ activates transporter gene expression via CAR, sexually dimorphic male and female Wistar-Kyoto (WKY) rats were treated with OPZ and mRNA levels quantified by bDNA signal amplification. OPZ induced Ugt1a6 and Ugt2b1 in males significantly higher than in females, indicating a CAR-dependent mechanism of induction. However, OPZ induced microsomal epoxide hydrolase, NAD(P)H quinone oxidoreductase, and Cyp3a1/23 equally in both genders, indicating a CAR-independent mechanism of induction of these genes. Similarly, the transporters Mdr1a, Mdr1b, Mrp3, and Mrp4 were induced by OPZ without any apparent difference between genders. In summary, OPZ coordinately increases multiple hepatic xenobiotic transporter mRNA levels, along with Phase-I and -II enzymes some of which may occur through CAR-dependent mechanisms.
• Clarke, J. D., Sharapova, T., Lake, A. D., Blomme, E., Maher, J., & Cherrington, N. J. (2013). Circulating microRNA 122 in the methionine- and choline-deficient mouse model of non-alcoholic steatohepatitis. Journal of Applied Toxicology.
  More info

  Abstract: Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) and is a major cause of liver cirrhosis and hepatic failure. The methionine choline-deficient diet (MCD) is a frequently used hepatotoxicity animal model of NASH that induces hepatic transaminase (ALT, AST) elevations and hepatobiliary histological changes similar to those observed in human NASH. Liver-specific microRNA-122 (miR-122) has been shown as a key regulator of cholesterol and fatty acid metabolism in adult liver, and has recently been proposed as a sensitive and specific circulating biomarker of hepatic injury. The purpose of this study was to assess miR-122 serum levels in mice receiving an MCD diet for 0, 3, 7, 14, 28 and 56 days and compare the performance vs. routine clinical chemistry when benchmarked against the histopathological liver findings. MiR-122 levels were quantified in serum using RT-qPCR. Both miR-122 and ALT/AST levels were significantly elevated in serum at all timepoints. MiR-122 levels increased on average by 40-fold after 3 days of initiating the MCD diet, whereas ALT and AST changes were 4.8- and 3.3-fold, respectively. In general, miR-122 levels remained elevated across all time points, whereas the ALT/AST increases were less robust but correlated with the progressive severity of NASH as assessed by histopathology. In conclusion, serum levels of miR-122 can potentially be used as a sensitive biomarker for the early detection of hepatotoxicity and can aid in monitoring the extent of NAFLD-associated liver injury in mouse efficacy models. © 2013 John Wiley & Sons, Ltd.
• Hardwick, R. N., Ferreira, D. W., More, V. R., Lake, A. D., Zhenqiang, L. u., Manautou, J. E., Slitt, A. L., & Cherrington, N. J. (2013). Altered UDP-glucuronosyltransferase and sulfotransferase expression and function during progressive stages of human nonalcoholic fatty liver diseases. Drug Metabolism and Disposition, 41(3), 554-561.
  More info

  PMID: 23223517;PMCID: PMC3583487;Abstract: The UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent major phase II drug-metabolizing enzymes that are also responsible for maintaining cellular homeostasis by metabolism of several endogenous molecules. Perturbations in the expression or function of these enzymes can lead to metabolic disorders and improper management of xenobiotics and endobiotics. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Because the liver plays a central role in the metabolism of xenobiotics, the purpose of the current study was to determine the effect of human NAFLD progression on the expression and function of UGTs and SULTs in normal, steatosis, NASH (fatty), and NASH (not fatty/cirrhosis) samples. We identified upregulation of UGT1A9, 2B10, and 3A1 and SULT1C4 mRNA in both stages of NASH, whereas UGT2A3, 2B15, and 2B28 and SULT1A1, 2B1, and 4A1 as well as 39-phosphoadenosine-59-phosphosulfate synthase 1 were increased in NASH (not fatty/ cirrhosis) only. UGT1A9 and 1A6 and SULT1A1 and 2A1 protein levels were decreased in NASH; however, SULT1C4 was increased. Measurement of the glucuronidation and sulfonation of acetaminophen (APAP) revealed no alterations in glucuronidation; however, SULT activity was increased in steatosis compared with normal samples, but then decreased in NASH compared with steatosis. In conclusion, the expression of specific UGT and SULT isoforms appears to be differentially regulated, whereas sulfonation of APAP is disrupted during progression of NAFLD. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Klein, D. M., Evans, K. K., Hardwick, R. N., Dantzler, W. H., Wright, S. H., & Cherrington, N. J. (2013). Basolateral uptake of nucleosides by sertoli cells is mediated primarily by equilibrative nucleoside transporter. Journal of Pharmacology and Experimental Therapeutics, 346(1), 121-129.
  More info

  PMID: 23639800;PMCID: PMC3684844;Abstract: The blood-testis barrier (BTB) prevents the entry of many xe-nobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiologic nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S- [(4-nitrophenyl)methyl]-6-thioinosine), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir disoproxil fumarate, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Lake, A. D., Novak, P., Shipkova, P., Aranibar, N., Robertson, D., Reily, M. D., Zhenqiang, L. u., Lehman-McKeeman, L. D., & Cherrington, N. J. (2013). Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease. Toxicology and Applied Pharmacology, 268(2), 132-140.
  More info

  PMID: 23391614;PMCID: PMC3627549;Abstract: Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the 'classical' (neutral) and 'alternative' (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. © 2013 Elsevier Inc.
• More, V. R., Cheng, Q., Donepudi, A. C., Buckley, D. B., Lu, Z. J., Cherrington, N. J., & Slitt, A. L. (2013). Alcohol cirrhosis alters nuclear receptor and drug transporter expression in human liver. Drug metabolism and disposition: the biological fate of chemicals, 41(5), 1148-55.
  More info

  Unsafe use of alcohol results in approximately 2.5 million deaths worldwide, with cirrhosis contributing to 16.6% of reported deaths. Serum insulin levels are often elevated in alcoholism and may result in diabetes, which is why alcoholic liver disease and diabetes often are present together. Because there is a sizable population with these diseases alone or in combination, the purpose of this study was to determine whether transporter expression in human liver is affected by alcoholic cirrhosis, diabetes, and alcoholic cirrhosis coexisting with diabetes. Transporters aid in hepatobiliary excretion of many drugs and toxic chemicals and can be determinants of drug-induced liver injury. Drug transporter expression and transcription factor-relative mRNA and protein expression in normal, diabetic, cirrhotic, and cirrhosis with diabetes human livers were quantified. Cirrhosis significantly increased ABCC4, 5, ABCG2, and solute carrier organic anion (SLCO) 2B1 mRNA expression and decreased SLCO1B3 mRNA expression in the liver. ABCC1, 3-5, and ABCG2 protein expression was also upregulated by alcoholic cirrhosis. ABCC3-5 and ABCG2 protein expression was also upregulated in diabetic cirrhosis. Cirrhosis increased nuclear factor E2-related factor 2 mRNA expression, whereas it decreased pregnane-X-receptor and farnesoid-X-receptor mRNA expression in comparison with normal livers. Hierarchical cluster analysis indicated that expressions of ABCC2, 3, and 6; SLCO1B1 and 1B3; and ABCC4 and 5 were more closely related in the livers from this cohort. Overall, alcoholic cirrhosis altered transporter expression in human liver.
• More, V. R., Cheng, Q., Donepudi, A. C., Buckley, D. B., Lu, Z. J., Cherrington, N. J., & Slitt, A. L. (2013). Alcohol cirrhosis alters nuclear receptor and drug transporter expression in human livers. Drug Metabolism and Disposition, 41(5), 1148-1155.
  More info

  PMID: 23462698;PMCID: PMC3629807;Abstract: Unsafe use of alcohol results in approximately 2.5 million deaths worldwide, with cirrhosis contributing to 16.6% of reported deaths. Serum insulin levels are often elevated in alcoholism and may result in diabetes, which is why alcoholic liver disease and diabetes often are present together. Because there is a sizable population with these diseases alone or in combination, the purpose of this study was to determine whether transporter expression in human liver is affected by alcoholic cirrhosis, diabetes, and alcoholic cirrhosis coexisting with diabetes. Transporters aid in hepatobiliary excretion of many drugs and toxic chemicals and can be determinants of drug-induced liver injury. Drug transporter expression and transcription factor-relative mRNA and protein expression in normal, diabetic, cirrhotic, and cirrhosis with diabetes human livers were quantified. Cirrhosis significantly increased ABCC4, 5, ABCG2, and solute carrier organic anion (SLCO) 2B1 mRNA expression and decreased SLCO1B3 mRNA expression in the liver. ABCC1, 3-5, and ABCG2 protein expression was also upregulated by alcoholic cirrhosis. ABCC3-5 and ABCG2 protein expression was also upregulated in diabetic cirrhosis. Cirrhosis increased nuclear factor E2-related factor 2 mRNA expression, whereas it decreased pregnane-X-receptor and farnesoid-X-receptor mRNA expression in comparison with normal livers. Hierarchical cluster analysis indicated that expressions of ABCC2, 3, and 6; SLCO1B1 and 1B3; and ABCC4 and 5 were more closely related in the livers from this cohort. Overall, alcoholic cirrhosis altered transporter expression in human liver. © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Yalcin, E. B., More, V., Neira, K. L., Lu, Z. J., Cherrington, N. J., Slitt, A. L., & King, R. S. (2013). Downregulation of sulfotransferase expression and activity in diseased human livers. Drug Metabolism and Disposition, 41(9), 1642-1650.
  More info

  PMID: 23775849;PMCID: PMC3876809;Abstract: Sulfotransferase (SULT) function has been well studied in healthy human subjects by quantifying mRNA and protein expression and determining enzyme activity with probe substrates. However, it is not well known if sulfotransferase activity changes in metabolic and liver disease, such as diabetes, steatosis, or cirrhosis. Sulfotransferases have significant roles in the regulation of hormones and excretion of xenobiotics. In the present study of normal subjects with nonfatty livers and patients with steatosis, diabetic cirrhosis, and alcoholic cirrhosis, we sought to determine SULT1A1, SULT2A1, SULT1E1, and SULT1A3 activity and mRNA and protein expression in human liver tissue. In general, sulfotransferase activity decreased significantly with severity of liver disease from steatosis to cirrhosis. Specifically, SULT1A1 and SULT1A3 activities were lower in disease states relative to nonfatty tissues. Alcoholic cirrhotic tissues further contained lower SULT1A1 and 1A3 activities than those affected by either of the two other disease states. SULT2A1, on the other hand, was only reduced in alcoholic cirrhotic tissues. SULT1E1 was reduced both in diabetic cirrhosis and in alcoholic cirrhosis tissues, relative to nonfatty liver tissues. In conclusion, the reduced levels of sulfotransferase expression and activity in diseased versus nondiseased liver tissue may alter the metabolism and disposition of xenobiotics and affect homeostasis of endobiotic sulfotransferase substrates. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Yalcin, E. B., More, V., Neira, K. L., Lu, Z. J., Cherrington, N. J., Slitt, A. L., & King, R. S. (2013). Downregulation of sulfotransferase expression and activity in diseased human livers. Drug metabolism and disposition: the biological fate of chemicals, 41(9), 1642-50.
  More info

  Sulfotransferase (SULT) function has been well studied in healthy human subjects by quantifying mRNA and protein expression and determining enzyme activity with probe substrates. However, it is not well known if sulfotransferase activity changes in metabolic and liver disease, such as diabetes, steatosis, or cirrhosis. Sulfotransferases have significant roles in the regulation of hormones and excretion of xenobiotics. In the present study of normal subjects with nonfatty livers and patients with steatosis, diabetic cirrhosis, and alcoholic cirrhosis, we sought to determine SULT1A1, SULT2A1, SULT1E1, and SULT1A3 activity and mRNA and protein expression in human liver tissue. In general, sulfotransferase activity decreased significantly with severity of liver disease from steatosis to cirrhosis. Specifically, SULT1A1 and SULT1A3 activities were lower in disease states relative to nonfatty tissues. Alcoholic cirrhotic tissues further contained lower SULT1A1 and 1A3 activities than those affected by either of the two other disease states. SULT2A1, on the other hand, was only reduced in alcoholic cirrhotic tissues. SULT1E1 was reduced both in diabetic cirrhosis and in alcoholic cirrhosis tissues, relative to nonfatty liver tissues. In conclusion, the reduced levels of sulfotransferase expression and activity in diseased versus nondiseased liver tissue may alter the metabolism and disposition of xenobiotics and affect homeostasis of endobiotic sulfotransferase substrates.
• Canet, M. J., Hardwick, R. N., Lake, A. D., Kopplin, M. J., Scheffer, G. L., Klimecki, W. T., Gandolfi, A. J., & Cherrington, N. J. (2012). Altered arsenic disposition in experimental nonalcoholic fatty liver disease. Drug Metabolism and Disposition, 40(9), 1817-1824.
  More info

  PMID: 22699396;PMCID: PMC3422539;Abstract: Nonalcoholic fatty liver disease (NAFLD) is represented by a spectrum of liver pathologies ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Liver damage sustained in the progressive stages of NAFLD may alter the ability of the liver to properly metabolize and eliminate xenobiotics. The purpose of the current study was to determine whether NAFLD alters the disposition of the environmental toxicant arsenic. C57BL/6 mice were fed either a high-fat or a methionine-choline-deficient diet to model simple steatosis and NASH, respectively. At the conclusion of the dietary regimen, all mice were given a single oral dose of either sodium arsenate or arsenic trioxide. Mice with NASH excreted significantly higher levels of total arsenic in urine (24 h) compared with controls. Total arsenic in the liver and kidneys of NASH mice was not altered; however, NASH liver retained significantly higher levels of the monomethyl arsenic metabolite, whereas dimethyl arsenic was retained significantly less in the kidneys of NASH mice. NASH mice had significantly higher levels of the more toxic trivalent form in their urine, whereas the pentavalent form was preferentially retained in the liver of NASH mice. Moreover, hepatic protein expression of the arsenic biotransformation enzyme arsenic (3+ oxidation state) methyltransferase was not altered in NASH animals, whereas protein expression of the membrane transporter multidrug resistance-associated protein 1 was increased, implicating cellular transport rather than biotransformation as a possible mechanism. These results suggest that NASH alters the disposition of arsenical species, which may have significant implications on the overall toxicity associated with arsenic in NASH. Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics.
• Cherrington, N. J., & Hardwick, R. N. (2012). Measuring Altered Disposition of Xenobiotics in Experimental Models of Liver Disease. Current Protocols in Toxicology, 52(1). doi:10.1002/0471140856.tx2301s52
• Cherrington, N., Canet, M. J., Hardwick, R. N., Lake, A. D., Kopplin, M. J., Scheffer, G. L., Klimecki, W. T., Gandolfi, A. J., & Cherrington, N. J. (2012). Altered arsenic disposition in experimental nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 40(9).
  More info

  Nonalcoholic fatty liver disease (NAFLD) is represented by a spectrum of liver pathologies ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Liver damage sustained in the progressive stages of NAFLD may alter the ability of the liver to properly metabolize and eliminate xenobiotics. The purpose of the current study was to determine whether NAFLD alters the disposition of the environmental toxicant arsenic. C57BL/6 mice were fed either a high-fat or a methionine-choline-deficient diet to model simple steatosis and NASH, respectively. At the conclusion of the dietary regimen, all mice were given a single oral dose of either sodium arsenate or arsenic trioxide. Mice with NASH excreted significantly higher levels of total arsenic in urine (24 h) compared with controls. Total arsenic in the liver and kidneys of NASH mice was not altered; however, NASH liver retained significantly higher levels of the monomethyl arsenic metabolite, whereas dimethyl arsenic was retained significantly less in the kidneys of NASH mice. NASH mice had significantly higher levels of the more toxic trivalent form in their urine, whereas the pentavalent form was preferentially retained in the liver of NASH mice. Moreover, hepatic protein expression of the arsenic biotransformation enzyme arsenic (3+ oxidation state) methyltransferase was not altered in NASH animals, whereas protein expression of the membrane transporter multidrug resistance-associated protein 1 was increased, implicating cellular transport rather than biotransformation as a possible mechanism. These results suggest that NASH alters the disposition of arsenical species, which may have significant implications on the overall toxicity associated with arsenic in NASH.
• Cherrington, N., Clarke, J. D., & Cherrington, N. J. (2012). Genetics or environment in drug transport: the case of organic anion transporting polypeptides and adverse drug reactions. Expert opinion on drug metabolism & toxicology, 8(3).
  More info

  Organic anion transporting polypeptide (OATP) uptake transporters are important for the disposition of many drugs and perturbed OATP activity can contribute to adverse drug reactions (ADRs). It is well documented that both genetic and environmental factors can alter OATP expression and activity. Genetic factors include single nucleotide polymorphisms (SNPs) that change OATP activity and epigenetic regulation that modify OATP expression levels. SNPs in OATPs contribute to ADRs. Environmental factors include the pharmacological context of drug-drug interactions and the physiological context of liver diseases. Liver diseases such as non-alcoholic fatty liver disease, cholestasis and hepatocellular carcinoma change the expression of multiple OATP isoforms. The role of liver diseases in the occurrence of ADRs is unknown.
• Cherrington, N., Hardwick, R. N., & Cherrington, N. J. (2012). Measuring altered disposition of xenobiotics in experimental models of liver disease. Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.], Chapter 23.
  More info

  Understanding the metabolic pathway and excretion mechanisms governing the disposition of a compound is essential to the safe use of pharmaceutical agents. Because the liver is the primary organ responsible for the metabolism and elimination of xenobiotics, chronic liver disease can have a significant effect on the disposition of many xenobiotics due to changes in the expression or function of drug metabolizing enzymes and transporters. Liver disease can result in increased retention of a xenobiotic within the body, causing greater exposure of the individual to a potentially harmful compound, which may lead to toxicity. On the other hand, liver disease may also up-regulate the elimination processes of a xenobiotic, accelerating its removal from the body. With regard to a pharmaceutical agent, enhanced elimination may result in a decreased pharmacologic effect. Such alterations may necessitate dosage adjustments to achieve the desired therapeutic outcome.
• Cherrington, N., Hardwick, R. N., Fisher, C. D., Street, S. M., Canet, M. J., & Cherrington, N. J. (2012). Molecular mechanism of altered ezetimibe disposition in nonalcoholic steatohepatitis. Drug metabolism and disposition: the biological fate of chemicals, 40(3).
  More info

  Ezetimibe (EZE) lowers serum lipid levels by blocking cholesterol uptake in the intestine. Disposition of EZE and its pharmacologically active glucuronide metabolite (EZE-GLUC) to the intestine is dependent on hepatobiliary efflux. Previous studies suggested that hepatic transporter expression and function may be altered during nonalcoholic steatohepatitis (NASH). The purpose of the current study was to determine whether NASH-induced changes in the expression and function of hepatic transporters result in altered disposition of EZE and EZE-GLUC. Rats fed a methionine- and choline-deficient (MCD) diet for 8 weeks were administered 10 mg/kg EZE either by intravenous bolus or oral gavage. Plasma and bile samples were collected over 2 h followed by terminal urine and tissue collection. EZE and EZE-GLUC concentrations were determined by liquid chromatography-tandem mass spectrometry. The sinusoidal transporter Abcc3 was induced in MCD rats, which correlated with increased plasma concentrations of EZE-GLUC, regardless of dosing method. Hepatic expression of the biliary transporters Abcc2 and Abcb1 was also increased in MCD animals, but the biliary efflux of EZE-GLUC was slightly diminished, whereas biliary bile acid concentrations were unaltered. The cellular localization of Abcc2 and Abcb1 appeared to be internalized away from the canalicular membrane in MCD livers, providing a mechanism for the shift to plasma drug efflux. The combination of induced expression and altered localization of efflux transporters in NASH shifts the disposition profile of EZE-GLUC toward plasma retention away from the site of action. This increased plasma retention of drugs in NASH may have implications for the pharmacological effect and safety of numerous drugs.
• Clarke, J. D., & Cherrington, N. J. (2012). Genetics or environment in drug transport: The case of organic anion transporting polypeptides and adverse drug reactions. Expert Opinion on Drug Metabolism and Toxicology, 8(3), 349-360.
  More info

  PMID: 22280100;PMCID: PMC3753226;Abstract: Introduction: Organic anion transporting polypeptide (OATP) uptake transporters are important for the disposition of many drugs and perturbed OATP activity can contribute to adverse drug reactions (ADRs). It is well documented that both genetic and environmental factors can alter OATP expression and activity. Genetic factors include single nucleotide polymorphisms (SNPs) that change OATP activity and epigenetic regulation that modify OATP expression levels. SNPs in OATPs contribute to ADRs. Environmental factors include the pharmacological context of drugdrug interactions and the physiological context of liver diseases. Liver diseases such as non-alcoholic fatty liver disease, cholestasis and hepatocellular carcinoma change the expression of multiple OATP isoforms. The role of liver diseases in the occurrence of ADRs is unknown. Areas covered: This article covers the roles OATPs play in ADRs when considered in the context of genetic or environmental factors. The reader will gain a greater appreciation for the current evidence regarding the salience and importance of each factor in OATP-mediated ADRs. Expert opinion: A SNP in a single OATP transporter can cause changes in drug pharmacokinetics and contribute to ADRs but, because of overlap in substrate specificities, there is potential for compensatory transport by other OATP isoforms. By contrast, the expression of multiple OATP isoforms is decreased in liver diseases, reducing compensatory transport and thereby increasing the probability of ADRs. To date, most research has focused on the genetic factors in OATP-mediated ADRs while the impact of environmental factors has largely been ignored. © 2012 Informa UK, Ltd.
• Hardwick, R. N., & Cherrington, N. J. (2012). Measuring altered disposition of xenobiotics in experimental models of liver disease. Current Protocols in Toxicology, 1(SUPPL.52).
  More info

  PMID: 22549269;Abstract: Understanding the metabolic pathway and excretion mechanisms governing the disposition of a compound is essential to the safe use of pharmaceutical agents. Because the liver is the primary organ responsible for the metabolism and elimination of xenobiotics, chronic liver disease can have a significant effect on the disposition of many xenobiotics due to changes in the expression or function of drug metabolizing enzymes and transporters. Liver disease can result in increased retention of a xenobiotic within the body, causing greater exposure of the individual to a potentially harmful compound, whichmay lead to toxicity. On the other hand, liver disease may also up-regulate the elimination processes of a xenobiotic, accelerating its removal from the body. With regard to a pharmaceutical agent, enhanced elimination may result in a decreased pharmacologic effect. Such alterations may necessitate dosage adjustments to achieve the desired therapeutic outcome. © 2012 by John Wiley & Sons, Inc.
• Hardwick, R. N., Fisher, C. D., Street, S. M., Canet, M. J., & Cherrington, N. J. (2012). Molecular mechanism of altered ezetimibe disposition in nonalcoholic steatohepatitis. Drug Metabolism and Disposition, 40(3), 450-460.
  More info

  PMID: 22112382;PMCID: PMC3286272;Abstract: Ezetimibe (EZE) lowers serum lipid levels by blocking cholesterol uptake in the intestine. Disposition of EZE and its pharmacologically active glucuronide metabolite (EZE-GLUC) to the intestine is dependent on hepatobiliary efflux. Previous studies suggested that hepatic transporter expression and function may be altered during nonalcoholic steatohepatitis (NASH). The purpose of the current study was to determine whether NASH-induced changes in the expression and function of hepatic transporters result in altered disposition of EZE and EZE-GLUC. Rats fed a methionine- and choline-deficient (MCD) diet for 8 weeks were administered 10 mg/kg EZE either by intravenous bolus or oral gavage. Plasma and bile samples were collected over 2 h followed by terminal urine and tissue collection. EZE and EZE-GLUC concentrations were determined by liquid chromatography-tandem mass spectrometry. The sinusoidal transporter Abcc3 was induced in MCD rats, which correlated with increased plasma concentrations of EZE-GLUC, regardless of dosing method. Hepatic expression of the biliary transporters Abcc2 and Abcb1 was also increased in MCD animals, but the biliary efflux of EZE-GLUC was slightly diminished, whereas biliary bile acid concentrations were unaltered. The cellular localization of Abcc2 and Abcb1 appeared to be internalized away from the canalicular membrane in MCD livers, providing a mechanism for the shift to plasma drug efflux. The combination of induced expression and altered localization of efflux transporters in NASH shifts the disposition profile of EZE-GLUC toward plasma retention away from the site of action. This increased plasma retention of drugs in NASH may have implications for the pharmacological effect and safety of numerous drugs. Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics.
• Jelinek, D. A., Maghsoodi, B., Borbon, I. A., Hardwick, R. N., Cherrington, N. J., & Erickson, R. P. (2012). Genetic variation in the mouse model of Niemann Pick C1 affects female, as well as male, adiposity, and hepatic bile transporters but has indeterminate effects on caveolae. Gene, 491(2), 128-134.
  More info

  PMID: 22020183;PMCID: PMC3258670;Abstract: We have previously shown that male Npc1 heterozygous mice (Npc1 +/-), as compared to homozygous wild-type mice (Npc1 +/+), both maintained on the "lean" BALB/cJ genetic background, become obese on a high fat but not on a low fat diet. We have now extended this result for female heterozygous mice. When fed high-fat diet, the Npc1 +/- white adipose weight is also increased in females, therefore following the same trend as males. Bile transporters which had previously been found to be altered in Npc1 -/- mice on a high fat diet, showed related, but small, changes in mRNA levels but large changes in protein expression. We have addressed the possible role of caveolae in these differences. It has long been known that caveolin 1 is increased in the liver (sex not specified) of Npc1 +/- (compared to Npc1 +/+ and Npc1 -/-) mice and in heterozygous cultured skin fibroblasts of NPC1 carriers. We now find that caveolin 1 is increased in male, but not female liver and female, but not male adipose tissue. The caveolin 1 increase was not accompanied by changes in another caveolar protein, polymerase1 and transcript release factor (Ptrf). The numbers of caveolae in female adipose cells could not be correlated with levels of caveolae. Thus, we conclude that Npc1 affects female as well as male obesity and bile transporters but that effects on caveolin 1 are not discernible. © 2011 Elsevier B.V.
• Jelinek, D. A., Maghsoodi, B., Borbon, I. A., Hardwick, R. N., Cherrington, N. J., & Erickson, R. P. (2012). Genetic variation in the mouse model of Niemann Pick C1 affects female, as well as male, adiposity, and hepatic bile transporters but has indeterminate effects on caveolae. Gene, 491(2), 128-34.
  More info

  We have previously shown that male Npc1 heterozygous mice (Npc1(+/-)), as compared to homozygous wild-type mice (Npc1(+/+)), both maintained on the "lean" BALB/cJ genetic background, become obese on a high fat but not on a low fat diet. We have now extended this result for female heterozygous mice. When fed high-fat diet, the Npc1(+/-) white adipose weight is also increased in females, therefore following the same trend as males. Bile transporters which had previously been found to be altered in Npc1(-/-) mice on a high fat diet, showed related, but small, changes in mRNA levels but large changes in protein expression. We have addressed the possible role of caveolae in these differences. It has long been known that caveolin 1 is increased in the liver (sex not specified) of Npc1(+/-) (compared to Npc1(+/+) and Npc1(-/-)) mice and in heterozygous cultured skin fibroblasts of NPC1 carriers. We now find that caveolin 1 is increased in male, but not female liver and female, but not male adipose tissue. The caveolin 1 increase was not accompanied by changes in another caveolar protein, polymerase1 and transcript release factor (Ptrf). The numbers of caveolae in female adipose cells could not be correlated with levels of caveolae. Thus, we conclude that Npc1 affects female as well as male obesity and bile transporters but that effects on caveolin 1 are not discernible.
• Cheng, Q., Taguchi, K., Aleksunes, L. M., Manautou, J. E., Cherrington, N. J., Yamamoto, M., & Slitt, A. L. (2011). Constitutive activation of nuclear factor-E2-related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte-specific deletion of Kelch-like ECH-associated protein 1. Journal of Biochemical and Molecular Toxicology, 25(5), 320-329.
  More info

  PMID: 21538727;PMCID: PMC3710738;Abstract: Chemicals that activate nuclear factor-E2-related factor 2 (Nrf2) often increase multidrug-resistance-associated protein (Mrp) expression in liver. Hepatocyte-specific deletion of Kelch-like ECH-associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte-specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild-type and hepatocyte-specific Keap1-null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate-cysteine ligase catalytic subunit expression were increased in livers of Keap1-null mice. Organic anion-transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild-type mice. By contrast, Mrp 1-5 mRNA and protein levels were increased in Keap1-null mouse livers, with Mrp4 expression being more than 15-fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions. © 2011 Wiley Periodicals, Inc.
• Cherrington, N., Hardwick, R. N., Fisher, C. D., Canet, M. J., Scheffer, G. L., & Cherrington, N. J. (2011). Variations in ATP-binding cassette transporter regulation during the progression of human nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 39(12).
  More info

  Transporters located on the sinusoidal and canalicular membranes of hepatocytes regulate the efflux of drugs and metabolites into blood and bile, respectively. Changes in the expression or function of these transporters during liver disease may lead to a greater risk of adverse drug reactions. Nonalcoholic fatty liver disease (NAFLD) is a progressive condition encompassing the relatively benign steatosis and the more severe, inflammatory state of nonalcoholic steatohepatitis (NASH). Here, we present an analysis of the effect of NAFLD progression on the major ATP-binding cassette (ABC) efflux transport proteins ABCC1-6, ABCB1, and ABCG2. Human liver samples diagnosed as normal, steatotic, NASH (fatty), and NASH (not fatty) were analyzed. Increasing trends in mRNA expression of ABCC1, ABCC4-5, ABCB1, and ABCG2 were found with NAFLD progression, whereas protein levels of all transporters exhibited increasing trends with disease progression. Immunohistochemical staining of ABCC3, ABCB1, and ABCG2 revealed no alterations in cellular localization during NAFLD progression. ABCC2 staining revealed an alternative mechanism of regulation in NASH in which the transporter appears to be internalized away from the canalicular membrane. This correlated with a preferential shift in the molecular mass of ABCC2 from 200 to 180 kDa in NASH, which has been shown to be associated with a loss of glycosylation and internalization of the protein. These data demonstrate increased expression of multiple efflux transporters as well as altered cellular localization of ABCC2 in NASH, which may have profound effects on the ability of patients with NASH to eliminate drugs in an appropriate manner.
• Cherrington, N., Merrell, M. D., & Cherrington, N. J. (2011). Drug metabolism alterations in nonalcoholic fatty liver disease. Drug metabolism reviews, 43(3).
  More info

  Drug-metabolizing enzymes play a vital role in the elimination of the majority of therapeutic drugs. The major organ involved in drug metabolism is the liver. Chronic liver diseases have been identified as a potential source of significant interindividual variation in metabolism. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the United States, affecting between 60 and 90 million Americans, yet the vast majority of NAFLD patients are undiagnosed. NAFLD encompasses a spectrum of pathologies, ranging from steatosis to nonalcoholic steatohepatitis and fibrosis. Numerous animal studies have investigated the effects of NAFLD on hepatic gene expression, observing significant alterations in mRNA, protein, and activity levels. Information on the effects of NAFLD in human patients is limited, though several significant investigations have recently been published. Significant alterations in the activity of drug-metabolizing enzymes may affect the clearance of therapeutic drugs, with the potential to result in adverse drug reactions. With the enormous prevalence of NAFLD, it is conceivable that every drug currently on the market is being given to patients with NAFLD. The current review is intended to present the results from both animal models and human patients, summarizing the observed alterations in the expression and activity of the phase I and II drug-metabolizing enzymes.
• Hardwick, R. N., Fisher, C. D., Canet, M. J., Scheffer, G. L., & Cherrington, N. J. (2011). Variations in ATP-binding cassette transporter regulation during the progression of human nonalcoholic fatty liver disease. Drug Metabolism and Disposition, 39(12), 2395-2402.
  More info

  PMID: 21878559;PMCID: PMC3226375;Abstract: Transporters located on the sinusoidal and canalicular membranes of hepatocytes regulate the efflux of drugs and metabolites into blood and bile, respectively. Changes in the expression or function of these transporters during liver disease may lead to a greater risk of adverse drug reactions. Nonalcoholic fatty liver disease (NAFLD) is a progressive condition encompassing the relatively benign steatosis and the more severe, inflammatory state of nonalcoholic steatohepatitis (NASH). Here, we present an analysis of the effect of NAFLD progression on the major ATP-binding cassette (ABC) efflux transport proteins ABCC1-6, ABCB1, and ABCG2. Human liver samples diagnosed as normal, steatotic, NASH (fatty), and NASH (not fatty) were analyzed. Increasing trends in mRNA expression of ABCC1, ABCC4-5, ABCB1, and ABCG2 were found with NAFLD progression, whereas protein levels of all transporters exhibited increasing trends with disease progression. Immunohistochemical staining of ABCC3, ABCB1, and ABCG2 revealed no alterations in cellular localization during NAFLD progression. ABCC2 staining revealed an alternative mechanism of regulation in NASH in which the transporter appears to be internalized away from the canalicular membrane. This correlated with a preferential shift in the molecular mass of ABCC2 from 200 to 180 kDa in NASH, which has been shown to be associated with a loss of glycosylation and internalization of the protein. These data demonstrate increased expression of multiple efflux transporters as well as altered cellular localization of ABCC2 in NASH, which may have profound effects on the ability of patients with NASH to eliminate drugs in an appropriate manner. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.
• Lake, A. D., Novak, P., Fisher, C. D., Jackson, J. P., Hardwick, R. N., Billheimer, D. D., Klimecki, W. T., & Cherrington, N. J. (2011). Analysis of global and absorption, distribution, metabolism, and elimination gene expression in the progressive stages of human nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 39(10), 1954-60.
  More info

  Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups-normal, steatosis, and NASH-was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes down-regulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease-compromised hepatocytes.
• Merrell, M. D., & Cherrington, N. J. (2011). Drug metabolism alterations in nonalcoholic fatty liver disease. Drug Metabolism Reviews, 43(3), 317-334.
  More info

  PMID: 21612324;PMCID: PMC3753221;Abstract: Drug-metabolizing enzymes play a vital role in the elimination of the majority of therapeutic drugs. The major organ involved in drug metabolism is the liver. Chronic liver diseases have been identified as a potential source of significant interindividual variation in metabolism. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the United States, affecting between 60 and 90 million Americans, yet the vast majority of NAFLD patients are undiagnosed. NAFLD encompasses a spectrum of pathologies, ranging from steatosis to nonalcoholic steatohepatitis and fibrosis. Numerous animal studies have investigated the effects of NAFLD on hepatic gene expression, observing significant alterations in mRNA, protein, and activity levels. Information on the effects of NAFLD in human patients is limited, though several significant investigations have recently been published. Significant alterations in the activity of drug-metabolizing enzymes may affect the clearance of therapeutic drugs, with the potential to result in adverse drug reactions. With the enormous prevalence of NAFLD, it is conceivable that every drug currently on the market is being given to patients with NAFLD. The current review is intended to present the results from both animal models and human patients, summarizing the observed alterations in the expression and activity of the phase I and II drug-metabolizing enzymes. © 2011 Informa Healthcare USA, Inc.
• Cherrington, N., Hardwick, R. N., Fisher, C. D., Canet, M. J., Lake, A. D., & Cherrington, N. J. (2010). Diversity in antioxidant response enzymes in progressive stages of human nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 38(12).
  More info

  Nonalcoholic fatty liver disease (NAFLD), which occurs in approximately 17 to 40% of Americans, encompasses progressive stages of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH). Inflammation and oxidative stress are known characteristics of NAFLD; however, the precise mechanisms occurring during disease progression remain unclear. The purpose of the current study was to determine whether the expression or function of enzymes involved in the antioxidant response, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione transferase (GST), and glutamate cysteine ligase, are altered in the progression of human NAFLD. Human livers staged as normal, steatotic, NASH (fatty), and NASH (not fatty) were obtained from the Liver Tissue Cell Distribution System. NQO1 mRNA, protein, and activity tended to increase with disease progression. mRNA levels of the GST isoforms A1, A2, A4, M3, and P1 increased with NAFLD progression. Likewise, GST A and P protein increased with progression; however, GST M protein levels tended to decrease. Of interest, total GST activity toward the substrate 1-chloro-2,4-dinitrobenzene decreased with NAFLD progression. GSH synthesis does not seem to be significantly dysregulated in NAFLD progression; however, the GSH/oxidized glutathione redox ratio seemed to be reduced with disease severity, indicating the presence of oxidative stress and depletion of GSH throughout progression of NAFLD. Malondialdehyde concentrations were significantly increased with disease progression, further indicating the presence of oxidative stress. Nuclear immunohistochemical staining of nuclear factor E2-related factor 2 (Nrf2), an indicator of activation of the transcription factor, was evident in all stages of NAFLD. The current data suggest that Nrf2 activation occurs in response to disease progression followed by induction of specific Nrf2 targets, whereas functionality of specific antioxidant defense enzymes seems to be impaired as NAFLD progresses.
• Erickson, R. P., Merrell, M. D., Cherrington, N. J., & A-kader, H. H. (2010). S1840 Acetaminophen Disposition: A Novel Marker for Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology, 138(5), S-800. doi:10.1016/s0016-5085(10)63687-6
• Genter, M. B., Krishan, M., Augustine, L. M., & Cherrington, N. J. (2010). Drug transporter expression and localization in rat nasal respiratory and olfactory mucosa and olfactory bulb. Drug Metabolism and Disposition, 38(10), 1644-1647.
  More info

  PMID: 20660103;PMCID: PMC2957163;Abstract: Uptake of drugs and other xenobiotics from the nasal cavity and into either the brain or systemic circulation can occur through several different mechanisms, including paracellular transport and movement along primary olfactory nerve axons, which extend from the nasal cavity to the olfactory bulb of the brain. The present study was conducted to expand knowledge on a third means of uptake, namely the expression of drug transporters in the rat nasal epithelium. We used branched DNA technology to compare the level of expression of nine transporters [(equilibrative nucleoside transporters (ENT)1 and ENT2; organic cation transporter (OCT)1, 2, and 3; OCTN1; organic anion-transporting polypeptide (OATP)3; and multidrug resistance (MRP)1 and MRP4] in nasal respiratory mucosa, olfactory mucosa, and olfactory bulb to the level of expression of these transporters in the liver and kidney. Transporters with high expression in the nasal respiratory mucosa or olfactory tissues were immunolocalized by immunohistochemistry. ENT1 and ENT2 expression was relatively high in nasal epithelia and olfactory bulb, which may explain the uptake of intranasally administered nucleoside derivatives observed by other investigators. OATP3 immunoreactivity was high in olfactory epithelium and olfactory nerve bundles, which suggests that substrates transported by OATP3 may be candidates for intranasal administration. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.
• Genter, M. B., Krishan, M., Augustine, L. M., & Cherrington, N. J. (2010). Drug transporter expression and localization in rat nasal respiratory and olfactory mucosa and olfactory bulb. Drug metabolism and disposition: the biological fate of chemicals, 38(10), 1644-7.
  More info

  Uptake of drugs and other xenobiotics from the nasal cavity and into either the brain or systemic circulation can occur through several different mechanisms, including paracellular transport and movement along primary olfactory nerve axons, which extend from the nasal cavity to the olfactory bulb of the brain. The present study was conducted to expand knowledge on a third means of uptake, namely the expression of drug transporters in the rat nasal epithelium. We used branched DNA technology to compare the level of expression of nine transporters [(equilibrative nucleoside transporters (ENT)1 and ENT2; organic cation transporter (OCT)1, 2, and 3; OCTN1; organic anion-transporting polypeptide (OATP)3; and multidrug resistance (MRP)1 and MRP4] in nasal respiratory mucosa, olfactory mucosa, and olfactory bulb to the level of expression of these transporters in the liver and kidney. Transporters with high expression in the nasal respiratory mucosa or olfactory tissues were immunolocalized by immunohistochemistry. ENT1 and ENT2 expression was relatively high in nasal epithelia and olfactory bulb, which may explain the uptake of intranasally administered nucleoside derivatives observed by other investigators. OATP3 immunoreactivity was high in olfactory epithelium and olfactory nerve bundles, which suggests that substrates transported by OATP3 may be candidates for intranasal administration.
• Hardwick, R. N., Fisher, C. D., Canet, M. J., Lake, A. D., & Cherrington, N. J. (2010). Diversity in antioxidant response enzymes in progressive stages of human nonalcoholic fatty liver disease. Drug Metabolism and Disposition, 38(12), 2293-2301.
  More info

  PMID: 20805291;PMCID: PMC2993454;Abstract: Nonalcoholic fatty liver disease (NAFLD), which occurs in approximately 17 to 40% of Americans, encompasses progressive stages of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH). Inflammation and oxidative stress are known characteristics of NAFLD; however, the precise mechanisms occurring during disease progression remain unclear. The purpose of the current study was to determine whether the expression or function of enzymes involved in the antioxidant response, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione transferase (GST), and glutamate cysteine ligase, are altered in the progression of human NAFLD. Human livers staged as normal, steatotic, NASH (fatty), and NASH (not fatty) were obtained from the Liver Tissue Cell Distribution System. NQO1 mRNA, protein, and activity tended to increase with disease progression. mRNA levels of the GST isoforms A1, A2, A4, M3, and P1 increased with NAFLD progression. Likewise, GST A and P protein increased with progression; however, GST M protein levels tended to decrease. Of interest, total GST activity toward the substrate 1-chloro-2,4-dinitrobenzene decreased with NAFLD progression. GSH synthesis does not seem to be significantly dysregulated in NAFLD progression; however, the GSH/oxidized glutathione redox ratio seemed to be reduced with disease severity, indicating the presence of oxidative stress and depletion of GSH throughout progression of NAFLD. Malondialdehyde concentrations were significantly increased with disease progression, further indicating the presence of oxidative stress. Nuclear immunohistochemical staining of nuclear factor E2-related factor 2 (Nrf2), an indicator of activation of the transcription factor, was evident in all stages of NAFLD. The current data suggest that Nrf2 activation occurs in response to disease progression followed by induction of specific Nrf2 targets, whereas functionality of specific antioxidant defense enzymes seems to be impaired as NAFLD progresses. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.
• Beilke, L. D., Aleksunes, L. M., Holland, R. D., Besselsen, D. G., Beger, R. D., Klaassen, C. D., & Cherrington, N. J. (2009). Constitutive androstane receptor-mediated changes in bile acid composition contributes to hepatoprotection from lithocholic acid-Induced liver injury in mice. Drug Metabolism and Disposition, 37(5), 1035-1045.
  More info

  PMID: 19196849;PMCID: PMC2683394;Abstract: Pharmacological activation of the constitutive androstane receptor (CAR) protects the liver during cholestasis. The current study evaluates how activation of CAR influences genes involved in bile acid biosynthesis as a mechanism of hepatoprotection during bile acid-induced liver injury. CAR activators phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or corn oil (CO) were administered to C57BL/6 wild-type (WT) and CAR knockout (CAR-null) mice before and during induction of intrahe- patic cholestasis using the secondary bile acid, lithocholic acid (LCA). In LCA-treated WT and all the CAR-null groups (excluding controls), histology revealed severe multifocal necrosis. This pathology was absent in WT mice pretreated with PB and TCPOBOP, indicating CAR-dependent hepatoprotection. Decreases in total hepatic bile acids and hepatic monohydroxy, dihydroxy, and trihy-droxy bile acids in PB- and TCPOBOP-pretreated WT mice correlated with hepatoprotection. In comparison, concentrations of mo- nohydroxylated and dihydroxylated bile acids were increased in all the treated CAR-null mice compared with CO controls. Along with several other enzymes (Cyp7b1, Cyp27a1, Cyp39a1), Cyp8b1 expression was increased in hepatoprotected mice, which could be suggestive of a shift in the bile acid biosynthesis pathway toward the formation of less toxic bile acids. In CAR-null mice, these changes in gene expression were not different among treatment groups. These results suggest CAR mediates a shift in bile acid biosynthesis toward the formation of less toxic bile acids, as well as a decrease in hepatic bile acid concentrations. We propose that these combined CAR-mediated effects may contribute to the hepatoprotection observed during LCA-induced liver injury.
• Beilke, L. D., Aleksunes, L. M., Olson, E. R., Besselsen, D. G., Klaassen, C. D., Dvorak, K., & Cherrington, N. J. (2009). Decreased apoptosis during CAR-mediated hepatoprotection against lithocholic acid-induced liver injury in mice. Toxicology Letters, 188(1), 38-44.
  More info

  PMID: 19433268;PMCID: PMC2681417;Abstract: Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that is regulated by the constitutive androstane receptor (CAR). Activation of CAR can protect the liver against bile acid-induced toxicity and it may have a role in cell death via apoptosis by altering expression of Bcl-2 family proteins such as myeloid cell leukemia-1 (Mcl-1). Our aim was to determine if activation of CAR reduces hepatocellular apoptosis during cholestasis as a mechanism of hepatoprotection. CAR+/+ (WT) and CAR-/- (CAR-null) mice were pre-treated with compounds known to activate CAR prior to induction of intrahepatic cholestasis using the secondary bile acid lithocholic acid (LCA). Pre-treatment with the CAR activators phenobarbital (PB) and TCPOBOP (TC), as well as the non-CAR activator pregnenolone 16α-carbontrile (PCN), protected against LCA-induced liver injury in WT mice, whereas liver injury was more extensive without CAR (CAR-null). Unexpectedly, expression of anti-apoptotic Mcl-1 and Bcl-xL was not increased in hepatoprotected mice. Compared to unprotected groups, apoptosis was decreased in hepatoprotected mice as evidenced by the absence of cleaved caspase 3 (cCasp3). In contrast to the cytoplasmic localization in the injured livers (LCA and oltipraz), Mcl-1 protein was localized in the nucleus of hepatoprotected livers to potentially promote cell survival. This study demonstrates that although apoptosis is reduced in hepatoprotected mice pre-treated with CAR and non-CAR activators; hepatoprotection is not directly a result of CAR-induced Mcl-1 expression. © 2009 Elsevier Ireland Ltd. All rights reserved.
• Campion, S. N., Tatis-Rios, C., Augustine, L. M., Goedken, M. J., Rooijen, N. v., Cherrington, N. J., & Manautou, J. E. (2009). Effect of allyl alcohol on hepatic transporter expression: Zonal patterns of expression and role of Kupffer cell function. Toxicology and Applied Pharmacology, 236(1), 49-58.
  More info

  PMID: 19371622;Abstract: During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH. © 2009 Elsevier Inc. All rights reserved.
• Campion, S. N., Tatis-Rios, C., Augustine, L. M., Goedken, M. J., van Rooijen, N., Cherrington, N. J., & Manautou, J. E. (2009). Effect of allyl alcohol on hepatic transporter expression: zonal patterns of expression and role of Kupffer cell function. Toxicology and applied pharmacology, 236(1), 49-58.
  More info

  During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH.
• Cherrington, N., Beilke, L. D., Aleksunes, L. M., Holland, R. D., Besselsen, D. G., Beger, R. D., Klaassen, C. D., & Cherrington, N. J. (2009). Constitutive androstane receptor-mediated changes in bile acid composition contributes to hepatoprotection from lithocholic acid-induced liver injury in mice. Drug metabolism and disposition: the biological fate of chemicals, 37(5).
  More info

  Pharmacological activation of the constitutive androstane receptor (CAR) protects the liver during cholestasis. The current study evaluates how activation of CAR influences genes involved in bile acid biosynthesis as a mechanism of hepatoprotection during bile acid-induced liver injury. CAR activators phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or corn oil (CO) were administered to C57BL/6 wild-type (WT) and CAR knockout (CAR-null) mice before and during induction of intrahepatic cholestasis using the secondary bile acid, lithocholic acid (LCA). In LCA-treated WT and all the CAR-null groups (excluding controls), histology revealed severe multifocal necrosis. This pathology was absent in WT mice pretreated with PB and TCPOBOP, indicating CAR-dependent hepatoprotection. Decreases in total hepatic bile acids and hepatic monohydroxy, dihydroxy, and trihydroxy bile acids in PB- and TCPOBOP-pretreated WT mice correlated with hepatoprotection. In comparison, concentrations of monohydroxylated and dihydroxylated bile acids were increased in all the treated CAR-null mice compared with CO controls. Along with several other enzymes (Cyp7b1, Cyp27a1, Cyp39a1), Cyp8b1 expression was increased in hepatoprotected mice, which could be suggestive of a shift in the bile acid biosynthesis pathway toward the formation of less toxic bile acids. In CAR-null mice, these changes in gene expression were not different among treatment groups. These results suggest CAR mediates a shift in bile acid biosynthesis toward the formation of less toxic bile acids, as well as a decrease in hepatic bile acid concentrations. We propose that these combined CAR-mediated effects may contribute to the hepatoprotection observed during LCA-induced liver injury.
• Cherrington, N., Beilke, L. D., Aleksunes, L. M., Olson, E. R., Besselsen, D. G., Klaassen, C. D., Dvorak, K., & Cherrington, N. J. (2009). Decreased apoptosis during CAR-mediated hepatoprotection against lithocholic acid-induced liver injury in mice. Toxicology letters, 188(1).
  More info

  Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that is regulated by the constitutive androstane receptor (CAR). Activation of CAR can protect the liver against bile acid-induced toxicity and it may have a role in cell death via apoptosis by altering expression of Bcl-2 family proteins such as myeloid cell leukemia-1 (Mcl-1). Our aim was to determine if activation of CAR reduces hepatocellular apoptosis during cholestasis as a mechanism of hepatoprotection. CAR(+/+) (WT) and CAR(-/-) (CAR-null) mice were pre-treated with compounds known to activate CAR prior to induction of intrahepatic cholestasis using the secondary bile acid lithocholic acid (LCA). Pre-treatment with the CAR activators phenobarbital (PB) and TCPOBOP (TC), as well as the non-CAR activator pregnenolone 16alpha-carbontrile (PCN), protected against LCA-induced liver injury in WT mice, whereas liver injury was more extensive without CAR (CAR-null). Unexpectedly, expression of anti-apoptotic Mcl-1 and Bcl-x(L) was not increased in hepatoprotected mice. Compared to unprotected groups, apoptosis was decreased in hepatoprotected mice as evidenced by the absence of cleaved caspase 3 (cCasp3). In contrast to the cytoplasmic localization in the injured livers (LCA and oltipraz), Mcl-1 protein was localized in the nucleus of hepatoprotected livers to potentially promote cell survival. This study demonstrates that although apoptosis is reduced in hepatoprotected mice pre-treated with CAR and non-CAR activators; hepatoprotection is not directly a result of CAR-induced Mcl-1 expression.
• Cherrington, N., Fisher, C. D., Lickteig, A. J., Augustine, L. M., Oude Elferink, R. P., Besselsen, D. G., Erickson, R. P., & Cherrington, N. J. (2009). Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats. European journal of pharmacology, 613(1-3).
  More info

  Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1beta). MC- rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC- diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC- rats when compared to control. Liver tissue from high-fat and MC- rats stained positive for IL-1beta, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity.
• Cherrington, N., Fisher, C. D., Lickteig, A. J., Augustine, L. M., Ranger-Moore, J., Jackson, J. P., Ferguson, S. S., & Cherrington, N. J. (2009). Hepatic cytochrome P450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 37(10).
  More info

  Members of the cytochrome P450 (P450) enzyme families CYP1, CYP2, and CYP3 are responsible for the metabolism of approximately 75% of all clinically relevant drugs. With the increased prevalence of nonalcoholic fatty liver disease (NAFLD), it is likely that patients with this disease represent an emerging population at significant risk for alterations in these important drug-metabolizing enzymes. The purpose of this study was to determine whether three progressive stages of human NALFD alter hepatic P450 expression and activity. Microsomes isolated from human liver samples diagnosed as normal, n = 20; steatosis, n = 11; nonalcoholic steatohepatitis (NASH) (fatty liver), n = 10; and NASH (no longer fatty), n = 11 were analyzed for P450 mRNA, protein, and enzyme activity. Microsomal CYP1A2, CYP2D6, and CYP2E1 mRNA levels were decreased with NAFLD progression, whereas CYP2A6, CYP2B6, and CYP2C9 mRNA expression increased. Microsomal protein expression of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 tended to decrease with NAFLD progression. Likewise, functional activity assays revealed decreasing trends in CYP1A2 (p = 0.001) and CYP2C19 (p = 0.05) enzymatic activity with increasing NAFLD severity. In contrast, activity of CYP2A6 (p = 0.001) and CYP2C9 (diclofenac, p = 0.0001; tolbutamide, p = 0.004) was significantly increased with NAFLD progression. Increased expression of proinflammatory cytokines tumor necrosis factor alpha and interleukin 1beta was observed and may be responsible for observed decreases in respective P450 activity. Furthermore, elevated CYP2C9 activity during NAFLD progression correlated with elevated hypoxia-induced factor 1alpha expression in the later stages of NAFLD. These results suggest that significant and novel changes occur in hepatic P450 activity during progressive stages of NAFLD.
• Fisher, C. D., Lickteig, A. J., Augustine, L. M., P.J., R., Besselsen, D. G., Erickson, R. P., & Cherrington, N. J. (2009). Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats. European Journal of Pharmacology, 613(1-3), 119-127.
  More info

  PMID: 19358839;PMCID: PMC2739623;Abstract: Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1β). MC- rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC- diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC- rats when compared to control. Liver tissue from high-fat and MC- rats stained positive for IL-1β, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity. © 2009 Elsevier B.V. All rights reserved.
• Fisher, C. D., Lickteig, A. J., Augustine, L. M., Ranger-Moore, J., Jackson, J. P., Ferguson, S. S., & Cherrington, N. J. (2009). Hepatic cytochrome P450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease. Drug Metabolism and Disposition, 37(10), 2087-2094.
  More info

  PMID: 19651758;PMCID: PMC2769034;Abstract: Members of the cytochrome P450 (P450) enzyme families CYP1, CYP2, and CYP3 are responsible for the metabolism of approximately 75% of all clinically relevant drugs. With the increased prevalence of nonalcoholic fatty liver disease (NAFLD), it is likely that patients with this disease represent an emerging population at significant risk for alterations in these important drug-metabolizing enzymes. The purpose of this study was to determine whether three progressive stages of human NALFD alter hepatic P450 expression and activity. Microsomes isolated from human liver samples diagnosed as normal, n = 20; steatosis, n = 11; nonalcoholic steatohepatitis (NASH) (fatty liver), n = 10; and NASH (no longer fatty), n = 11 were analyzed for P450 mRNA, protein, and enzyme activity. Microsomal CYP1A2, CYP2D6, and CYP2E1 mRNA levels were decreased with NAFLD progression, whereas CYP2A6, CYP2B6, and CYP2C9 mRNA expression increased. Microsomal protein expression of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 tended to decrease with NAFLD progression. Likewise, functional activity assays revealed decreasing trends in CYP1A2 (p = 0.001) and CYP2C19 (p = 0.05) enzymatic activity with increasing NAFLD severity. In contrast, activity of CYP2A6 (p = 0.001) and CYP2C9 (diclofenac, p = 0.0001; tolbutamide, p = 0.004) was significantly increased with NAFLD progression. Increased expression of proinflammatory cytokines tumor necrosis factor α and interleukin 1β was observed and may be responsible for observed decreases in respective P450 activity. Furthermore, elevated CYP2C9 activity during NAFLD progression correlated with elevated hypoxia-induced factor 1α expression in the later stages of NAFLD. These results suggest that significant and novel changes occur in hepatic P450 activity during progressive stages of NAFLD. Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics.
• Aleksunes, L. M., Augustine, L. M., Scheffer, G. L., Cherrington, N. J., & Manautou, J. E. (2008). Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment. Toxicology, 250(2-3), 82-8.
  More info

  The goal of this study was to identify alterations in mRNA and protein expression of various xenobiotic transport proteins in mouse kidney during cisplatin-induced acute renal failure. For this purpose, male C57BL/6J mice received a single dose of cisplatin (18 mg/kg, i.p.) or vehicle. Four days later, tissues were collected for assessment of plasma BUN, histopathological analysis of renal lesions, and mRNA and Western blot analysis of renal transporters including organic anion and cation transporters (Oat, Oct), organic anion transporting polypeptides (Oatp), multidrug resistance-associated proteins (Mrp), multidrug resistance proteins (Mdr), breast cancer resistance protein (Bcrp) and multidrug and toxin extrusion proteins (Mate). Cisplatin treatment caused necrosis of renal proximal tubules along with elevated plasma BUN and renal kidney injury molecule-1 mRNA expression. Cisplatin-induced renal injury increased mRNA and protein levels of the efflux transporters Mrp2, Mrp4, Mrp5, Mdr1a and Mdr1b. Uptake transporters Oatp2a1 and Oatp2b1 mRNA were also up-regulated following cisplatin. By contrast, expression of Oat1, Oat2, Oct2 and Oatp1a1 mRNA was reduced in cisplatin-treated mice. Expression of several uptake and efflux transporters was unchanged in cisplatin-treated mice. Apical staining of Mrp2 and Mrp4 proteins was enhanced in proximal tubules from cisplatin-treated mice. Collectively, these expression patterns suggest coordinated regulation of uptake and efflux pathways during cisplatin-induced renal injury. Reduced expression of basolateral and apical uptake transporters along with enhanced transcription of export transporters likely represents an adaptation to lower intracellular accumulation of chemicals, prevent their reabsorption and enhance urinary clearance.
• Aleksunes, L. M., Augustine, L. M., Scheffer, G. L., Cherrington, N. J., & Manautou, J. E. (2008). Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment. Toxicology, 250(2-3), 82-88.
  More info

  PMID: 18640236;PMCID: PMC2570650;Abstract: The goal of this study was to identify alterations in mRNA and protein expression of various xenobiotic transport proteins in mouse kidney during cisplatin-induced acute renal failure. For this purpose, male C57BL/6J mice received a single dose of cisplatin (18 mg/kg, i.p.) or vehicle. Four days later, tissues were collected for assessment of plasma BUN, histopathological analysis of renal lesions, and mRNA and Western blot analysis of renal transporters including organic anion and cation transporters (Oat, Oct), organic anion transporting polypeptides (Oatp), multidrug resistance-associated proteins (Mrp), multidrug resistance proteins (Mdr), breast cancer resistance protein (Bcrp) and multidrug and toxin extrusion proteins (Mate). Cisplatin treatment caused necrosis of renal proximal tubules along with elevated plasma BUN and renal kidney injury molecule-1 mRNA expression. Cisplatin-induced renal injury increased mRNA and protein levels of the efflux transporters Mrp2, Mrp4, Mrp5, Mdr1a and Mdr1b. Uptake transporters Oatp2a1 and Oatp2b1 mRNA were also up-regulated following cisplatin. By contrast, expression of Oat1, Oat2, Oct2 and Oatp1a1 mRNA was reduced in cisplatin-treated mice. Expression of several uptake and efflux transporters was unchanged in cisplatin-treated mice. Apical staining of Mrp2 and Mrp4 proteins was enhanced in proximal tubules from cisplatin-treated mice. Collectively, these expression patterns suggest coordinated regulation of uptake and efflux pathways during cisplatin-induced renal injury. Reduced expression of basolateral and apical uptake transporters along with enhanced transcription of export transporters likely represents an adaptation to lower intracellular accumulation of chemicals, prevent their reabsorption and enhance urinary clearance. © 2008 Elsevier Ireland Ltd. All rights reserved.
• Aleksunes, L. M., Slitt, A. L., Maher, J. M., Augustine, L. M., Goedken, M. J., Chan, J. Y., Cherrington, N. J., Klaassen, C. D., & Manautou, J. E. (2008). Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2. Toxicology and Applied Pharmacology, 226(1), 74-83.
  More info

  PMID: 17935745;PMCID: PMC2214834;Abstract: The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1-4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2 dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 h after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals. © 2007 Elsevier Inc. All rights reserved.
• Aleksunes, L. M., Slitt, A. L., Maher, J. M., Augustine, L. M., Goedken, M. J., Chan, J. Y., Cherrington, N. J., Klaassen, C. D., & Manautou, J. E. (2008). Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2. Toxicology and applied pharmacology, 226(1), 74-83.
  More info

  The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1-4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2 dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 h after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.
• Augustine, L. M., Fisher, C. D., Lickteig, A. J., Aleksunes, L. M., Slitt, A. L., & Cherrington, N. J. (2008). Gender divergent expression of Nqo1 in Sprague Dawley and August Copenhagen x Irish rats. Journal of Biochemical and Molecular Toxicology, 22(2), 93-100.
  More info

  PMID: 18418895;Abstract: In the mammalian liver, there is an abundance of enzymes that function to enable the safe and efficient elimination of potentially harmful xenobiotics that are encountered through environmental exposure. A variety of factors, including gender and genetic polymorphisms, contribute to the variation between an individual system's detoxification capacity and thus its ability to protect itself against oxidative stress, cellular damage, cell death, etc. NAD(P)H:quinone oxidoreducatase 1 (Nqo1) is an antioxidant enzyme that plays a major role in reducing reactive electrophiles, thereby protecting cells from free-radical damage and oxidative stress. The goal of this study was to determine the gender-specific expression and inducibility of Nqo1 in the Sprague Dawley (SD) and August Copenhagen x Irish (ACI) rat strains, two strains that are commonly used in drug metabolism and drug-induced enzyme induction, toxicity, and carcinogenesis studies. Nqo1 mRNA, protein, and activity levels were determined through 96 h in SD and ACI males and females following treatment with known Nqo1 inducers oltipraz and butylated hydroxyanisole. In the SD strain, gender dimorphic expression of Nqo1 was observed with female mRNA, protein, and activity levels being significantly higher than in males. In contrast, there were minimal differences in Nqo1 mRNA, protein, and activity levels between ACI males and females. The gender dimorphic expression of Nqo1 in the SD rats was maintained through the course of induction, with female-induced levels greater than male-induced levels indicating that SD females may have a greater capacity to protect against oxidative stress and thus a decreased susceptibility to carcinogens. © 2008 Wiley Periodicals, Inc.
• Beilke, L. D., Besselsen, D. G., Cheng, Q., Kulkarni, S., Slitt, A. I., & Cherrington, N. J. (2008). Minimal role of hepatic transporters in the hepatoprotection against LCA-induced intrahepatic cholestasis. Toxicological Sciences, 102(1), 196-204.
  More info

  PMID: 18032408;Abstract: The multidrug resistance-associated proteins (Mrps) are a family of adenosine triphosphate-dependent transporters that facilitate the movement of various compounds, including bile acids, out of hepatocytes. The current study was conducted to determine whether induction of these transporters alters bile acid disposition as a means of hepatoprotection during bile acid - induced cholestasis. Lithocholic acid (LCA) was used to induce intrahepatic cholestasis. C57BL/6 mice were pretreated with corn oil (CO) or known transporter inducers, phenobarbital (PB), oltipraz (OPZ), or TCPOBOP (TC) for 3 days prior to cotreatment with LCA and inducer for 4 days. Histopathology revealed that PB and TC pretreatments provide a protective effect from LCA-induced toxicity, whereas OPZ pretreatment did not. Both PB/LCA and TC/LCA cotreatment groups also had significantly lower alanine aminotransferase values than the LCA-only group. In TC/LCA cotreated mice compared with LCA only, messenger RNA (mRNA) expression of uptake transporters Ntcp and Oatp4 was significantly increased, as were sinusoidal efflux transporters Mrp3 and Mrp4. Although in PB/LCA cotreated mice, the only significant change compared with LCA-only treatment was an increase in uptake transporter Oatp4. Oatp1 was reduced in all groups compared with CO controls. No significant changes in mRNA expression were observed in Oatp2, Bsep, Mrp2, Bcrp, Mrp1, Mrp5, or Mrp6. Mrp4 protein expression was induced in the OPZ/LCA and TC/LCA cotreated groups, whereas Mrp3 protein levels remained unchanged between groups. Protein expression of Mrp1 and Mrp5 was increased in the unprotected LCA-only and OPZ/LCA mice. Thus, transporter expression did not correlate with histologic hepatoprotection, however, there was a correlation between hepatoprotection and significantly reduced total liver bile acids in the PB/LCA and TC/LCA cotreated mice compared with LCA only. In conclusion, changes in transporter expression did not correlate with hepatoprotection, and therefore, transport may not play a critical role in the observed hepatoprotection from LCA-induced cholestasis in the C57BL/6 mouse. © The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
• Campion, S. N., Johnson, R., Aleksunes, L. M., Goedken, M. J., Rooijen, N. V., Scheffer, G. L., Cherrington, N. J., & Manautou, J. E. (2008). Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function. American Journal of Physiology - Gastrointestinal and Liver Physiology, 295(2), G294-G304.
  More info

  PMID: 18556419;PMCID: PMC2519859;Abstract: During acetaminophen (APAP) hepatotoxicity, increased expression of multidrug resistance-associated proteins 2, 3, and 4 (Mrp2-4) occurs. Mrp4 is the most significantly upregulated transporter in mouse liver following APAP treatment. Although the expression profiles of liver transporters following APAP hepatotoxicity are well characterized, the regulatory mechanisms contributing to these changes remain unknown. We hypothesized that Kupffer cell-derived mediators participate in the regulation of hepatic transporters during APAP toxicity. To investigate this, C57BL/6J mice were pretreated with clodronate liposomes (0.1 ml iv) to deplete Kupffer cells and then challenged with APAP (500 mg/kg ip). Liver injury was assessed by plasma alanine aminotransferase and hepatic transporter protein expression was determined by Western blot and immunohistochemistry. Depletion of Kupffer cells by liposomal clodronate increased susceptibility to APAP hepatotoxicity. Although increased expression of several efflux transporters was observed after APAP exposure, only Mrp4 was found to be differentially regulated following Kupffer cell depletion. At 48 and 72 h after APAP dosing, Mrp4 levels were increased by 10- and 33-fold, respectively, in mice receiving empty liposomes. Immunohistochemistry revealed Mrp4 staining confined to centrilobular hepatocytes. Remarkably, Kupffer cell depletion completely prevented Mrp4 induction by APAP. Elevated plasma levels of TNF-α and IL-1β were also prevented by Kupffer cell depletion. These findings show that Kupffer cells protect the liver from APAP toxicity and that Kupffer cell mediators released in response to APAP are likely responsible for the induction of Mrp4. Copyright © 2008 the American Physiological Society.
• Campion, S. N., Johnson, R., Aleksunes, L. M., Goedken, M. J., van Rooijen, N., Scheffer, G. L., Cherrington, N. J., & Manautou, J. E. (2008). Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function. American journal of physiology. Gastrointestinal and liver physiology, 295(2), G294-304.
  More info

  During acetaminophen (APAP) hepatotoxicity, increased expression of multidrug resistance-associated proteins 2, 3, and 4 (Mrp2-4) occurs. Mrp4 is the most significantly upregulated transporter in mouse liver following APAP treatment. Although the expression profiles of liver transporters following APAP hepatotoxicity are well characterized, the regulatory mechanisms contributing to these changes remain unknown. We hypothesized that Kupffer cell-derived mediators participate in the regulation of hepatic transporters during APAP toxicity. To investigate this, C57BL/6J mice were pretreated with clodronate liposomes (0.1 ml iv) to deplete Kupffer cells and then challenged with APAP (500 mg/kg ip). Liver injury was assessed by plasma alanine aminotransferase and hepatic transporter protein expression was determined by Western blot and immunohistochemistry. Depletion of Kupffer cells by liposomal clodronate increased susceptibility to APAP hepatotoxicity. Although increased expression of several efflux transporters was observed after APAP exposure, only Mrp4 was found to be differentially regulated following Kupffer cell depletion. At 48 and 72 h after APAP dosing, Mrp4 levels were increased by 10- and 33-fold, respectively, in mice receiving empty liposomes. Immunohistochemistry revealed Mrp4 staining confined to centrilobular hepatocytes. Remarkably, Kupffer cell depletion completely prevented Mrp4 induction by APAP. Elevated plasma levels of TNF-alpha and IL-1beta were also prevented by Kupffer cell depletion. These findings show that Kupffer cells protect the liver from APAP toxicity and that Kupffer cell mediators released in response to APAP are likely responsible for the induction of Mrp4.
• Cheng, Q., Aleksunes, L. M., Manautou, J. E., Cherrington, N. J., Scheffer, G. L., Yamasaki, H., & Slitt, A. L. (2008). Drug-metabolizing enzyme and transporter expression in a mouse model of diabetes and obesity. Molecular Pharmaceutics, 5(1), 77-91.
  More info

  PMID: 18189363;Abstract: Obesity and type II diabetes pose a serious human health risk. Obese or diabetic patients usually take prescription drugs that require hepatic and renal metabolism and transport, and these patients sometimes display different pharmacokinetics of these drugs. Therefore, mRNA and protein expression of drug-metabolizing enzymes (DMEs) and transporters was measured in livers and kidneys of adult wild-type and ob/ob mice, which model obesity and diabetes. mRNA expression of numerous DMEs increased by at least 2-fold in livers of male ob/ob mice, including Cyp4a14, Cyp2b10, NAD(P)H:quinone oxidoreductase 1 (Nqo1), and sulfotransferase 2a1/2. In general, expression of uptake transporters was decreased in livers of ob/ob mice, namely organic anion-transporting polypeptides (Oatps) and sodium/taurocholate cotransporting polypeptide (Ntcp). In particular, Oatp1a1 mRNA and protein expression in livers of ob/ob mice was diminished to
• Cherrington, N., Beilke, L. D., Besselsen, D. G., Cheng, Q., Kulkarni, S., Slitt, A. L., & Cherrington, N. J. (2008). Minimal role of hepatic transporters in the hepatoprotection against LCA-induced intrahepatic cholestasis. Toxicological sciences : an official journal of the Society of Toxicology, 102(1).
  More info

  The multidrug resistance-associated proteins (Mrps) are a family of adenosine triphosphate-dependent transporters that facilitate the movement of various compounds, including bile acids, out of hepatocytes. The current study was conducted to determine whether induction of these transporters alters bile acid disposition as a means of hepatoprotection during bile acid-induced cholestasis. Lithocholic acid (LCA) was used to induce intrahepatic cholestasis. C57BL/6 mice were pretreated with corn oil (CO) or known transporter inducers, phenobarbital (PB), oltipraz (OPZ), or TCPOBOP (TC) for 3 days prior to cotreatment with LCA and inducer for 4 days. Histopathology revealed that PB and TC pretreatments provide a protective effect from LCA-induced toxicity, whereas OPZ pretreatment did not. Both PB/LCA and TC/LCA cotreatment groups also had significantly lower alanine aminotransferase values than the LCA-only group. In TC/LCA cotreated mice compared with LCA only, messenger RNA (mRNA) expression of uptake transporters Ntcp and Oatp4 was significantly increased, as were sinusoidal efflux transporters Mrp3 and Mrp4. Although in PB/LCA cotreated mice, the only significant change compared with LCA-only treatment was an increase in uptake transporter Oatp4. Oatp1 was reduced in all groups compared with CO controls. No significant changes in mRNA expression were observed in Oatp2, Bsep, Mrp2, Bcrp, Mrp1, Mrp5, or Mrp6. Mrp4 protein expression was induced in the OPZ/LCA and TC/LCA cotreated groups, whereas Mrp3 protein levels remained unchanged between groups. Protein expression of Mrp1 and Mrp5 was increased in the unprotected LCA-only and OPZ/LCA mice. Thus, transporter expression did not correlate with histologic hepatoprotection, however, there was a correlation between hepatoprotection and significantly reduced total liver bile acids in the PB/LCA and TC/LCA cotreated mice compared with LCA only. In conclusion, changes in transporter expression did not correlate with hepatoprotection, and therefore, transport may not play a critical role in the observed hepatoprotection from LCA-induced cholestasis in the C57BL/6 mouse.
• Cherrington, N., Fisher, C. D., Jackson, J. P., Lickteig, A. J., Augustine, L. M., & Cherrington, N. J. (2008). Drug metabolizing enzyme induction pathways in experimental non-alcoholic steatohepatitis. Archives of toxicology, 82(12).
  More info

  Non-alcoholic steatohepatitis (NASH) is a disease that compromises hepatic function and the capacity to metabolize numerous drugs. Aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor-E2 related factor 2 (Nrf2) are xenobiotic activated transcription factors that regulate induction of a number of drug metabolizing enzymes (DMEs). The purpose of the current study was to determine whether experimental NASH alters the xenobiotic activation of these transcription factors and induction of downstream DME targets Cyp1A1, Cyp2B10, Cyp3A11, Cyp4A14 and NAD(P)H:quinone oxidoreductase 1 (Nqo1), respectively. Mice fed normal rodent chow or methionine-choline-deficient (MCD) diet for 8 weeks were then treated with microsomal enzyme inducers beta-naphoflavone (BNF), 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), pregnenolone-16alpha-carbonitrile (PCN), clofibrate (CFB) or oltipraz (OPZ), known activators of AhR, CAR, PXR, PPARalpha and Nrf2, respectively. Results of this study show that (1) Hepatic PXR mRNA levels were significantly increased (1.4-fold) in mice fed MCD diet, while AhR, CAR, PPARalpha and Nrf2 were not affected. (2) The MCD diet did not alter hepatic inducibility of Cyp1A1, Cyp2B10, Cyp3A11 mRNA levels by their respective microsomal inducers. (3) Constitutive levels of Cyp4A14 mRNA were significantly increased in mice fed the MCD diet, yet further induction by clofibrate was not observed. (4) Hepatic Nqo1 mRNA levels were significantly increased by the MCD diet; however, additional induction of Nqo1 was still achievable following treatment with the Nrf2 activator OPZ.
• Cherrington, N., Lickteig, A. J., Cheng, X., Augustine, L. M., Klaassen, C. D., & Cherrington, N. J. (2008). Tissue distribution, ontogeny and induction of the transporters Multidrug and toxin extrusion (MATE) 1 and MATE2 mRNA expression levels in mice. Life sciences, 83(1-2).
  More info

  Transporters are expressed in a wide variety of tissues where they perform the critical function of enabling anionic and cationic chemicals of exogenous and endogenous origin to cross otherwise impermeable cell membranes. The Multidrug and toxin extrusion (MATE) transporters mediate cellular efflux of a variety of organic cations, including many drugs. The purpose of the current study was to determine (1) constitutive expression levels of MATE mRNA in various tissues, (2) whether there are gender differences in the expression of MATEs, (3) the ontogenic expression pattern of MATE1 in kidney and (4) whether MATEs are pharmacologically inducible in liver via activation of known transcription factors. In both male and female mice, MATE1 mRNA levels were highest in the kidney, where male expression was higher than female. MATE2 mRNA expression levels were the highest in the testis, where high expression was localized to Sertoli cells, a critical cell type of the blood testis barrier. In female mice, MATE2 mRNA levels were expressed most highly in the colon. The ontogenic pattern of expression of MATE1 mRNA in the kidneys of both males and females was gradual, with levels increasing steadily from prenatal day -2 to 45 days of age, and a gender difference appearing at day 30. Of the transcription factor activators examined (AhR, CAR, Nrf2, PPARalpha and PXR), none were capable of altering MATE1 or MATE2. The current findings support a potential role for MATE1 and MATE2 in a wide range of tissues and, notably, a unique role for MATE2 in the blood-testis barrier.
• Cherrington, N., Merrell, M. D., Jackson, J. P., Augustine, L. M., Fisher, C. D., Slitt, A. L., Maher, J. M., Huang, W., Moore, D. D., Zhang, Y., Klaassen, C. D., & Cherrington, N. J. (2008). The Nrf2 activator oltipraz also activates the constitutive androstane receptor. Drug metabolism and disposition: the biological fate of chemicals, 36(8).
  More info

  Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2-related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2-/- mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR-/- mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively.
• Das, P. C., Cao, Y., Rose, R. L., Cherrington, N., & Hodgson, E. (2008). Enzyme induction and cytotoxicity in human hepatocytes by chlorpyrifos and N,N-diethyl-m-toluamide (DEET). Drug Metabolism and Drug Interactions, 23(3-4), 237-260.
  More info

  PMID: 19326769;Abstract: Xenobiotics, including drugs and environmental chemicals, can influence cytochrome P450 (CYP) levels by altering the transcription of CYP genes. To minimize potential drug-pesticide and pesticide-pesticide interactions it is important to evaluate the potential of pesticides to induce CYP isoforms and to cause cytotoxicity in humans. The present study was designed to examine chlorpyrifos and DEET mediated induction of CYP isoforms and also to characterize their potential cytotoxic effects on primary human hepatocytes. DEET significantly induced CYP3A4, CYP2B6, CYP2A6 and CYP1A2 mRNA expression while chlorpyrifos induced CYP1A1, CYP1A2 and CYP3A4 mRNA, and to a lesser extent, CYP1B1 and CYP2B6 mRNA in primary human hepatocytes. Chlorpyrifos and DEET also mediated the expression of CYP isoforms, particularly CYP3A4, CYP2B6 and CYP1A1, as shown by CYP3A4-specific protein expression, testosterone metabolism and CYP1A1-specific activity assays. DEET is a mild, while chlorpyrifos is a relatively potent, inducer of adenylate kinase and caspase-3/7, an indicator of apoptosis, while inducing 15-20% and 25-30% cell death, respectively. Therefore, DEET and chlorpyrifos mediated induction of CYP mRNA and functional CYP isoforms together with their cytotoxic potential in human hepatocytes suggests that exposure to chlorpyrifos and/or DEET should be considered in human health impact analysis. © Freund Publishing House Ltd., 2008.
• Das, P. C., Cao, Y., Rose, R. L., Cherrington, N., & Hodgson, E. (2008). Enzyme induction and cytotoxicity in human hepatocytes by chlorpyrifos and N,N-diethyl-m-toluamide (DEET). Drug metabolism and drug interactions, 23(3-4), 237-60.
  More info

  Xenobiotics, including drugs and environmental chemicals, can influence cytochrome P450 (CYP) levels by altering the transcription of CYP genes. To minimize potential drug-pesticide and pesticide-pesticide interactions it is important to evaluate the potential of pesticides to induce CYP isoforms and to cause cytotoxicity in humans. The present study was designed to examine chlorpyrifos and DEET mediated induction of CYP isoforms and also to characterize their potential cytotoxic effects on primary human hepatocytes. DEET significantly induced CYP3A4, CYP2B6, CYP2A6 and CYP1A2 mRNA expression while chlorpyrifos induced CYP1A1, CYP1A2 and CYP3A4 mRNA, and to a lesser extent, CYP1B1 and CYP2B6 mRNA in primary human hepatocytes. Chlorpyrifos and DEET also mediated the expression of CYP isoforms, particularly CYP3A4, CYP2B6 and CYP1A1, as shown by CYP3A4-specific protein expression, testosterone metabolism and CYP1Al-specific activity assays. DEET is a mild, while chlorpyrifos is a relatively potent, inducer of adenylate kinase and caspase-3/7, an indicator of apoptosis, while inducing 15-20% and 25-30% cell death, respectively. Therefore, DEET and chlorpyrifos mediated induction of CYP mRNA and functional CYP isoforms together with their cytotoxic potential in human hepatocytes suggests that exposure to chlorpyrifos and/or DEET should be considered in human health impact analysis.
• Das, P. C., Streit, T. M., Cao, Y., Rose, R. L., Cherrington, N., Ross, M. K., Wallace, A. D., & Hodgson, E. (2008). Pyrethroids: Cytotoxicity and induction of CYP isoforms in human hepatocytes. Drug Metabolism and Drug Interactions, 23(3-4), 211-236.
  More info

  PMID: 19326768;Abstract: Deltamethrin [(S)-α-cyano-3-phenoxybenzyl-cis-(1R,3R)-3(2,2-dibromovinyl) (2,2-dimethyl-cyclopropane-carboxylate] and permethrin [3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlorovinyl) -2,2-dimethyl-cyclopropanecarboxylate] are pyrethroid insecticides used in agriculture, public health and military deployments. Pyrethroids are known to be capable of inducing cytochrome P450 (CYP) 2B1/2B2, CYP1A1 and overall CYP content in rat liver. The objectives of this study were to evaluate the potential of deltamethrin and permethrin to cause cytotoxicity and to induce CYP isoforms in human hepatocytes. Permethrin and deltamethrin showed dose-dependent effects on adenylate kinase activity in HepG2 cells, in which 50 and 100 μM doses, respectively, induced a 3-5 fold increase in activity, and also induced adenylate kinase activity in primary human hepatocytes. An approximately 3-fold induction was noted at 200 μM deltamethrin and a 4-fold induction at 100 μM permethrin. Cytotoxicity was noted in HepG2 cells following 48-72 h exposure to 100 or 200 μM deltamethrin and permethrin, respectively. Dose-dependent induction of caspase-3/7 was initiated by 12.5 μM deltamethrin or by 3.125 μM permethrin. Actinomycin D, a positive control for induction of caspase 3/7, induced caspase-3/7, an effect completely abrogated by the specific inhibitor Z-DEVD-FMK. At 100 μM deltamethrin 2-3 fold induction of CYP1A1 and CYP2B6 mRNA was observed, while at the same time an ∼25-fold induction of CYP3A4 was noted. Permethrin-mediated CYP induction was much less potent, 4-fold or less for CYP1A1, CYP3A4, CYP3A5, CYP2B6 and CYP2A6. It has also been shown that these pyrethroids are ligands for the pregnane X receptor (PXR). © Freund Publishing House Ltd., 2008.
• Das, P. C., Streit, T. M., Cao, Y., Rose, R. L., Cherrington, N., Ross, M. K., Wallace, A. D., & Hodgson, E. (2008). Pyrethroids: cytotoxicity and induction of CYP isoforms in human hepatocytes. Drug metabolism and drug interactions, 23(3-4), 211-36.
  More info

  Deltamethrin [(S)-alpha-cyano-3-phenoxybenzyl-cis-(1 R,3R)-3(2,2-dibromovinyl)(2,2-dimethyl-cyclopropane-carboxylate] and permethrin [3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate] are pyrethroid insecticides used in agriculture, public health and military deployments. Pyrethroids are known to be capable of inducing cytochrome P450 (CYP) 2B1/2B2, CYP1A1 and overall CYP content in rat liver. The objectives of this study were to evaluate the potential of deltamethrin and permethrin to cause cytotoxicity and to induce CYP isoforms in human hepatocytes. Permethrin and deltamethrin showed dose-dependent effects on adenylate kinase activity in HepG2 cells, in which 50 and 100 microM doses, respectively, induced a 3-5 fold increase in activity, and also induced adenylate kinase activity in primary human hepatocytes. An approximately 3-fold induction was noted at 200 microM deltamethrin and a 4-fold induction at 100 microM permethrin. Cytotoxicity was noted in HepG2 cells following 48-72 h exposure to 100 or 200 microM deltamethrin and permethrin, respectively. Dose-dependent induction of caspase-3/7 was initiated by 12.5 microM deltamethrin or by 3.125 microM permethrin. Actinomycin D, a positive control for induction of caspase 3/7, induced caspase-3/7, an effect completely abrogated by the specific inhibitor Z-DEVD-FMK. At 100 microM deltamethrin 2-3 fold induction of CYP1A1 and CYP2B6 mRNA was observed, while at the same time an approximately 25-fold induction of CYP3A4 was noted. Permethrin-mediated CYP induction was much less potent, 4-fold or less for CYP1A1, CYP3A4, CYP3A5, CYP2B6 and CYP2A6. It has also been shown that these pyrethroids are ligands for the pregnane X receptor (PXR).
• Fisher, C. D., Jackson, J. P., Lickteig, A. J., Augustine, L. M., & Cherrington, N. J. (2008). Drug metabolizing enzyme induction pathways in experimental non-alcoholic steatohepatitis. Archives of Toxicology, 82(12), 959-964.
  More info

  PMID: 18488193;Abstract: Non-alcoholic steatohepatitis (NASH) is a disease that compromises hepatic function and the capacity to metabolize numerous drugs. Aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARα), and nuclear factor-E2 related factor 2 (Nrf2) are xenobiotic activated transcription factors that regulate induction of a number of drug metabolizing enzymes (DMEs). The purpose of the current study was to determine whether experimental NASH alters the xenobiotic activation of these transcription factors and induction of downstream DME targets Cyp1A1, Cyp2B10, Cyp3A11, Cyp4A14 and NAD(P)H:quinone oxidoreductase 1 (Nqo1), respectively. Mice fed normal rodent chow or methionine-choline-deficient (MCD) diet for 8 weeks were then treated with microsomal enzyme inducers β-naphoflavone (BNF), 1,4-bis-[2-(3,5- dichloropyridyloxy)] benzene (TCPOBOP), pregnenolone-16α-carbonitrile (PCN), clofibrate (CFB) or oltipraz (OPZ), known activators of AhR, CAR, PXR, PPARα and Nrf2, respectively. Results of this study show that (1) Hepatic PXR mRNA levels were significantly increased (1.4-fold) in mice fed MCD diet, while AhR, CAR, PPARα and Nrf2 were not affected. (2) The MCD diet did not alter hepatic inducibility of Cyp1A1, Cyp2B10, Cyp3A11 mRNA levels by their respective microsomal inducers. (3) Constitutive levels of Cyp4A14 mRNA were significantly increased in mice fed the MCD diet, yet further induction by clofibrate was not observed. (4) Hepatic Nqo1 mRNA levels were significantly increased by the MCD diet; however, additional induction of Nqo1 was still achievable following treatment with the Nrf2 activator OPZ. © 2008 Springer-Verlag.
• Lickteig, A. J., Cheng, X., Augustine, L. M., Klaassen, C. D., & Cherrington, N. J. (2008). Tissue distribution, ontogeny and induction of the transporters Multidrug and toxin extrusion (MATE) 1 and MATE2 mRNA expression levels in mice. Life Sciences, 83(1-2), 59-64.
  More info

  PMID: 18573474;PMCID: PMC2494953;Abstract: Transporters are expressed in a wide variety of tissues where they perform the critical function of enabling anionic and cationic chemicals of exogenous and endogenous origin to cross otherwise impermeable cell membranes. The Multidrug and toxin extrusion (MATE) transporters mediate cellular efflux of a variety of organic cations, including many drugs. The purpose of the current study was to determine (1) constitutive expression levels of MATE mRNA in various tissues, (2) whether there are gender differences in the expression of MATEs, (3) the ontogenic expression pattern of MATE1 in kidney and (4) whether MATEs are pharmacologically inducible in liver via activation of known transcription factors. In both male and female mice, MATE1 mRNA levels were highest in the kidney, where male expression was higher than female. MATE2 mRNA expression levels were the highest in the testis, where high expression was localized to Sertoli cells, a critical cell type of the blood testis barrier. In female mice, MATE2 mRNA levels were expressed most highly in the colon. The ontogenic pattern of expression of MATE1 mRNA in the kidneys of both males and females was gradual, with levels increasing steadily from prenatal day - 2 to 45 days of age, and a gender difference appearing at day 30. Of the transcription factor activators examined (AhR, CAR, Nrf2, PPARα and PXR), none were capable of altering MATE1 or MATE2. The current findings support a potential role for MATE1 and MATE2 in a wide range of tissues and, notably, a unique role for MATE2 in the blood-testis barrier. © 2008 Elsevier Inc. All rights reserved.
• Merrell, M. D., Augustine, L. M., Slitt, A. L., & Cherrington, N. J. (2008). Induction of drug metabolism enzymes and transporters by oltipraz in rats. Journal of Biochemical and Molecular Toxicology, 22(2), 128-135.
  More info

  PMID: 18418891;Abstract: Coordinate regulation of Phase-I and -II enzymes with xenobiotic transporters has been shown after treatment with microsomal enzyme inducers. The chemopreventive agent oltipraz (OPZ) induces Phase-I and -II drug-metabolizing enzymes such as CYP2B and NQO1. The purpose of this study was to examine the regulation of drug-metabolizing enzymes and transporters in response to OPZ treatment and to investigate a potential role for constitutive androstane receptor (CAR) in OPZ-mediated induction. Sprague-Dawley rats treated with OPZ exhibited increased mRNA and protein levels of both Nqo1 and Cyp2b1/2 by 24 h. To examine whether OPZ activates transporter gene expression via CAR, sexually dimorphic male and female Wistar-Kyoto (WKY) rats were treated with OPZ and mRNA levels quantified by bDNA signal amplification. OPZ induced Ugt1a6 and Ugt2b1 in males significantly higher than in females, indicating a CAR-dependent mechanism of induction. However, OPZ induced microsomal epoxide hydrolase, NAD(P)H quinone oxidoreductase, and Cyp3a1/23 equally in both genders, indicating a CAR-independent mechanism of induction of these genes. Similarly, the transporters Mdr1a, Mdr1b, Mrp3, and Mrp4 were induced by OPZ without any apparent difference between genders. In summary, OPZ coordinately increases multiple hepatic xenobiotic transporter mRNA levels, along with Phase-I and -II enzymes some of which may occur through CAR-dependent mechanisms. © 2008 Wiley Periodicals, Inc.
• Merrell, M. D., Jackson, J. P., Augustine, L. M., Fisher, C. D., Slitt, A. L., Maher, J. M., Huang, W., Moore, D. D., Zhang, Y., Klaassen, C. D., & Cherrington, N. J. (2008). The Nrf2 activator oltipraz also activates the constitutive androstane receptor. Drug Metabolism and Disposition, 36(8), 1716-1721.
  More info

  PMID: 18474683;PMCID: PMC3693743;Abstract: Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2-related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2-/- mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR-/- mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics.
• Aleksunes, L. M., Augustine, L. M., Cherrington, N. J., & Manautou, J. E. (2007). Influence of acetaminophen vehicle on regulation of transporter gene expression during hepatotoxicity. Journal of Toxicology and Environmental Health - Part A: Current Issues, 70(21), 1870-1872.
  More info

  PMID: 17934960;Abstract: Researchers who study acetaminophen (APAP) hepatotoxicity use either a 50% propylene glycol solution or saline as a diluent. Previous studies demonstrated differential expression of hepatobiliary transporter mRNA in mice treated with a toxic dose of APAP dissolved in 50% propylene glycol. The purpose of this study was to determine whether using saline as a diluent for APAP alters regulation of transporter gene expression during hepatotoxicity. Male C57BL/6J mice received acetaminophen (APAP 400 mg/kg, ip in saline) or saline (20 ml/kg). Plasma and liver samples were collected at 24 and 48 h for assessment of alanine aminotransferase (ALT) activity and gene expression. It was determined that plasma ALT activity was elevated at 24 and 48 h after APAP administration. Using the branched DNA signal amplification assay, reductions in organic anion-transporting polypeptides Oatp1a1, Oatp1b2, sodium/taurocholate- cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep) mRNA were observed in APAP-treated mice. In contrast, multidrug resistance-associated proteins Mrp1, Mrp2, Mrp3, and Mrp4, as well as multidrug resistance proteins Mdr1a and Mdr1b genes, were increased following APAP. No changes in Oatp1a4, Mdr2, or breast cancer resistance protein (Bcrp) mRNA were observed. Alterations in transporter gene expression in this study were similar to those reported previously using propylene glycol as diluent. With the exceptions of Oatp1a1, Ntcp, and Mrp1, these data mirror previous results suggesting that the solution used to dissolve APAP may alter the susceptibility of mice to hepatotoxicity, but only minimally change the regulation of transporter gene expression. Copyright © Taylor & Francis Group, LLC.
• Aleksunes, L. M., Augustine, L. M., Cherrington, N. J., & Manautou, J. E. (2007). Influence of acetaminophen vehicle on regulation of transporter gene expression during hepatotoxicity. Journal of toxicology and environmental health. Part A, 70(21), 1870-2.
  More info

  Researchers who study acetaminophen (APAP) hepatotoxicity use either a 50% propylene glycol solution or saline as a diluent. Previous studies demonstrated differential expression of hepatobiliary transporter mRNA in mice treated with a toxic dose of APAP dissolved in 50% propylene glycol. The purpose of this study was to determine whether using saline as a diluent for APAP alters regulation of transporter gene expression during hepatotoxicity. Male C57BL/6J mice received acetaminophen (APAP 400 mg/kg, i.p. in saline) or saline (20 ml/kg). Plasma and liver samples were collected at 24 and 48 h for assessment of alanine aminotransferase (ALT) activity and gene expression. It was determined that plasma ALT activity was elevated at 24 and 48 h after APAP administration. Using the branched DNA signal amplification assay, reductions in organic anion-transporting polypeptides Oatp1a1, Oatp1b2, sodium/taurocholate-cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep) mRNA were observed in APAP-treated mice. In contrast, multidrug resistance-associated proteins Mrp1, Mrp2, Mrp3, and Mrp4, as well as multidrug resistance proteins Mdr1a and Mdr1b genes, were increased following APAP. No changes in Oatp1a4, Mdr2, or breast cancer resistance protein (Bcrp) mRNA were observed. Alterations in transporter gene expression in this study were similar to those reported previously using propylene glycol as diluent. With the exceptions of Oatp1a1, Ntcp, and Mrp1, these data mirror previous results suggesting that the solution used to dissolve APAP may alter the susceptibility of mice to hepatotoxicity, but only minimally change the regulation of transporter gene expression.
• Barnes, S. N., Aleksunes, L. M., Augustine, L., Scheffer, G. L., Goedken, M. J., Jakowski, A. B., Pruimboom-Brees, I. M., Cherrington, N. J., & Manautou, J. E. (2007). Induction of hepatobiliary efflux transporters in acetaminophen-induced acute liver failure cases. Drug Metabolism and Disposition, 35(10), 1963-1969.
  More info

  PMID: 17627974;Abstract: Alterations in transporter expression may represent a compensatory mechanism of damaged hepatocytes to reduce accumulation of potentially toxic compounds. The present study was conducted to investigate the expression of hepatobiliary efflux transporters in livers from patients after toxic acetaminophen (APAP) ingestion, with livers from patients with primary biliary cirrhosis (PBC) serving as positive controls. mRNA and protein expression of multidrug resistance-associated protein (MRP) 1-6, multidrug resistance protein (MDR) 1-3/P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) in normal (n = 6), APAP overdose (n = 5), and PBC (n = 6) human liver samples were determined by branched DNA and Western blot analysis, respectively. Double immunohistochemical staining of P-gp and proliferating cell nuclear antigen (PCNA), a marker of proliferation, was performed on paraffin-embedded tissue sections. Compared with normal liver specimens, MRP1 and MRP4 mRNA levels were elevated after APAP overdose and in PBC. Up-regulation of MRP5, MDR1, and BCRP mRNA occurred in PBC livers. Protein levels of MRP4, MRP5, BCRP, and P-gp were increased in both disease states, with MRP1 and MRP3 protein also being induced in PBC. Increased P-gp protein was confirmed immunohistochemically and was found to localize to areas of PCNA-positive hepatocytes, which were detected in APAP overdose and PBC livers. The findings from this study demonstrate that hepatic efflux transporter expression is up-regulated in cases of APAP-induced liver failure and PBC. This adaptation may aid in reducing retention of byproducts of cellular injury and bile constituents within hepatocytes. The close proximity of P-gp and PCNA-positive hepatocytes during liver injury suggests that along with cell regeneration, increased efflux transporter expression is a critical response to hepatic damage to protect the liver from additional insult. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.
• Barnes, S. N., Aleksunes, L. M., Augustine, L., Scheffer, G. L., Goedken, M. J., Jakowski, A. B., Pruimboom-Brees, I. M., Cherrington, N. J., & Manautou, J. E. (2007). Induction of hepatobiliary efflux transporters in acetaminophen-induced acute liver failure cases. Drug metabolism and disposition: the biological fate of chemicals, 35(10), 1963-9.
  More info

  Alterations in transporter expression may represent a compensatory mechanism of damaged hepatocytes to reduce accumulation of potentially toxic compounds. The present study was conducted to investigate the expression of hepatobiliary efflux transporters in livers from patients after toxic acetaminophen (APAP) ingestion, with livers from patients with primary biliary cirrhosis (PBC) serving as positive controls. mRNA and protein expression of multidrug resistance-associated protein (MRP) 1-6, multidrug resistance protein (MDR) 1-3/P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) in normal (n = 6), APAP overdose (n = 5), and PBC (n = 6) human liver samples were determined by branched DNA and Western blot analysis, respectively. Double immunohistochemical staining of P-gp and proliferating cell nuclear antigen (PCNA), a marker of proliferation, was performed on paraffin-embedded tissue sections. Compared with normal liver specimens, MRP1 and MRP4 mRNA levels were elevated after APAP overdose and in PBC. Up-regulation of MRP5, MDR1, and BCRP mRNA occurred in PBC livers. Protein levels of MRP4, MRP5, BCRP, and P-gp were increased in both disease states, with MRP1 and MRP3 protein also being induced in PBC. Increased P-gp protein was confirmed immunohistochemically and was found to localize to areas of PCNA-positive hepatocytes, which were detected in APAP overdose and PBC livers. The findings from this study demonstrate that hepatic efflux transporter expression is up-regulated in cases of APAP-induced liver failure and PBC. This adaptation may aid in reducing retention of byproducts of cellular injury and bile constituents within hepatocytes. The close proximity of P-gp and PCNA-positive hepatocytes during liver injury suggests that along with cell regeneration, increased efflux transporter expression is a critical response to hepatic damage to protect the liver from additional insult.
• Cherrington, N., Fisher, C. D., Augustine, L. M., Maher, J. M., Nelson, D. M., Slitt, A. L., Klaassen, C. D., Lehman-McKeeman, L. D., & Cherrington, N. J. (2007). Induction of drug-metabolizing enzymes by garlic and allyl sulfide compounds via activation of constitutive androstane receptor and nuclear factor E2-related factor 2. Drug metabolism and disposition: the biological fate of chemicals, 35(6).
  More info

  Garlic oil (GO) contains several linear sulfur compounds, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), that induce drug-metabolizing enzymes such as CYP2B and NAD(P)H quinone oxidoreductase 1 (NQO1). CYP2B and NQO1 are primarily regulated by constitutive androstane receptor (CAR) and nuclear factor E2-related factor 2 (Nrf2) transcription factors, respectively. The purpose of this study was to determine whether GO and its specific constituents induce these two enzymes via CAR and Nrf2 activation. Female Wistar-Kyoto (WKY) rats express little CAR protein and exhibit less induction of CYP2B1/2 than males. GO, DAS, and DADS, but not DATS, induced CYP2B1/2 mRNA levels to a greater extent in WKY males than in females, suggesting CAR activation. Conversely, DAS induced NQO1 levels equally in WKY males and females, indicating CAR-independent induction in rats. DAS, but not GO, DADS, or DATS, induced CYP2B10 mRNA levels 530-fold in wild-type (WT) mice, whereas this induction was attenuated in CAR(-/-) mice. DAS induced NQO1 in WT and CAR(-/-) mice equally, suggesting CAR-independent induction in mice. DAS induced NQO1 5-fold in WT mice, whereas induction was completely absent in Nrf2(-/-) mice, indicating DAS also activates Nrf2. DAS induction of CYP2B10 mRNA was independent of Nrf2 presence or absence. In in vivo transcription assays, DAS activated the human CYP2B6 promoter, and the antioxidant response element of the human NQO1 promoter, respectively. These studies indicate that GO constituents, particularly DAS, activate CAR and Nrf2 to induce drug-metabolizing enzymes.
• Cherrington, N., Lickteig, A. J., Fisher, C. D., Augustine, L. M., & Cherrington, N. J. (2007). Genes of the antioxidant response undergo upregulation in a rodent model of nonalcoholic steatohepatitis. Journal of biochemical and molecular toxicology, 21(4).
  More info

  Nonalcoholic fatty liver disease encompasses a spectrum of hepatic pathologies ranging from simple fatty liver to an inflammatory state known as nonalcoholic steatohepatitis (NASH). NASH is also characterized by severe hepatic oxidative stress. The goal of this study was to determine whether genes of the antioxidant response are induced in rodent models of nonalcoholic fatty liver disease. To simulate simple fatty liver and NASH, respectively, male Sprague-Dawley rats were fed a high-fat (HF) or a methionine and choline-deficient (MCD) diet for 8 weeks. Key marker genes of the antioxidant response that are known to undergo upregulation via activation of Nuclear Factor Erythroid 2-Related Factor 2 were measured using the branched DNA signal amplification assay. Messenger RNA levels of the antioxidant response, including NAD(P)H:quinone oxidoreductase-1 (Nqo1), Glutamate cysteine ligase catalytic (Gclc), and Heme oxygenase-1 (Ho-1), were significantly induced in MCD rat liver but not in HF rat liver. Furthermore, Nqo1 protein expression and activity underwent significant upregulation in MCD rat liver but not in HF rat liver. These data strongly indicate that the pathology induced by the MCD dietary model of NASH results in upregulation of the antioxidant response in rats.
• Cherrington, N., Lickteig, A. J., Fisher, C. D., Augustine, L. M., Aleksunes, L. M., Besselsen, D. G., Slitt, A. L., Manautou, J. E., & Cherrington, N. J. (2007). Efflux transporter expression and acetaminophen metabolite excretion are altered in rodent models of nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 35(10).
  More info

  Efflux transporters are responsible for the excretion of numerous xenobiotics and endobiotics and thus play an essential role in proper liver and kidney function. Nonalcoholic fatty liver diseases (NAFLDs) comprise a spectrum of disorders that range from simple fatty liver (SFL) to nonalcoholic steatohepatitis (NASH). Although the precise events leading to NAFLD are unclear, even less is known about the effects on efflux transporter expression and drug disposition. The purpose of this study was to determine the effect of NAFLD on efflux transporter expression in rat liver as well as on acetaminophen (APAP) metabolite excretion. To simulate SFL and NASH, rats were fed either a high-fat (HF) or a methionine- and choline-deficient (MCD) diet for 8 weeks. In the livers of MCD rats, there were striking increases in both mRNA and protein levels of multidrug resistance-associated protein (Mrp) 3, Mrp4, and breast cancer resistance protein, as well as increased Mrp2 protein. After administration of a nontoxic dose of APAP, biliary concentrations of APAP-sulfate, APAP-glucuronide (APAP-GLUC), and APAP-glutathione were reduced in MCD rats. The effects of the HF diet on both transporter expression and APAP disposition were by comparison far less dramatic than the MCD diet-induced alterations. Whereas APAP-sulfate levels were also decreased in MCD rat plasma, the levels of the Mrp3 substrate APAP-GLUC were elevated. Urinary elimination of APAP metabolites was identical between groups, except for APAP-GLUC, the concentration of which was 80% higher in MCD rats. These studies correlate increased hepatic Mrp3 protein in the MCD model of NASH with increased urinary elimination of APAP-GLUC. Furthermore, the proportional shift in elimination of APAP metabolites from bile to urine indicates that MCD-induced alterations in efflux transporter expression can affect the route of drug elimination.
• Cherrington, N., Lickteig, A. J., Slitt, A. L., Arkan, M. C., Karin, M., & Cherrington, N. J. (2007). Differential regulation of hepatic transporters in the absence of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and nuclear factor-kappaB in two models of cholestasis. Drug metabolism and disposition: the biological fate of chemicals, 35(3).
  More info

  Hepatic transporters are responsible for uptake and efflux of bile acids and xenobiotics as an essential aspect of liver function. When normal vectorial transport of bile acids by the apical uptake and canalicular excretion transporters is disrupted, cholestasis ensues, leading to accumulation of toxic bile constituents and considerable hepatocellular damage. The purpose of this study was to assess the role of cytokines and nuclear factor-kappaB (NF-kappaB) in the transcriptional regulation of transporters in two models of cholestasis, lipopolysaccharide (LPS) administration and bile duct ligation (BDL). In wild-type (WT) and knockout mouse strains lacking tumor necrosis factor (TNF) receptor-1, interleukin (IL)-1 receptor I, IL-6, or inhibitor of kappaB(IkappaB) kinase beta, transporter mRNA levels in liver were determined using branched DNA signal amplification 16 h after LPS administration or 3 days after BDL. In WT mice, LPS administration tended to decrease mRNA levels of organic anion-transporting polypeptide (Oatp) 2, Na(+)-taurocholate cotransporting polypeptide (Ntcp), Oatp1, Oatp4, bile salt excretory protein (Bsep), multidrug resistance-associated protein (Mrp) 2, and Mrp6 compared with saline treatment, whereas it increased Mrp1, 3, and 5 levels. Similar changes were observed in each knockout strain after LPS administration. Conversely, BDL decreased only Oatp1 expression in WT mice, meanwhile increasing expression of Mrp1, 3, and 5 and Oatp2 expression in both WT and knockout strains. Because the transcriptional effects of BDL- and LPS-induced cholestasis reflect dissimilarity in hepatic transporter regulation, we conclude that these disparities are not due to the individual activity of TNF-alpha, IL-1, IL-6, or NF-kappaB but to the differences in the mechanism of cholestasis.
• Fisher, C. D., Augustine, L. M., Maher, J. M., Nelson, D. M., Slitt, A. L., Klaassen, C. D., Lehman-McKeeman, L. D., & Cherrington, N. J. (2007). Induction of drug-metabolizing enzymes by garlic and allyl sulfide compounds via activation of constitutive androstane receptor and nuclear factor E2-related factor 2. Drug Metabolism and Disposition, 35(6), 995-1000.
  More info

  PMID: 17353348;Abstract: Garlic oil (GO) contains several linear sulfur compounds, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), that induce drug-metabolizing enzymes such as CYP2B and NAD(P)H quinone oxidoreductase 1 (NQO1). CYP2B and NQO1 are primarily regulated by constitutive androstane receptor (CAR) and nuclear factor E2-related factor 2 (Nrf2) transcription factors, respectively. The purpose of this study was to determine whether GO and its specific constituents induce these two enzymes via CAR and Nrf2 activation. Female Wistar-Kyoto (WKY) rats express little CAR protein and exhibit less induction of CYP2B1/2 than males. GO, DAS, and DADS, but not DATS, induced CYP2B1/2 mRNA levels to a greater extent in WKY males than in females, suggesting CAR activation. Conversely, DAS induced NQO1 levels equally in WKY males and females, indicating CAR-independent induction in rats. DAS, but not GO, DADS, or DATS, induced CYP2B10 mRNA levels 530-fold in wild-type (WT) mice, whereas this induction was attenuated in CAR-/- mice. DAS induced NQO1 in WT and CAR-/- mice equally, suggesting CAR-independent induction in mice. DAS induced NQO1 5-fold in WT mice, whereas induction was completely absent in Nrf2-/- mice, indicating DAS also activates Nrf2. DAS induction of CYP2B10 mRNA was independent of Nrf2 presence or absence. In in vivo transcription assays, DAS activated the human CYP2B6 promoter, and the antioxidant response element of the human NQO1 promoter, respectively. These studies indicate that GO constituents, particularly DAS, activate CAR and Nrf2 to induce drug-metabolizing enzymes. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.
• Lickteig, A. J., Fisher, C. D., Augustine, L. M., & Cherrington, N. J. (2007). Genes of the antioxidant response undergo upregulation in a rodent model of nonalcoholic steatohepatitis. Journal of Biochemical and Molecular Toxicology, 21(4), 216-220.
  More info

  PMID: 17721935;Abstract: Nonalcoholic fatty liver disease encompasses a spectrum of hepatic pathologies ranging from simple fatty liver to an inflammatory state known as nonalcoholic steatohepatitis (NASH). NASH is also characterized by severe hepatic oxidative stress. The goal of this study was to determine whether genes of the antioxidant response are induced in rodent models of nonalcoholic fatty liver disease. To simulate simple fatty liver and NASH, respectively, male Sprague-Dawley rats were fed a high-fat (HF) or a methionine and choline-deficient (MCD) diet for 8 weeks. Key marker genes of the antioxidant response that are known to undergo upregulation via activation of Nuclear Factor Erythroid 2-Related Factor 2 were measured using the branched DNA signal amplification assay. Messenger RNA levels of the antioxidant response, including NAD(P)H:quinone oxidoreductase-1 (Nqo1), Glutamate cysteine ligase catalytic (Gclc), and Heme oxygenase-1 (Ho-1), were significantly induced in MCD rat liver but not in HF rat liver. Furthermore, Nqo1 protein expression and activity underwent significant upregulation in MCD rat liver but not in HF rat liver. These data strongly indicate that the pathology induced by the MCD dietary model of NASH results in upregulation of the antioxidant response in rats. © 2007 Wiley Periodicals, Inc.
• Lickteig, A. J., Fisher, C. D., Augustine, L. M., Aleksunes, L. M., Besselsen, D. G., Slitt, A. L., Manautou, J. E., & Cherrington, N. J. (2007). Efflux transporter expression and acetaminophen metabolite excretion are altered in rodent models of nonalcoholic fatty liver disease. Drug Metabolism and Disposition, 35(10), 1970-1978.
  More info

  PMID: 17640958;Abstract: Efflux transporters are responsible for the excretion of numerous xenobiotics and endobiotics and thus play an essential role in proper liver and kidney function. Nonalcoholic fatty liver diseases (NAFLDs) comprise a spectrum of disorders that range from simple fatty liver (SFL) to nonalcoholic steatohepatitis (NASH). Although the precise events leading to NAFLD are unclear, even less is known about the effects on efflux transporter expression and drug disposition. The purpose of this study was to determine the effect of NAFLD on efflux transporter expression in rat liver as well as on acetaminophen (APAP) metabolite excretion. To simulate SFL and NASH, rats were fed either a high-fat (HF) or a methionine- and choline-deficient (MCD) diet for 8 weeks. In the livers of MCD rats, there were striking increases in both mRNA and protein levels of multidrug resistance-associated protein (Mrp) 3, Mrp4, and breast cancer resistance protein, as well as increased Mrp2 protein. After administration of a nontoxic dose of APAP, biliary concentrations of APAP-sulfate, APAP-glucuronide (APAP-GLUC), and APAP-glutathione were reduced in MCD rats. The effects of the HF diet on both transporter expression and APAP disposition were by comparison far less dramatic than the MCD diet-induced alterations. Whereas APAP-sulfate levels were also decreased in MCD rat plasma, the levels of the Mrp3 substrate APAP-GLUC were elevated. Urinary elimination of APAP metabolites was identical between groups, except for APAP-GLUC, the concentration of which was 80% higher in MCD rats. These studies correlate increased hepatic Mrp3 protein in the MCD model of NASH with increased urinary elimination of APAP-GLUC. Furthermore, the proportional shift in elimination of APAP metabolites from bile to urine indicates that MCD-induced alterations in efflux transporter expression can affect the route of drug elimination. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.
• Lickteig, A. J., Slitt, A. L., Arkan, M. C., Karin, M., & Cherrington, N. J. (2007). Differential regulation of hepatic transporters in the absence of tumor necrosis factor-α, interleukin-1β, interleukin-6, and nuclear factor-κB in two models of cholestasis. Drug Metabolism and Disposition, 35(3), 402-409.
  More info

  PMID: 17151194;Abstract: Hepatic transporters are responsible for uptake and efflux of bile acids and xenobiotics as an essential aspect of liver function. When normal vectorial transport of bile acids by the apical uptake and canalicular excretion transporters is disrupted, cholestasis ensues, leading to accumulation of toxic bile constituents and considerable hepatocellular damage. The purpose of this study was to assess the role of cytokines and nuclear factor-κB (NF-κB) in the transcriptional regulation of transporters in two models of cholestasis, lipopolysaccharide (LPS) administration and bile duct ligation (BDL). In wild-type (WT) and knockout mouse strains lacking tumor necrosis factor (TNF) receptor-1, interleukin (IL)-1 receptor I, IL-6, or inhibitor of κB (IκB) kinase β, transporter mRNA levels in liver were determined using branched DNA signal amplification 16 h after LPS administration or 3 days after BDL. In WT mice, LPS administration tended to decrease mRNA levels of organic anion-transporting polypeptide (Oatp) 2, Na +-taurocholate cotransporting polypeptide (Ntcp), Oatp1, Oatp4, bile salt excretory protein (Bsep), multidrug resistance-associated protein (Mrp) 2, and Mrp6 compared with saline treatment, whereas it increased Mrp1, 3, and 5 levels. Similar changes were observed in each knockout strain after LPS administration. Conversely, BDL decreased only Oatp1 expression in WT mice, meanwhile increasing expression of Mrp1, 3, and 5 and Oatp2 expression in both WT and knockout strains. Because the transcriptional effects of BDL- and LPS-induced cholestasis reflect dissimilarity in hepatic transporter regulation, we conclude that these disparities are not due to the individual activity of TNF-α, IL-1, IL-6, or NF-κB but to the differences in the mechanism of cholestasis. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.
• Slitt, A. L., Allen, K., Morrone, J., Aleksunes, L. M., Chen, C., Maher, J. M., Manautou, J. E., Cherrington, N. J., & Klaassen, C. D. (2007). Regulation of transporter expression in mouse liver, kidney, and intestine during extrahepatic cholestasis. Biochimica et Biophysica Acta - Biomembranes, 1768(3), 637-647.
  More info

  PMID: 17141734;Abstract: It is hypothesized that during cholestasis, the liver, kidney, and intestine alter gene expression to prevent BA accumulation; enhance urinary excretion of BA; and decrease BA absorption, respectively. To test this hypothesis, mice were subjected to either sham or bile-duct ligation (BDL) surgery and liver, kidney, duodenum, ileum, and serum samples were collected at 1, 3, 7, and 14 days after surgery. Serum total BA concentrations were 1-5 μmol/l in sham-operated mice and were elevated at 1, 3, 7, and 14 days after BDL, respectively. BDL decreased liver Ntcp, Oatp1a1, 1a5, and 1b2 mRNA expression and increased Bsep, Oatp1a4, and Mrp1-5 mRNA levels. In kidney, BDL decreased Oatp1a1 and increased Mrp1-5 mRNA levels. In intestine, BDL increased Mrp3 and Ibat mRNA levels in ileum. BDL increased Mrp1, 3, 4, and 5 protein expression in mouse liver. These data indicate that the compensatory regulation of transporters in liver, kidney, and intestine is unable to fully compensate for the loss of hepatic BA excretion because serum BA concentration remained elevated after 14 days of BDL. Additionally, hepatic and renal Oatp and Mrp genes are regulated similarly during extrahepatic cholestasis, and may suggest that transporter expression is regulated not to remove bile constituents from the body, but instead to remove bile constituents from tissues. © 2006 Elsevier B.V. All rights reserved.
• Slitt, A. L., Allen, K., Morrone, J., Aleksunes, L. M., Chen, C., Maher, J. M., Manautou, J. E., Cherrington, N. J., & Klaassen, C. D. (2007). Regulation of transporter expression in mouse liver, kidney, and intestine during extrahepatic cholestasis. Biochimica et biophysica acta, 1768(3), 637-47.
  More info

  It is hypothesized that during cholestasis, the liver, kidney, and intestine alter gene expression to prevent BA accumulation; enhance urinary excretion of BA; and decrease BA absorption, respectively. To test this hypothesis, mice were subjected to either sham or bile-duct ligation (BDL) surgery and liver, kidney, duodenum, ileum, and serum samples were collected at 1, 3, 7, and 14 days after surgery. Serum total BA concentrations were 1-5 mumol/l in sham-operated mice and were elevated at 1, 3, 7, and 14 days after BDL, respectively. BDL decreased liver Ntcp, Oatp1a1, 1a5, and 1b2 mRNA expression and increased Bsep, Oatp1a4, and Mrp1-5 mRNA levels. In kidney, BDL decreased Oatp1a1 and increased Mrp1-5 mRNA levels. In intestine, BDL increased Mrp3 and Ibat mRNA levels in ileum. BDL increased Mrp1, 3, 4, and 5 protein expression in mouse liver. These data indicate that the compensatory regulation of transporters in liver, kidney, and intestine is unable to fully compensate for the loss of hepatic BA excretion because serum BA concentration remained elevated after 14 days of BDL. Additionally, hepatic and renal Oatp and Mrp genes are regulated similarly during extrahepatic cholestasis, and may suggest that transporter expression is regulated not to remove bile constituents from the body, but instead to remove bile constituents from tissues.
• Zhang, X., Cherrington, N. J., & Wright, S. H. (2007). Molecular identification and functional characterization of rabbit MATE1 and MATE2-K. American Journal of Physiology - Renal Physiology, 293(1), F360-F370.
  More info

  PMID: 17442726;Abstract: An electroneutral organic cation (OC)/proton exchanger in the apical membrane of proximal tubules mediates the final step of renal OC excretion. Two members of the multidrug and toxin extrusion family, MATE1 and MATE2-K, were recently identified in human and rodent kidney and proposed to be the molecular basis of renal OC/H+ exchange. To take advantage of the comparative value of the large database on the kinetic and selectivity characteristics of OC/H+ exchange that exists for rabbit kidney, we cloned rbMATE1 and rbMATE2-K. The rabbit homologs have 75% (MATE1) and 74% (MATE2-K) amino acid identity to their human counterparts (and 51% identity with each other). rbMATE1 and rbMATE2-K exhibited H+ gradient-dependent uptake and efflux of tetraethylammonium (TEA) when expressed in Chinese hamster ovary cells. Both transporters displayed similar affinities for selected compounds [IC 50 values within 2-fold for TEA, 1-methyl-4-phenylpyridinium, and quinidine] and very different affinities for others (IC50 values differing by 8- to 80-fold for choline and cimetidine, respectively). These results indicate that rbMATE1 and rbMATE2-K are multispecific OC/H+ exchangers with similar, but distinct, functional characteristics. Overall, the selectivity of MATE1 and MATE2-K correlated closely with that observed in rabbit renal brush-border membrane vesicles. Copyright © 2007 the American Physiological Society.
• Zhang, X., Cherrington, N. J., & Wright, S. H. (2007). Molecular identification and functional characterization of rabbit MATE1 and MATE2-K. American journal of physiology. Renal physiology, 293(1), F360-70.
  More info

  An electroneutral organic cation (OC)/proton exchanger in the apical membrane of proximal tubules mediates the final step of renal OC excretion. Two members of the multidrug and toxin extrusion family, MATE1 and MATE2-K, were recently identified in human and rodent kidney and proposed to be the molecular basis of renal OC/H(+) exchange. To take advantage of the comparative value of the large database on the kinetic and selectivity characteristics of OC/H(+) exchange that exists for rabbit kidney, we cloned rbMATE1 and rbMATE2-K. The rabbit homologs have 75% (MATE1) and 74% (MATE2-K) amino acid identity to their human counterparts (and 51% identity with each other). rbMATE1 and rbMATE2-K exhibited H(+) gradient-dependent uptake and efflux of tetraethylammonium (TEA) when expressed in Chinese hamster ovary cells. Both transporters displayed similar affinities for selected compounds [IC(50) values within 2-fold for TEA, 1-methyl-4-phenylpyridinium, and quinidine] and very different affinities for others (IC(50) values differing by 8- to 80-fold for choline and cimetidine, respectively). These results indicate that rbMATE1 and rbMATE2-K are multispecific OC/H(+) exchangers with similar, but distinct, functional characteristics. Overall, the selectivity of MATE1 and MATE2-K correlated closely with that observed in rabbit renal brush-border membrane vesicles.
• Aleksunes, L. M., Slitt, A. L., Maher, J. M., Dieter, M. Z., Knight, T. R., Goedken, M., Cherrington, N. J., Chan, J. Y., Klaassen, C. D., & Manautou, J. E. (2006). Nuclear factor-E2-related factor 2 expression in liver is critical for induction of NAD(P)H:quinone oxidoreductase 1 during cholestasis. Cell Stress and Chaperones, 11(4), 356-363.
  More info

  PMID: 17278884;PMCID: PMC1759988;Abstract: Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqo1 mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism. © Cell Stress Society International 2006.
• Aleksunes, L. M., Slitt, A. L., Maher, J. M., Dieter, M. Z., Knight, T. R., Goedken, M., Cherrington, N. J., Chan, J. Y., Klaassen, C. D., & Manautou, J. E. (2006). Nuclear factor-E2-related factor 2 expression in liver is critical for induction of NAD(P)H:quinone oxidoreductase 1 during cholestasis. Cell stress & chaperones, 11(4), 356-63.
  More info

  Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqol mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism.
• Das, P. C., Cao, Y., Cherrington, N., Hodgson, E., & Rose, R. L. (2006). Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes. Chemico-Biological Interactions, 164(3), 200-214.
  More info

  PMID: 17084830;Abstract: Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 μM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to ∼55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 μM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6β-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 μM fipronil followed by dramatically declining activity measurements at 10 and 25 μM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 μM fipronil followed by decreasing activities at 25 and 50 μM. For fipronil sulfone, cytotoxic effects increased throughout the dose range. The trypan blue assay indicated that cytotoxic effects contributing to an increase of greater than 10% of control values was indicated at doses above 12.5 μM. However, fipronil sulfone induced cytotoxic effects at lower doses. The possibility that cytotoxic effects were due to apoptosis was indicated by significant time- and dose-dependent induction of caspase-3/7 activity in both HepG2 cells and human hepatocytes. Fipronil mediated activation of caspase-3/7 in concurrence with compromised ATP production and viability are attributed to apoptotic cell death. © 2006 Elsevier Ireland Ltd. All rights reserved.
• Das, P. C., Cao, Y., Cherrington, N., Hodgson, E., & Rose, R. L. (2006). Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes. Chemico-biological interactions, 164(3), 200-14.
  More info

  Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For fipronil sulfone, cytotoxic effects increased throughout the dose range. The trypan blue assay indicated that cytotoxic effects contributing to an increase of greater than 10% of control values was indicated at doses above 12.5 microM. However, fipronil sulfone induced cytotoxic effects at lower doses. The possibility that cytotoxic effects were due to apoptosis was indicated by significant time- and dose-dependent induction of caspase-3/7 activity in both HepG2 cells and human hepatocytes. Fipronil mediated activation of caspase-3/7 in concurrence with compromised ATP production and viability are attributed to apoptotic cell death.
• Groves, C. E., Suhre, W. B., Cherrington, N. J., & Wright, S. H. (2006). Sex differences in the mRNA, protein, and functional expression of organic anion transporter (Oat) 1, Oat3, and organic cation transporter (Oct) 2 in rabbit renal proximal tubules. Journal of Pharmacology and Experimental Therapeutics, 316(2), 743-752.
  More info

  PMID: 16249371;Abstract: Sex differences in transport of the organic anion (OA) substrate p-aminohippurate (PAH) and the organic cation (OC) substrate tetraethylammonium (TEA) have been recognized for sometime. In the rat kidney, androgens up-regulate and estrogens down-regulate PAH and TEA transport, which correlate with similar changes in mRNA and protein expression for the renal basolateral membrane transporters organic anion transporter (Oat) 1 and organic cation transporter (Oct) 2. However, these sex differences are not readily demonstrated in other species. The present study characterizes the kinetics of basolateral membrane PAH, estrone sulfate (ES), and TEA uptake in renal proximal tubule (RPT) suspensions isolated from female and male rabbits to compare functional expression of transport with mRNA and protein expression for rbOat1, rbOat3, and rbOct2. Although rbOat1-rbOat3 mRNA expression exhibited developmental differences, no sex differences in mRNA levels were observed. Oat1 and Oat3 protein expression in RPT suspensions also was similar between adult female and male rabbits. In contrast, rbOct1 and rbOct2 mRNA levels did not show developmental differences, but rbOct2 mRNA expression was greater in adult male than female rabbits. However, the sex difference in rbOct2 mRNA level did not translate to rbOct2 protein expression. Importantly, functional expression of Oat1, Oat3, and Oct2 transport as measured by kinetics (Jmax and Kt) of PAH, ES, and TEA uptake was similar between adult male and female rabbits, and correlated with rbOat1, rbOat3, and rbOct2 protein expression. Thus, unlike rodents, rabbit renal OA and OC transport does not exhibit sex differences, pointing to the need for caution in extrapolating transport-related sex differences between species. Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics.
• Groves, C. E., Suhre, W. B., Cherrington, N. J., & Wright, S. H. (2006). Sex differences in the mRNA, protein, and functional expression of organic anion transporter (Oat) 1, Oat3, and organic cation transporter (Oct) 2 in rabbit renal proximal tubules. The Journal of pharmacology and experimental therapeutics, 316(2), 743-52.
  More info

  Sex differences in transport of the organic anion (OA) substrate p-aminohippurate (PAH) and the organic cation (OC) substrate tetraethylammonium (TEA) have been recognized for some time. In the rat kidney, androgens up-regulate and estrogens down-regulate PAH and TEA transport, which correlate with similar changes in mRNA and protein expression for the renal basolateral membrane transporters organic anion transporter (Oat) 1 and organic cation transporter (Oct) 2. However, these sex differences are not readily demonstrated in other species. The present study characterizes the kinetics of basolateral membrane PAH, estrone sulfate (ES), and TEA uptake in renal proximal tubule (RPT) suspensions isolated from female and male rabbits to compare functional expression of transport with mRNA and protein expression for rbOat1, rbOat3, and rbOct2. Although rbOat1-rbOat3 mRNA expression exhibited developmental differences, no sex differences in mRNA levels were observed. Oat1 and Oat3 protein expression in RPT suspensions also was similar between adult female and male rabbits. In contrast, rbOct1 and rbOct2 mRNA levels did not show developmental differences, but rbOct2 mRNA expression was greater in adult male than female rabbits. However, the sex difference in rbOct2 mRNA level did not translate to rbOct2 protein expression. Importantly, functional expression of Oat1, Oat3, and Oct2 transport as measured by kinetics (J(max) and K(t)) of PAH, ES, and TEA uptake was similar between adult male and female rabbits, and correlated with rbOat1, rbOat3, and rbOct2 protein expression. Thus, unlike rodents, rabbit renal OA and OC transport does not exhibit sex differences, pointing to the need for caution in extrapolating transport-related sex differences between species.
• Maher, J. M., Cherrington, N. J., Slitt, A. L., & Klaassen, C. D. (2006). Tissue distribution and induction of the rat multidrug resistance- associated proteins 5 and 6. Life Sciences, 78(19), 2219-2225.
  More info

  PMID: 16260000;Abstract: Multidrug resistance-associated proteins (Mrps) are ATP-dependent transporters which transport a wide variety of anionic and cationic compounds. The purpose of this study was to determine the tissue distribution of Mrp5 and 6 in male and female Sprague-Dawley rats in various tissues, and to investigate whether the expression is altered by cholestasis or administration of microsomal enzyme inducers (MEIs). These MEIs activate six different transcriptionally- mediated pathways, and their effects on Mrp5 and Mrp6 expression were determined. The effects of bile-duct ligation, a cholestasis model, on Mrp5 and 6 expression in male rats were quantified. Mrp5 had marked expression in adrenal gland, and moderate expression in cerebral cortex, cerebellum, and stomach. The MEIs polychlorinated biphenyl (PCB)126, phenobarbital, and PCB99 slightly repressed Mrp5, but no single class of receptor agonists induced or repressed Mrp5. Bile-duct ligation tended to increase Mrp5 expression, but was not statistically significant at a 3 day timepoint. Mrp6 expression was highest in intestine, liver, and kidney. Mrp6 was slightly repressed by phenobarbital, dexamethasone, and isoniazid, but no one class of receptor agonists induced or repressed Mrp6, and expression was also unchanged bile-duct ligation. In conclusion, Mrp5 in rats is most highly expressed in the adrenal gland, whereas Mrp6 is mainly expressed in excretory organs (liver, intestine, and kidney), suggesting markedly different functions. Hepatic mRNA levels of Mrp5 or Mrp6 do not seem to be coordinately regulated along with Phase I enzymes via receptor-mediated pathways, and are not part of the hepatoprotective upregulation of basolateral transporters that occurs during cholestasis. © 2005 Elsevier Inc. All rights reserved.
• Maher, J. M., Cherrington, N. J., Slitt, A. L., & Klaassen, C. D. (2006). Tissue distribution and induction of the rat multidrug resistance-associated proteins 5 and 6. Life sciences, 78(19), 2219-25.
  More info

  Multidrug resistance-associated proteins (Mrps) are ATP-dependent transporters which transport a wide variety of anionic and cationic compounds. The purpose of this study was to determine the tissue distribution of Mrp5 and 6 in male and female Sprague-Dawley rats in various tissues, and to investigate whether the expression is altered by cholestasis or administration of microsomal enzyme inducers (MEIs). These MEIs activate six different transcriptionally-mediated pathways, and their effects on Mrp5 and Mrp6 expression were determined. The effects of bile-duct ligation, a cholestasis model, on Mrp5 and 6 expression in male rats were quantified. Mrp5 had marked expression in adrenal gland, and moderate expression in cerebral cortex, cerebellum, and stomach. The MEIs polychlorinated biphenyl (PCB)126, phenobarbital, and PCB99 slightly repressed Mrp5, but no single class of receptor agonists induced or repressed Mrp5. Bile-duct ligation tended to increase Mrp5 expression, but was not statistically significant at a 3 day timepoint. Mrp6 expression was highest in intestine, liver, and kidney. Mrp6 was slightly repressed by phenobarbital, dexamethasone, and isoniazid, but no one class of receptor agonists induced or repressed Mrp6, and expression was also unchanged bile-duct ligation. In conclusion, Mrp5 in rats is most highly expressed in the adrenal gland, whereas Mrp6 is mainly expressed in excretory organs (liver, intestine, and kidney), suggesting markedly different functions. Hepatic mRNA levels of Mrp5 or Mrp6 do not seem to be coordinately regulated along with Phase I enzymes via receptor-mediated pathways, and are not part of the hepatoprotective upregulation of basolateral transporters that occurs during cholestasis.
• Slitt, A. L., Cherrington, N. J., Dieter, M. Z., Aleksunes, L. M., Scheffer, G. L., Huang, W., Moore, D. D., & Klaassen, C. D. (2006). trans-Stilbene oxide induces expression of genes involved in metabolism and transport in mouse liver via CAR and Nrf2 transcription factors. Molecular Pharmacology, 69(5), 1554-1563.
  More info

  PMID: 16449384;Abstract: trans-Stilbene oxide (TSO) induces drug metabolizing enzymes in rat and mouse liver. TSO is considered a phenobarbital-like compound because it induces Cyp2B mRNA expression in liver. Phenobarbital increases Cyp2B expression in liver via activation of the constitutive androstane receptor (CAR). The purpose of this study was to determine whether TSO induces gene expression in mouse liver via CAR activation. TSO increased CAR nuclear localization in mouse liver, activated the human Cyp2B6 promoter in liver in vivo, and activated a reporter plasmid that contains five nuclear receptor 1 (NR1) binding sites in HepG2 cells. TSO administration increased expression of Cyp2b10, NAD(P)H:quinone oxidoreductase (Nqo1), epoxide hydrolase, heme oxygenase-1, UDP- glucuronosyltransferase (Ugt) 1a6 and 2b5, and multidrug resistance-associated proteins (Mrp) 2 and 3 mRNA in livers from male mice. Cyp2b10 and epoxide hydrolase induction by TSO was decreased in livers from CAR-null mice, compared with wild-type mice, suggesting CAR involvement. In contrast, TSO administration induced Nqo1 and Mrp3 mRNA expression equally in livers from wild-type and CAR-null mice, suggesting that TSO induces expression of some genes through a mechanism independent of CAR. TSO increased nuclear staining of the transcription factor Nrf2 in liver, and activated an antioxidant/electrophile response element luciferase reporter construct that was transfected into HepG2 cells. In summary, in mice, TSO increases Cyp2b10 and epoxide hydrolase expression in mice via CAR, and potentially induces Nqo1 and Mrp3 expression via Nrf2. Moreover, our data demonstrate that a single compound can activate both CAR and Nrf2 transcription factors in liver. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics.
• Slitt, A. L., Cherrington, N. J., Dieter, M. Z., Aleksunes, L. M., Scheffer, G. L., Huang, W., Moore, D. D., & Klaassen, C. D. (2006). trans-Stilbene oxide induces expression of genes involved in metabolism and transport in mouse liver via CAR and Nrf2 transcription factors. Molecular pharmacology, 69(5), 1554-63.
  More info

  trans-Stilbene oxide (TSO) induces drug metabolizing enzymes in rat and mouse liver. TSO is considered a phenobarbital-like compound because it induces Cyp2B mRNA expression in liver. Phenobarbital increases Cyp2B expression in liver via activation of the constitutive androstane receptor (CAR). The purpose of this study was to determine whether TSO induces gene expression in mouse liver via CAR activation. TSO increased CAR nuclear localization in mouse liver, activated the human Cyp2B6 promoter in liver in vivo, and activated a reporter plasmid that contains five nuclear receptor 1 (NR1) binding sites in HepG2 cells. TSO administration increased expression of Cyp2b10, NAD(P)H:quinone oxidoreductase (Nqo1), epoxide hydrolase, heme oxygenase-1, UDP-glucuronosyl-transferase (Ugt) 1a6 and 2b5, and multidrug resistance-associated proteins (Mrp) 2 and 3 mRNA in livers from male mice. Cyp2b10 and epoxide hydrolase induction by TSO was decreased in livers from CAR-null mice, compared with wild-type mice, suggesting CAR involvement. In contrast, TSO administration induced Nqo1 and Mrp3 mRNA expression equally in livers from wild-type and CAR-null mice, suggesting that TSO induces expression of some genes through a mechanism independent of CAR. TSO increased nuclear staining of the transcription factor Nrf2 in liver, and activated an antioxidant/electrophile response element luciferase reporter construct that was transfected into HepG2 cells. In summary, in mice, TSO increases Cyp2b10 and epoxide hydrolase expression in mice via CAR, and potentially induces Nqo1 and Mrp3 expression via Nrf2. Moreover, our data demonstrate that a single compound can activate both CAR and Nrf2 transcription factors in liver.
• Slitt, A. L., Cherrington, N. J., Fisher, C. D., Negishi, M., & Klaassen, C. D. (2006). Induction of genes for metabolism and transport by trans-stilbene oxide in livers of Sprague-Dawley and Wistar-Kyoto rats. Drug Metabolism and Disposition, 34(7), 1190-1197.
  More info

  PMID: 16621935;Abstract: trans-Stilbene oxide (TSO) is a synthetic proestrogen that induces phase I and II drug-metabolizing enzymes in rat liver. The purpose of this study was to determine whether TSO also induces transporter expression in rat liver and whether gene induction in rat liver after TSO occurs in a constitutive androstane receptor (CAR)-dependent manner. Total RNA was isolated from male rat livers after treatment with TSO for up to 4 days (200 mg/kg, i.p., twice daily), and the mRNA levels for each gene were quantified. CYP2B1/2, CYP3A1, epoxide hydrolase, heme oxygenase-1, UGT1A6, UGT2B1, multiple drug resistance protein (Mdr) 1a and 1b, as well as multidrug resistance-associated protein (Mrp) 2, 3, and 4 mRNA were increased in livers after TSO treatment. To determine whether TSO activates gene expression in a CAR-dependent manner, male and female Wistar-Kyoto (WKY) rats were treated with TSO for 3 days. TSO induced CYP2B1/2, UGT2B1, and Mdr1b in males more than in females, suggesting that TSO could increase their expression via CAR. Conversely, TSO induced CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 similarly in both genders, indicating that induction of these genes occurs independently of CAR. TSO treatment also increased the activity of a CAR binding element luciferase reporter construct in HepG2 cells transfected with rat CAR and in mouse liver. Additionally, TSO increased antioxidant response element/electrophile response element luciferase reporter construct activity in HepG2 cells. In conclusion, in WKY rat liver, TSO increases CYP2B1/2, UGT2B1, and Mdr1b mRNA expression in a gender-dependent manner and CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 in a gender-independent manner.
• Slitt, A. L., Cherrington, N. J., Fisher, C. D., Negishi, M., & Klaassen, C. D. (2006). Induction of genes for metabolism and transport by trans-stilbene oxide in livers of Sprague-Dawley and Wistar-Kyoto rats. Drug metabolism and disposition: the biological fate of chemicals, 34(7), 1190-7.
  More info

  trans-Stilbene oxide (TSO) is a synthetic proestrogen that induces phase I and II drug-metabolizing enzymes in rat liver. The purpose of this study was to determine whether TSO also induces transporter expression in rat liver and whether gene induction in rat liver after TSO occurs in a constitutive androstane receptor (CAR)-dependent manner. Total RNA was isolated from male rat livers after treatment with TSO for up to 4 days (200 mg/kg, i.p., twice daily), and the mRNA levels for each gene were quantified. CYP2B1/2, CYP3A1, epoxide hydrolase, heme oxygenase-1, UGT1A6, UGT2B1, multiple drug resistance protein (Mdr) 1a and 1b, as well as multidrug resistance-associated protein (Mrp) 2, 3, and 4 mRNA were increased in livers after TSO treatment. To determine whether TSO activates gene expression in a CAR-dependent manner, male and female Wistar-Kyoto (WKY) rats were treated with TSO for 3 days. TSO induced CYP2B1/2, UGT2B1, and Mdr1b in males more than in females, suggesting that TSO could increase their expression via CAR. Conversely, TSO induced CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 similarly in both genders, indicating that induction of these genes occurs independently of CAR. TSO treatment also increased the activity of a CAR binding element luciferase reporter construct in HepG2 cells transfected with rat CAR and in mouse liver. Additionally, TSO increased antioxidant response element/electrophile response element luciferase reporter construct activity in HepG2 cells. In conclusion, in WKY rat liver, TSO increases CYP2B1/2, UGT2B1, and Mdr1b mRNA expression in a gender-dependent manner and CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 in a gender-independent manner.
• Aleksunes, L. M., Sliit, A. M., Cherrington, N. J., Thibodeau, M. S., Klaassen, C. D., & Manautou, J. E. (2005). Differential expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride. Toxicological Sciences, 83(1), 44-52.
  More info

  PMID: 15496496;Abstract: Drug-metabolizing enzymes and membrane transporters are responsible for the detoxication and elimination of xenobiotics from the body. The goal of this study was to identify alterations in mRNA expression of various transport and detoxication proteins in mouse liver after administration of the hepatotoxicants, acetaminophen or carbon tetrachloride. Therefore, male C57BL/6 J mice received acetaminophen (APAP, 200, 300, or 400 mg/kg, ip) or carbon tetrachloride (CCl4, 10 or 25 μl/kg, ip). Plasma and liver samples were collected at 6, 24, and 48 h for assessment of alanine aminotransferase (ALT) activity, total RNA isolation, and histopathological analysis of injury. Heme oxygenase-1 (Ho-1), NAD(P)H quinone oxidoreductase-1 (Nqo1), organic anion-transporting polypeptides (Oatp1a1, 1a4 and 1b2),sodium/taurocholate-cotransporting polypeptide (Ntcp), and multidrug resistance-associated protein (Mrp 1-6) mRNA levels in liver were determined using the branched DNA signal amplification assay. Hepatotoxic doses of APAP and CCl4 increased Ho-1 and Nqo1 mRNA levels by 22- and 2.5-fold, respectively, and reduced Oatp1a1, 1a4, and Ntcp mRNA levels in liver. By contrast, expression of Mrps 1-4 was increased after treatment with APAP and CCl4. Notably, a marked elevation of Mrp4 mRNA expression was observed 24 h after APAP 400 mg/kg (5-fold) and CCl4 25 μl/kg (37-fold). Collectively, these expression patterns suggest a coordinated regulation of both transport and detoxification genes during liver injury. This reduction in expression of uptake transporters, as well as enhanced transcription of detoxication enzymes and export transporters may limit the accumulation of potentially toxic products in hepatocytes. © Society of Toxicology 2005; all rights reserved.
• Aleksunes, L. M., Slitt, A. M., Cherrington, N. J., Thibodeau, M. S., Klaassen, C. D., & Manautou, J. E. (2005). Differential expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride. Toxicological sciences : an official journal of the Society of Toxicology, 83(1), 44-52.
  More info

  Drug-metabolizing enzymes and membrane transporters are responsible for the detoxication and elimination of xenobiotics from the body. The goal of this study was to identify alterations in mRNA expression of various transport and detoxication proteins in mouse liver after administration of the hepatotoxicants, acetaminophen or carbon tetrachloride. Therefore, male C57BL/6 J mice received acetaminophen (APAP, 200, 300, or 400 mg/kg, ip) or carbon tetrachloride (CCl4, 10 or 25 microl/kg, ip). Plasma and liver samples were collected at 6, 24, and 48 h for assessment of alanine aminotransferase (ALT) activity, total RNA isolation, and histopathological analysis of injury. Heme oxygenase-1 (Ho-1), NAD(P)H quinone oxidoreductase-1 (Nqo1), organic anion-transporting polypeptides (Oatp1a1, 1a4 and 1b2), sodium/taurocholate-cotransporting polypeptide (Ntcp), and multidrug resistance-associated protein (Mrp 1-6) mRNA levels in liver were determined using the branched DNA signal amplification assay. Hepatotoxic doses of APAP and CCl4 increased Ho-1 and Nqo1 mRNA levels by 22- and 2.5-fold, respectively, and reduced Oatp1a1, 1a4, and Ntcp mRNA levels in liver. By contrast, expression of Mrps 1-4 was increased after treatment with APAP and CCl4. Notably, a marked elevation of Mrp4 mRNA expression was observed 24 h after APAP 400 mg/kg (5-fold) and CCl4 25 microl/kg (37-fold). Collectively, these expression patterns suggest a coordinated regulation of both transport and detoxification genes during liver injury. This reduction in expression of uptake transporters, as well as enhanced transcription of detoxication enzymes and export transporters may limit the accumulation of potentially toxic products in hepatocytes.
• Augustine, L. M., Markelewicz Jr., R. J., Boekelheide, K., & Cherrington, N. J. (2005). Xenobiotic and endobiotic transporter MRNA expression in the blood-testis barrier. Drug Metabolism and Disposition, 33(1), 182-189.
  More info

  PMID: 15494472;Abstract: A major function of xenobiotic and endobiotic transporters is to move a wide range of organic substances across cell membranes. Sertoli cells play an important role in protecting developing germ cells by forming a physiological barrier, limiting exposure to potentially toxic substrates, or conversely, facilitating uptake of xenobiotics within the testis. The aim of this study was to quantitatively determine the constitutive expression of various transporters in isolated Sertoli cells from adult Sprague-Dawley rats. The following mRNA levels were measured in isolated Sertoli cells by the branched DNA signal amplification method, multidrug resistance (Mdr) protein 1a, 1b, and 2; multiple drug resistance protein (Mrp) 1, 2, 3, 4, 5, 6, 7, and 8; sodium taurocholate cotransporting polypeptide; bile salt excretory protein; ileal bile acid transporter; AbcG5 and AbcG8; organic anion transporting polypeptide (Oatp) 1, 2, 3, 4, 5, 9, and 12; prostaglandin transporter (Pgt); testis-specific transporter (Tst) 1 and Tst2; organic anion transporter (Oat) 1, 2, 3, and K; organic cation transporter (Oct) 1, 2, 3, N1, and N2; divalent metal transporter (Dmt) 1, Menke's, and Wilson's; zinc transporter (Znt) 1; equilibrative nucleoside transporter (Ent) 1 and 2; concentrative nucleoside transporter (Cnt) 1 and 2; and peptide transporter (Pept) 1 and 2. Levels were also determined in whole testis, liver, kidney, and ileum to provide a reference for determining relative expression levels. Mrp8, Tst1 and 2, and Ent1 and 2 were expressed in Sertoli cells at higher levels than in liver, kidney, or ileum, whereas Mrp1, 5, and 7, Mdr2, Oatp3, Oat2, OctN2, Dmt1, Menke's, Wilson's, and Znt1 were all significantly expressed in Sertoli cells, but Sertoli cell expression was not the tissue of highest expression. The remaining transporters were expressed at low levels in isolated Sertoli cells. Additionally, expression levels of Mrp1, Mrp7, Mrp8, Tst1, Tst2, OctN2, Wilson's, Znt1, Ent1, and Ent2 were greater in isolated Sertoli cells than in whole testis. Constitutive expression of transporters in Sertoli cells may provide an insight into the range of xenobiotics that can potentially be transported by Sertoli cells and thereby provide a mechanistic understanding of blood-testis barrier function.
• Cherrington, N., Augustine, L. M., Markelewicz, R. J., Boekelheide, K., & Cherrington, N. J. (2005). Xenobiotic and endobiotic transporter mRNA expression in the blood-testis barrier. Drug metabolism and disposition: the biological fate of chemicals, 33(1).
  More info

  A major function of xenobiotic and endobiotic transporters is to move a wide range of organic substances across cell membranes. Sertoli cells play an important role in protecting developing germ cells by forming a physiological barrier, limiting exposure to potentially toxic substrates, or conversely, facilitating uptake of xenobiotics within the testis. The aim of this study was to quantitatively determine the constitutive expression of various transporters in isolated Sertoli cells from adult Sprague-Dawley rats. The following mRNA levels were measured in isolated Sertoli cells by the branched DNA signal amplification method, multidrug resistance (Mdr) protein 1a, 1b, and 2; multiple drug resistance protein (Mrp) 1, 2, 3, 4, 5, 6, 7, and 8; sodium taurocholate cotransporting polypeptide; bile salt excretory protein; ileal bile acid transporter; AbcG5 and AbcG8; organic anion transporting polypeptide (Oatp) 1, 2, 3, 4, 5, 9, and 12; prostaglandin transporter (Pgt); testis-specific transporter (Tst) 1 and Tst2; organic anion transporter (Oat) 1, 2, 3, and K; organic cation transporter (Oct) 1, 2, 3, N1, and N2; divalent metal transporter (Dmt) 1, Menke's, and Wilson's; zinc transporter (Znt) 1; equilibrative nucleoside transporter (Ent) 1 and 2; concentrative nucleoside transporter (Cnt) 1 and 2; and peptide transporter (Pept) 1 and 2. Levels were also determined in whole testis, liver, kidney, and ileum to provide a reference for determining relative expression levels. Mrp8, Tst1 and 2, and Ent1 and 2 were expressed in Sertoli cells at higher levels than in liver, kidney, or ileum, whereas Mrp1, 5, and 7, Mdr2, Oatp3, Oat2, OctN2, Dmt1, Menke's, Wilson's, and Znt1 were all significantly expressed in Sertoli cells, but Sertoli cell expression was not the tissue of highest expression. The remaining transporters were expressed at low levels in isolated Sertoli cells. Additionally, expression levels of Mrp1, Mrp7, Mrp8, Tst1, Tst2, OctN2, Wilson's, Znt1, Ent1, and Ent2 were greater in isolated Sertoli cells than in whole testis. Constitutive expression of transporters in Sertoli cells may provide an insight into the range of xenobiotics that can potentially be transported by Sertoli cells and thereby provide a mechanistic under standing of blood-testis barrier function.
• Cherrington, N., Erickson, R. P., Bhattacharyya, A., Hunter, R. J., Heidenreich, R. A., & Cherrington, N. J. (2005). Liver disease with altered bile acid transport in Niemann-Pick C mice on a high-fat, 1% cholesterol diet. American journal of physiology. Gastrointestinal and liver physiology, 289(2).
  More info

  Cholestatic hepatitis is frequently found in Niemann-Pick C (NPC) disease. We studied the influence of diet and the low density lipoprotein receptor (LDLR, Ldlr in mice, known to be the source of most of the stored cholesterol) on liver disease in the mouse model of NPC. Npc1-/- mice of both sexes, with or without the Ldlr knockout, were fed a 18% fat, 1% cholesterol ("high-fat") diet and were evaluated by chemical and histological methods. Bile acid transporters [multidrug resistance protein (Mrps) 1-5; Ntcp, Bsep, and OatP1, 2, and 4] were quantitated by real-time RT-PCR. Many mice died prematurely (within 6 wk) with hepatomegaly. Histopathology showed an increase in macrophage and hepatocyte lipids independent of Ldlr genotype. Non-zone-dependent diffuse fibrosis was found in the surviving mice. Serum alanine aminotransferase was elevated in Npc1-/- mice on the regular diet and frequently became markedly elevated with the high-fat diet. Serum cholesterol was increased in the controls but not the Npc1-/- mice on the high-fat diet; it was massively increased in the Ldlr-/- mice. Esterified cholesterol was greatly increased by the high-fat diet, independent of Ldlr genotype. gamma-Glutamyltransferase was also elevated in Npc1-/- mice, more so on the high-fat diet. Mrps 1-5 were elevated in Npc1-/- liver and became more elevated with the high-fat diet; Ntcp, Bsep, and OatP2 were elevated in Npc1-/- liver and were suppressed by the high-fat diet. In conclusion, Npc1-/- mice on a high-fat diet provide an animal model of NPC cholestatic hepatitis and indicate a role for altered bile acid transport in its pathogenesis.
• Erickson, R. P., Bhattacharyya, A., Hunter, R. J., Heidenreich, R. A., & Cherrington, N. J. (2005). Liver disease with altered bile acid transport in Niemann-Pick C mice on a high-fat, 1% cholesterol diet. American Journal of Physiology - Gastrointestinal and Liver Physiology, 289(2 52-2), G300-G307.
  More info

  PMID: 15790756;Abstract: Cholestatic hepatitis is frequently found in Niemann-Pick C (NPC) disease. We studied the influence of diet and the low density lipoprotein receptor (LDLR, Ldlr in mice, known to be the source of most of the stored cholesterol) on liver disease in the mouse model of NPC. Npc1-/- mice of both sexes, with or without the Ldlr knockout, were fed a 18% fat, 1% cholesterol ("high-fat") diet and were evaluated by chemical and histological methods. Bile acid transporters [multidrug resistance protein (Mrps) 1-5; Ntcp, Bsep, and OatP1, 2, and 4] were quantitated by real-time RT-PCR. Many mice died prematurely (within 6 wk) with hepatomegaly. Histopathology showed an increase in macrophage and hepatocyte lipids independent of Ldlr genotype. Non-zone-dependent diffuse fibrosis was found in the surviving mice. Serum alanine aminotransferase was elevated in Npc1-/- mice on the regular diet and frequently became markedly elevated with the high-fat diet. Serum cholesterol was increased in the controls but not the Npc1-/- mice on the high-fat diet; it was massively increased in the Ldlr-/- mice. Esterified cholesterol was greatly increased by the high-fat diet, independent of Ldlr genotype. γ-Glutamyltransferase was also elevated in Npc1 -/- mice, more so on the high-fat diet. Mrps 1-5 were elevated in Npc1-/- liver and became more elevated with the high-fat diet; Ntcp, Bsep, and OatP2 were elevated in Npc1-/- liver and were suppressed by the high-fat diet. In conclusion, Npc1-/- mice on a high-fat diet provide an animal model of NPC cholestatic hepatitis and indicate a role for altered bile acid transport in its pathogenesis. Copyright © 2005 the American Physiological Society.
• Maher, J. M., Slitt, A. L., Cherrington, N. J., Cheng, X., & Klaassen, C. D. (2005). Tissue distribution and hepatic and renal ontogeny of the multidrug resistance-associated protein (MRP) family in mice. Drug Metabolism and Disposition, 33(7), 947-955.
  More info

  PMID: 15802388;Abstract: Analysis of the mouse genome has revealed eight multidrug resistance-associated (Mrp) transporters, with mouse homologs for all human MRPs except MRP8. Whereas MRP expression in tissues of humans and rats has been examined, no characterization exists for mice. Furthermore, the ontogeny of mouse Mrps is unknown, and such knowledge may be helpful in understanding age-related pharmacokinetics. Therefore, the purpose of this study was to quantitatively determine 1) expression of the Mrp family in 12 different tissues, 2) gender variations in Mrp expression in liver and kidney, and 3) whether Mrp expression is altered during development. Highest expression of the Mrp family members is as follows: Mrp1 in testes, ovary, and placenta; Mrp2 in intestine, followed by liver and kidney; Mrp3 in large intestine; Mrp4 in kidney; Mrp5 in brain, followed by lung and stomach; Mrp6 in liver; Mrp7 in testes, intestine, and kidney; and Mrp9 solely in testes. Gender differences in Mrp expression were observed: Mrp1, 3, and 4 in kidney, as well as Mrp1 and 4 in liver were female-predominant. Ontogeny of the four Mrps expressed in liver was as follows: Mrp2 and Mrp4 were expressed at adult levels at birth; Mrp3 reached adult levels at day 30, and Mrp6 was not expressed until day 10. In kidney, Mrp1 and Mrp5 were expressed at adult levels at birth, whereas Mrp2, 3, 4, and 6 generally increased over time. In conclusion, marked differences in expression of the individual Mrp family members exist in various tissues, with age, and with gender. Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics.
• Maher, J. M., Slitt, A. L., Cherrington, N. J., Cheng, X., & Klaassen, C. D. (2005). Tissue distribution and hepatic and renal ontogeny of the multidrug resistance-associated protein (Mrp) family in mice. Drug metabolism and disposition: the biological fate of chemicals, 33(7), 947-55.
  More info

  Analysis of the mouse genome has revealed eight multidrug resistance-associated (Mrp) transporters, with mouse homologs for all human MRPs except MRP8. Whereas MRP expression in tissues of humans and rats has been examined, no characterization exists for mice. Furthermore, the ontogeny of mouse Mrps is unknown, and such knowledge may be helpful in understanding age-related pharmacokinetics. Therefore, the purpose of this study was to quantitatively determine 1) expression of the Mrp family in 12 different tissues, 2) gender variations in Mrp expression in liver and kidney, and 3) whether Mrp expression is altered during development. Highest expression of the Mrp family members is as follows: Mrp1 in testes, ovary, and placenta; Mrp2 in intestine, followed by liver and kidney; Mrp3 in large intestine; Mrp4 in kidney; Mrp5 in brain, followed by lung and stomach; Mrp6 in liver; Mrp7 in testes, intestine, and kidney; and Mrp9 solely in testes. Gender differences in Mrp expression were observed: Mrp1, 3, and 4 in kidney, as well as Mrp1 and 4 in liver were female-predominant. Ontogeny of the four Mrps expressed in liver was as follows: Mrp2 and Mrp4 were expressed at adult levels at birth; Mrp3 reached adult levels at day 30, and Mrp6 was not expressed until day 10. In kidney, Mrp1 and Mrp5 were expressed at adult levels at birth, whereas Mrp2, 3, 4, and 6 generally increased over time. In conclusion, marked differences in expression of the individual Mrp family members exist in various tissues, with age, and with gender.
• Rose, R. L., Tang, J., Choi, J., Cao, Y., Usmani, A., Cherrington, N., & Hodgson, E. (2005). Pesticide metabolism in humans, including polymorphisms. Scandinavian Journal of Work, Environment and Health, 31(SUPPL. 1), 156-163.
  More info

  PMID: 16190164;Abstract: Recent epidemiologic studies involving Gulf War veterans or agricultural workers suggest that pesticide-pesticide or pesticide-drug interactions may be related to Gulf-War-related illnesses or elevated cancer risks, respectively. Metabolic interactions are one of many potential mechanisms requiring exploration in humans. The goal of the studies is to characterize important metabolic profiles of selected pesticides and examine potential interactions to characterize human risks associated with exposure. Pesticides examined using human liver microsomes and cytosolic fractions included chlorpyrifos, carbaryl and permethrin. The metabolic pathways involved include cytochrome P450 monooxygenases (CYP), esterases, and alcohol and aldehyde dehydrogenases. Specific isoforms and some polymorphic enzymes were characterized. Pesticide-pesticide interactions with metabolizing enzymes were demonstrated. Exposure of human hepatocytes to chlorpyrifos and permethrin demonstrated their potential to induce CYP isoforms using the bDNA (branched deoxyribonucleic acid) assay [used to monitor mRNA (messenger ribonucleic acid) levels]. These studies suggest that knowledge of human metabolic pathways will provide information that can aid the risk assessment process.
• Rose, R. L., Tang, J., Choi, J., Cao, Y., Usmani, A., Cherrington, N., & Hodgson, E. (2005). Pesticide metabolism in humans, including polymorphisms. Scandinavian journal of work, environment & health, 31 Suppl 1, 156-63; discussion 119-22.
  More info

  Recent epidemiologic studies involving Gulf War veterans or agricultural workers suggest that pesticide-pesticide or pesticide-drug interactions may be related to Gulf-War-related illnesses or elevated cancer risks, respectively. Metabolic interactions are one of many potential mechanisms requiring exploration in humans. The goal of the studies is to characterize important metabolic profiles of selected pesticides and examine potential interactions to characterize human risks associated with exposure. Pesticides examined using human liver microsomes and cytosolic fractions included chlorpyrifos, carbaryl and permethrin. The metabolic pathways involved include cytochrome P450 monooxygenases (CYP), esterases, and alcohol and aldehyde dehydrogenases. Specific isoforms and some polymorphic enzymes were characterized. Pesticide-pesticide interactions with metabolizing enzymes were demonstrated. Exposure of human hepatocytes to chlorpyrifos and permethrin demonstrated their potential to induce CYP isoforms using the bDNA (branched deoxyribonucleic acid) assay [used to monitor mRNA (messenger ribonucleic acid) levels]. These studies suggest that knowledge of human metabolic pathways will provide information that can aid the risk assessment process.
• Cherrington, N. J., Slitt, A. L., Li, N., & Klaassen, C. D. (2004). Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats. Drug metabolism and disposition: the biological fate of chemicals, 32(7), 734-41.
  More info

  The function of hepatic transporters is to move organic substances across sinusoidal and canalicular membranes. During extrahepatic cholestasis, transporters involved in the movement of substances from blood to bile, such as sodium/taurocholate-cotransporting polypeptide (Ntcp) and multidrug resistance protein 2 (Mrp2), are down-regulated, whereas others that transport chemicals from liver to blood, such as Mrp3, are up-regulated. Unlike extrahepatic cholestasis, where transporter expression responds to the stress of accumulating bile constituents, lipopolysaccharide (LPS)-induced intrahepatic cholestasis may be directly caused by alterations in transporter expression. The aim of this study was to quantitatively determine the effect of LPS on transporter expression and study the mechanism(s) by which LPS alters mRNA levels of major hepatic transporters in Sprague-Dawley rats. Hepatic mRNA levels of Mrp2, Mrp6, multiple drug resistance protein 1a (Mdr1a), organic anion-transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, Ntcp, bile salt export pump, organic cation transporter 1 (Oct1), and organic anion transporter 3 (Oat3) were dramatically decreased, beginning approximately 6 h after LPS administration, whereas Mrp5 and Oat2 levels were unchanged. In contrast, LPS increased mRNA levels of Mrp1, Mrp3, and Mdr1b concurrently with the down-regulated transporters. Pretreatment with dexamethasone, which decreases the release of cytokines, reversed the reduction of Mdr1a, Oatp1, Oatp2, Oct1, and Ntcp mRNA following LPS administration. Furthermore, dexamethasone pretreatment also prevented the LPS-mediated increase in Mrp1, Mrp3, and Mdr1b, whereas pretreatment with aminoguanidine or gadolinium chloride, an inhibitor of inducible nitric oxide synthetase and a Kupffer cell toxicant, respectively, had no effect on the LPS-induced changes. The concurrent repression and induction of various transporters, as well as dexamethasone abatement of both LPS-mediated repression and induction, indicates that these responses may be mediated through similar pathways.
• Cherrington, N. J., Slitt, A. L., Ning, L. i., & Klaassen, C. D. (2004). Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats. Drug Metabolism and Disposition, 32(7), 734-741.
  More info

  PMID: 15205389;Abstract: The function of hepatic transporters is to move organic substances across sinusoidal and canalicular membranes. During extrahepatic cholestasis, transporters involved in the movement of substances from blood to bile, such as sodium/taurocholate-cotransporting polypeptide (Ntcp) and multidrug resistance protein 2 (Mrp2), are down-regulated, whereas others that transport chemicals from liver to blood, such as Mrp3, are up-regulated. Unlike extrahepatic cholestasis, where transporter expression responds to the stress of accumulating bile constituents, lipopolysaccharide (LPS)-induced intrahepatic cholestasis may be directly caused by alterations in transporter expression. The aim of this study was to quantitatively determine the effect of LPS on transporter expression and study the mechanism(s) by which LPS alters mRNA levels of major hepatic transporters in Sprague-Dawley rats. Hepatic mRMA levels of Mrp2, Mrp6, multiple drug resistance protein to (Mdr1a), organic anion-transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, Ntcp, bile salt export pump, organic cation transporter 1 (Oct1), and organic anion transporter 3 (Oat3) were dramatically decreased, beginning approximately 6 h after LPS administration, whereas Mrp5 and Oat2 levels were unchanged. In contrast, LPS increased mRNA levels of Mrp1, Mrp3, and Mdr1b concurrently with the down-regulated transporters. Pretreatment with dexamethasone, which decreases the release of cytokines, reversed the reduction of Mdr1a, Oatp1, Oatp2, Oct1, and Ntcp mRNA following LPS administration. Furthermore, dexamethasone pretreatment also prevented the LPS-mediated increase in Mrp1, Mrp3, and Mdr1b, whereas pretreatment with aminoguanidine or gadolinium chloride, an inhibitor of inducible nitric oxide synthetase and a Kupffer cell toxicant, respectively, had no effect on the LPS-induced changes. The concurrent repression and induction of various transporters, as well as dexamethasone abatement of both LPS-mediated repression and induction, indicates that these responses may be mediated through similar pathways.
• Li, N., Choudhuri, S., Cherrington, N. J., & Klaassen, C. D. (2004). Down-regulation of mouse organic anion-transporting polypeptide 4 (Oatp4; Oatp1b2; Slc21a10) mRNA by lipopolysaccharide through the toll-like receptor 4 (TLR4). Drug metabolism and disposition: the biological fate of chemicals, 32(11), 1265-71.
  More info

  Lipopolysaccharide (LPS) causes a systemic reaction known as sepsis, which is frequently associated with cholestasis. Many biological effects produced by LPS are thought to be mediated by Toll-like receptor 4 (TLR4). Organic anion-transporting polypeptide 4 (Oatp4; Slc21a10) mediates hepatic uptake of bile acids and other organic anions. The purpose of this study was to determine 1) whether LPS decreases Oatp4 mRNA levels; 2) the role of TLR4 in the LPS-induced down-regulation of Oatp4; and 3) the time course of serum concentrations of tumor necrosis factor alpha, interleukin (IL) 1beta, and IL-6 after LPS administration. For the dose-response study, LPS (1 mg/kg i.p.) produced a significant decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice, and higher doses produced slightly greater decreases. However, none of the doses of LPS examined significantly decreased Oatp4 mRNA levels in TLR4-mutant C3H/HeJ mice. For the time-response study, LPS (5 mg/kg i.p.) produced a rapid decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice. The maximal decrease in Oatp4 mRNA levels (80%) was observed 12 h after LPS administration and returned to control levels thereafter. In contrast, LPS did not produce a significant decrease in Oatp4 mRNA levels at any time in TLR4-mutant C3H/HeJ mice. These findings demonstrate that LPS decreases Oatp4 mRNA levels in mice, and the decrease is mediated through TLR4.
• Ning, L. i., Choudhuri, S., Cherrington, N. J., & Klaassen, C. D. (2004). Down-regulation of mouse organic anion-transporting polypeptide 4 (Oatp4; Oatp1b2; Slc21a10) mRNA by lipopolysaccharide through the toll-like receptor 4 (TLR4). Drug Metabolism and Disposition, 32(11), 1265-1271.
  More info

  PMID: 15483194;Abstract: Lipopolysaccharide (LPS) causes a systemic reaction known as sepsis, which is frequently associated with cholestasis. Many biological effects produced by LPS are thought to be mediated by Toll-like receptor 4 (TLR4). Organic anion-transporting polypeptide 4 (Oatp4; Slc21a10) mediates hepatic uptake of bile acids and other organic anions. The purpose of this study was to determine 1) whether LPS decreases Oatp4 mRNA levels; 2) the role of TLR4 in the LPS-induced down-regulation of Oatp4; and 3) the time course of serum concentrations of tumor necrosis factor α, interleukin (IL) 1β, and IL-6 after LPS administration. For the dose-response study, LPS (1 mg/kg i.p.) produced a significant decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice, and higher doses produced slightly greater decreases. However, none of the doses of LPS examined significantly decreased Oatp4 mRNA levels in TLR4-mutant C3H/HeJ mice. For the time-response study, LPS (5 mg/kg i.p.) produced a rapid decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice. The maximal decrease in Oatp4 mRNA levels (80%) was observed 12 h after LPS administration and returned to control levels thereafter. In contrast, LPS did not produce a significant decrease in Oatp4 mRNA levels at any time in TLR4-mutant C3H/HeJ mice. These findings demonstrate that LPS decreases Oatp4 mRNA levels in mice, and the decrease is mediated through TLR4.
• Wright, S. H., Evans, K. K., Zhang, X., Cherrington, N. J., Sitar, D. S., & Dantzler, W. H. (2004). Functional map of TEA transport activity in isolated rabbit renal proximal tubules. American Journal of Physiology - Renal Physiology, 287(3 56-3), F442-F451.
  More info

  PMID: 15126247;Abstract: The organic cation (OC) transporters OCT1 and OCT2 are suspected of mediating substrate entry from the blood into proximal tubule cells as the first step in renal secretion of OCs. We examined the contribution of each process in different rabbit renal proximal tubule (RPT) segments, taking advantage of the fact that rabbit orthologs of OCT1 and OCT2 can be distinguished by the high affinity of the former for tyramine (TYR) and of the latter for cimetidine (CIM). We verified that TEA uptake, for which both transporters share a similar affinity, is relatively constant in all three segments (apparent inhibitory constant of 33, 74, and 30 μM and maximal rate of mediated TEA uptake of 0.8, 1.0, and 1.2 pmol·mm-1 in S1, S2, and S3. respectively). In the S1 segment, TYR was a more effective inhibitor of TEA uptake than CIM (IC50 values of 39 and 328 μM, respectively), implicating OCT1 as the predominant pathway for TEA transport. The opposite profiles were noted in the S2 segment (IC50 values of 302 and 20 μM for TYR and CIM, respectively) and S3 segment (IC50 values of 2,900 and 54 μM TYR and CIM, respectively), suggesting that OCT2 is the predominant TEA transporter in the later portion of RPT, TEA sufficient to saturate OCT1 and OCT2 blocked only 37% of mediated amantadine transport in the S2 segment, confirming the functional presence of at least one additional OC transporter (perhaps OCT3). These data indicate that renal OC transport involves the concerted activity of a suite of transport processes.
• Wright, S. H., Evans, K. K., Zhang, X., Cherrington, N. J., Sitar, D. S., & Dantzler, W. H. (2004). Functional map of TEA transport activity in isolated rabbit renal proximal tubules. American journal of physiology. Renal physiology, 287(3), F442-51.
  More info

  The organic cation (OC) transporters OCT1 and OCT2 are suspected of mediating substrate entry from the blood into proximal tubule cells as the first step in renal secretion of OCs. We examined the contribution of each process in different rabbit renal proximal tubule (RPT) segments, taking advantage of the fact that rabbit orthologs of OCT1 and OCT2 can be distinguished by the high affinity of the former for tyramine (TYR) and of the latter for cimetidine (CIM). We verified that TEA uptake, for which both transporters share a similar affinity, is relatively constant in all three segments (apparent inhibitory constant of 33, 74, and 30 microM and maximal rate of mediated TEA uptake of 0.8, 1.0, and 1.2 pmol x mm(-1) x min(-1) in S1, S2, and S3, respectively). In the S1 segment, TYR was a more effective inhibitor of TEA uptake than CIM (IC50 values of 39 and 328 microM, respectively), implicating OCT1 as the predominant pathway for TEA transport. The opposite profiles were noted in the S2 segment (IC50 values of 302 and 20 microM for TYR and CIM, respectively) and S3 segment (IC50 values of 2,900 and 54 microM for TYR and CIM, respectively), suggesting that OCT2 is the predominant TEA transporter in the later portion of RPT. TEA sufficient to saturate OCT1 and OCT2 blocked only 37% of mediated amantadine transport in the S2 segment, confirming the functional presence of at least one additional OC transporter (perhaps OCT3). These data indicate that renal OC transport involves the concerted activity of a suite of transport processes.
• Buist, S. C., Cherrington, N. J., & Klaassen, C. D. (2003). Endocrine regulation of rat organic anion transporters. Drug metabolism and disposition: the biological fate of chemicals, 31(5), 559-64.
  More info

  Messenger RNA levels of rat organic anion transporter 1 (Oat1; Slc22a6) and Oat2 (Slc22a7) in kidney and Oat3 (Slc22a8) in liver are gender-predominant. Oat1 and Oat3 are male-predominant, whereas Oat2 is female-predominant. Gonadectomized and hypophysectomized (HX) rats were studied to determine whether sex steroids and/or growth hormone (GH) are responsible for these gender-divergent patterns. GH was administered to HX rats by two daily injections (simulating male secretion) or continuous infusion (simulating female secretion). Oat1 mRNA levels, normally higher in male than female kidney, were lowered by gonadectomy and HX in male rats, but not in females. Additionally, GH injections or infusion did not alter Oat1 levels in HX rats. Oat2 mRNA levels, typically much higher in female than in male kidney, were unaffected by gonadectomy. However, HX dramatically decreased Oat2 in female kidney without altering male levels. GH administered by continuous infusion increased Oat2 in kidneys of both HX male and female rats, whereas injections had no affect. Gonadectomy reduced Oat3 mRNA levels in male livers without affecting levels in female livers. In contrast, HX decreased male and elevated female Oat3 mRNA. GH injections did not significantly change Oat3 mRNA levels in HX rats, but infusion decreased Oat3 mRNA in liver. In conclusion, androgens, but not GH, are responsible for the Oat1 mRNA gender difference in kidney; the female GH secretion pattern is responsible for the Oat2 mRNA gender difference in kidney; and both androgens and the female GH secretion pattern are responsible for the Oat3 mRNA gender difference in liver.
• C., S., Cherrington, N. J., & Klaassen, C. D. (2003). Endocrine regulation of rat organic anion transporters. Drug Metabolism and Disposition, 31(5), 559-564.
  More info

  PMID: 12695343;Abstract: Messenger RNA levels of rat organic anion transporter 1 (Oat1; Slc22a6) and Oat2 (Slc22a7) in kidney and Oat3 (Slc22a8) in liver are gender-predominant. Oat1 and Oat3 are male-predominant, whereas Oat2 is female-predominant. Gonadectomized and hypophysectomized (HX) rats were studied to determine whether sex steroids and/or growth hormone (GH) are responsible for these gender-divergent patterns. GH was administered to HX rats by two daily injections (simulating male secretion) or continuous infusion (simulating female secretion). Oat1 mRNA levels, normally higher in male than female kidney, were lowered by gonadectomy and HX in male rats, but not in females. Additionally, GH injections or infusion did not alter Oat1 levels in HX rats. Oat2 mRNA levels, typically much higher in female than in male kidney, were unaffected by gonadectomy. However, HX dramatically decreased Oat2 in female kidney without altering male levels. GH administered by continuous infusion increased Oat2 in kidneys of both HX male and female rats, whereas injections had no affect. Gonadectomy reduced Oat3 mRNA levels in male livers without affecting levels in female livers. In contrast, HX decreased male and elevated female Oat3 mRNA. GH injections did not significantly change Oat3 mRNA levels in HX rats, but infusion decreased Oat3 mRNA in liver. In conclusion, androgens, but not GH, are responsible for the Oat1 mRNA gender difference in kidney; the female GH secretion pattern is responsible for the Oat2 mRNA gender difference in kidney; and both androgens and the female GH secretion pattern are responsible for the Oat3 mRNA gender difference in liver.
• Cherrington, N. J., Slitt, A. L., Maher, J. M., Zhang, X., Zhang, J., Huang, W., Wan, Y. Y., Moore, D. D., & Klaassen, C. D. (2003). Induction of multidrug resistance protein 3 (MRP3) in vivo is independent of constitutive androstane receptor. Drug Metabolism and Disposition, 31(11), 1315-1319.
  More info

  PMID: 14570762;Abstract: We previously demonstrated that multidrug resistance protein 3 (Mrp3/ABCC3) is induced in rat liver by phenobarbital (PB) and several other microsomal enzyme inducers that induce cytochrome P450 2B (CYP2B). CYP2B is induced by constitutive androstane receptor (CAR)-retinoid X receptor (RXR) heterodimer binding to a phenobarbital-responsive promoter element in the CYP2B promoter. Hepatic mRNA levels of CYP2B and Mrp3 were measured in three models of altered CAR activity to determine whether CAR is also involved in the induction of Mrp3. In Wistar Kyoto rats, where males express higher CAR protein levels than females, the induction of CYP2B1/2 was significantly higher in males than in females by PB, diallyl sulfide, and trans-stilbene oxide but not oltipraz. Mrp3 was induced by each of these treatments, but in contrast to CYP2B1/2, to a similar magnitude in males and females. In male hepatocyte-specific RXRα-/- mice, CYP2B10 was not induced by diallyl sulfide or oltipraz but remained inducible by PB and trans-stilbene oxide after considering the decrease in basal CYP2B10 expression. Mrp3, however, was induced by PB, diallyl sulfide, trans-stilbene oxide and oltipraz in both wild-type and RXRα-/- mice. Additionally, constitutive expression of Mrp3 was significantly reduced in RXRα-/- mice. In CAR-/- mice, the robust induction of CYP2B10 by PB was completely absent. However, Mrp3 was equally induced both in wild-type and CAR -/- mice by PB. These data clearly demonstrate that induction of hepatic Mrp3 by PB and other microsomal enzyme inducers is CAR-independent and implies a role for RXRα in the constitutive expression of Mrp3.
• Cherrington, N. J., Slitt, A. L., Maher, J. M., Zhang, X., Zhang, J., Huang, W., Wan, Y. Y., Moore, D. D., & Klaassen, C. D. (2003). Induction of multidrug resistance protein 3 (mrp3) in vivo is independent of constitutive androstane receptor. Drug metabolism and disposition: the biological fate of chemicals, 31(11), 1315-9.
  More info

  We previously demonstrated that multidrug resistance protein 3 (Mrp3/ABCC3) is induced in rat liver by phenobarbital (PB) and several other microsomal enzyme inducers that induce cytochrome P450 2B (CYP2B). CYP2B is induced by constitutive androstane receptor (CAR)-retinoid X receptor (RXR) heterodimer binding to a phenobarbital-responsive promoter element in the CYP2B promoter. Hepatic mRNA levels of CYP2B and Mrp3 were measured in three models of altered CAR activity to determine whether CAR is also involved in the induction of Mrp3. In Wistar Kyoto rats, where males express higher CAR protein levels than females, the induction of CYP2B1/2 was significantly higher in males than in females by PB, diallyl sulfide, and trans-stilbene oxide but not oltipraz. Mrp3 was induced by each of these treatments, but in contrast to CYP2B1/2, to a similar magnitude in males and females. In male hepatocyte-specific RXRalpha-/- mice, CYP2B10 was not induced by diallyl sulfide or oltipraz but remained inducible by PB and trans-stilbene oxide after considering the decrease in basal CYP2B10 expression. Mrp3, however, was induced by PB, diallyl sulfide, trans-stilbene oxide and oltipraz in both wild-type and RXRalpha-/- mice. Additionally, constitutive expression of Mrp3 was significantly reduced in RXRalpha-/- mice. In CAR-/- mice, the robust induction of CYP2B10 by PB was completely absent. However, Mrp3 was equally induced both in wild-type and CAR-/- mice by PB. These data clearly demonstrate that induction of hepatic Mrp3 by PB and other microsomal enzyme inducers is CAR-independent and implies a role for RXRalpha in the constitutive expression of Mrp3.
• Choudhuri, S., Cherrington, N. J., Li, N., & Klaassen, C. D. (2003). Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats. Drug metabolism and disposition: the biological fate of chemicals, 31(11), 1337-45.
  More info

  The aim of this study was to quantitatively determine the constitutive expression levels of various transporter mRNAs in rat choroid plexus. To provide a reference for the relative expression levels, the expression of various transporter mRNAs in choroid plexus were compared with that in liver, kidney, and ileum. The mRNA levels of multidrug resistance protein (Mrp)1, 2, 3, 4, 5, and 6; multidrug resistance (Mdr)1a, 1b, and 2; organic anion transporting polypeptide (Oatp)1, 2, 3, 4, 5, 9, 12, and Oat-K (1/2); organic anion transporter (Oat)1, 2, and 3; organic cation transporter (Oct)1, 2, 3, N1, and N2; bile acid transporters sodium taurocholate cotransporting polypeptide (Ntcp), bile salt excretory protein (Bsep), and ileal bile acid transporter (Ibat); divalent metal transporter 1 (DMT1), Menke's and Wilson's metal transporters; equilibrative nucleotide transporters (Ent) 1 and 2, and constitutive nucleotide transporters (Cnt)1 and 2; peptide transporters (Pept)1 and 2; as well as ATP-binding cassette (Abc)G5 and 8 were measured in choroid plexus by the branched DNA signal amplification method. Mrp1, 4, and 5, Oatp3, Menke's transporter, DMT1, Ent1, and Pept2 mRNAs were expressed in choroid plexus at higher levels than in liver, kidney, or ileum. OctN1 and N2, Oatp2, Oat2 and 3, and Cnt1 and 2 mRNAs expressions were detectable in choroid plexus, but the levels were lower compared with that in liver, kidney, or ileum. The remaining transporters [Mrp2, Mrp3, Oct1, Oct2, Oatp1, Oatp4, Oatp5, Oatp12, Oat-K (1/2), Ntcp, Bsep, Ibat, Mdr1a, Mdr1b, Mdr2, Oat1, Ent2, Pept1, AbcG5, AbcG8] were expressed at very low levels in choroid plexus. The constitutive expression levels of different transporters in choroid plexus may provide an insight into the range of xenobiotics that can potentially be transported by the choroid plexus, thereby providing a means of xenobiotic detoxification in the brain.
• Choudhuri, S., Cherrington, N. J., Ning, L. i., & Klaassen, C. D. (2003). Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats. Drug Metabolism and Disposition, 31(11), 1337-1345.
  More info

  PMID: 14570765;Abstract: The aim of this study was to quantitatively determine the constitutive expression levels of various transporter mRNAs in rat choroid plexus. To provide a reference for the relative expression levels, the expression of various transporter mRNAs in choroid plexus were compared with that in liver, kidney, and ileum. The mRNA levels of multidrug resistance protein (Mrp)1, 2, 3, 4, 5, and 6; multidrug resistance (Mdr)1a, 1b, and 2; organic anion transporting polypeptide (Oatp)1, 2, 3, 4, 5, 9, 12, and Oat-K (1/2); organic anion transporter (Oat)1, 2, and 3; organic cation transporter (Oct)1, 2, 3, N1, and N2; bile acid transporters sodium taurocholate cotransporting polypeptide (Ntcp), bile salt excretory protein (Bsep), and ileal bile acid transporter (Ibat); divalent metal transporter 1 (DMT1), Menke's and Wilson's metal transporters; equilibrative nucleotide transporters (Ent) 1 and 2, and constitutive nucleotide transporters (Cnt)1 and 2; peptide transporters (Pept)1 and 2; as well as ATP-binding cassette (Abc)G5 and 8 were measured in choroid plexus by the branched DNA signal amplification method. Mrp1, 4, and 5, Oatp3, Menke's transporter, DMT1, Ent1, and Pept2 mRNAs were expressed in choroid plexus at higher levels than in liver, kidney, or ileum. OctN1 and N2, Oatp2, Oat2 and 3, and Cnt1 and 2 mRNAs expressions were detectable in choroid plexus, but the levels were lower compared with that in liver, kidney, or ileum. The remaining transporters [Mrp2, Mrp3, Oct1, Oct2, Oatp1, Oatp4, Oatp5, Oatp12, Oat-K (1/2), Ntcp, Bsep, Ibat, Mdr1a, Mdr1b, Mdr2, Oat1, Ent2, Pept1, AbcG5, AbcG8] were expressed at very low levels in choroid plexus. The constitutive expression levels of different transporters in choroid plexus may provide an insight into the range of xenobiotics that can potentially be transported by the choroid plexus, thereby providing a means of xenobiotic detoxification in the brain.
• Shelby, M. K., Cherrington, N. J., Vansell, N. R., & Klaassen, C. D. (2003). Tissue mRNA expression of the rat UDP-glucuronosyltransferase gene family. Drug Metabolism and Disposition, 31(3), 326-333.
  More info

  PMID: 12584160;Abstract: UDP-Glucuronosyltransferases (UGTs) are phase II biotransformation enzymes that glucuronidate numerous endobiotic and xenobiotic substrates. Glucuronidation increases the water solubility of the substrate and facilitates renal and biliary excretion of the resulting glucuronide conjugate. UGTs have been divided into two gene families, UGT1 and UGT2. Tissue distribution of UGTs has not been thoroughly examined, and such data could provide insight into the importance of individual UGT isoforms in specific tissues and to the pharmacokinetics and target organ toxicity of UGT substrates. Therefore, the aim of this study was to determine mRNA levels of rat UGT1 and UGT2 family members in liver, kidney, lung, stomach, duodenum, jejunum, ileum, large intestine, cerebellum, and cerebral cortex, as well as nasal epithelium for UGT2A1. Tissue levels of UGT mRNA were detected using branched DNA signal amplification analysis. Three UGT isoforms, UGT1A1, UGT1A6, and UGT2B12, were detected in many tissues, whereas distribution of other UGT isoforms was more tissue-specific. For example, UGT2A1 was detected predominantly in nasal epithelium. Additionally, UGT1A5, UGT2B1, UGT2B2, UGT2B3, and UGT2B6 were detected primarily in liver. Furthermore, detection of UGT1A2, UGT1A3, UGT1A7, and UGT2B8 was somewhat specific to gastro-intestinal (GI) tract. However, not all of these UGTs were detected in all portions of the GI tract. UGT1A8 was unique in that it was barely detectable in any of the tissues examined. In conclusion, some UGT isoforms were expressed in multiple tissues, whereas other UGT isoforms were predominantly expressed in a certain tissue such as nasal epithelium, liver, or GI tract.
• Shelby, M. K., Cherrington, N. J., Vansell, N. R., & Klaassen, C. D. (2003). Tissue mRNA expression of the rat UDP-glucuronosyltransferase gene family. Drug metabolism and disposition: the biological fate of chemicals, 31(3), 326-33.
  More info

  UDP-Glucuronosyltransferases (UGTs) are phase II biotransformation enzymes that glucuronidate numerous endobiotic and xenobiotic substrates. Glucuronidation increases the water solubility of the substrate and facilitates renal and biliary excretion of the resulting glucuronide conjugate. UGTs have been divided into two gene families, UGT1 and UGT2. Tissue distribution of UGTs has not been thoroughly examined, and such data could provide insight into the importance of individual UGT isoforms in specific tissues and to the pharmacokinetics and target organ toxicity of UGT substrates. Therefore, the aim of this study was to determine mRNA levels of rat UGT1 and UGT2 family members in liver, kidney, lung, stomach, duodenum, jejunum, ileum, large intestine, cerebellum, and cerebral cortex, as well as nasal epithelium for UGT2A1. Tissue levels of UGT mRNA were detected using branched DNA signal amplification analysis. Three UGT isoforms, UGT1A1, UGT1A6, and UGT2B12, were detected in many tissues, whereas distribution of other UGT isoforms was more tissue-specific. For example, UGT2A1 was detected predominantly in nasal epithelium. Additionally, UGT1A5, UGT2B1, UGT2B2, UGT2B3, and UGT2B6 were detected primarily in liver. Furthermore, detection of UGT1A2, UGT1A3, UGT1A7, and UGT2B8 was somewhat specific to gastrointestinal (GI) tract. However, not all of these UGTs were detected in all portions of the GI tract. UGT1A8 was unique in that it was barely detectable in any of the tissues examined. In conclusion, some UGT isoforms were expressed in multiple tissues, whereas other UGT isoforms were predominantly expressed in a certain tissue such as nasal epithelium, liver, or GI tract.
• Slitt, A. L., Cherrington, N. J., Maher, J. M., & Klaassen, C. D. (2003). Induction of multidrug resistance protein 3 in rat liver is associated with altered vectorial excretion of acetaminophen metabolites. Drug Metabolism and Disposition, 31(9), 1176-1186.
  More info

  PMID: 12920174;Abstract: Treatment with the microsomal enzyme inducer trans-stilbene oxide (TSO) can decrease biliary excretion of acetaminophen-glucuronide (AA-GLUC) and increase efflux of AA-GLUC into blood. The hepatic canalicular multidrug resistance protein (Mrp) 2 and sinusoidal protein Mrp3 transport AA-GLUC conjugates into bile and blood, respectively. Thus, TSO-induced alterations in the vectorial excretion of AA-GLUC may occur via increased hepatic Mrp3 levels. The goal of this study was to determine whether TSO, diallyl sulfide (DAS), and oltipraz (OLT) treatments can up-regulate Mrp3 protein expression, and whether treatment with DAS and OLT can correspondingly increase hepatovascular efflux of AA metabolites. Rats were administered phenobarbital, TSO, DAS, OLT, or vehicle for 4 days. Interestingly, all of the chemicals increased the plasma concentration and urinary excretion of AA-GLUC and decreased its biliary excretion. In control animals, approximately 77% and 23% of AA-GLUC was excreted into bile or urine, respectively, whereas with inducer-pretreated animals, 68% was excreted into urine. Correspondingly, all of the compounds increased hepatic Mrp3 mRNA levels by 13- to 37-fold and protein levels by 2- to 6-fold, respectively. In conclusion, these studies correlate increased Mrp3 protein levels in liver with increased hepatovascular excretion of AA-GLUC and suggest that induction of Mrp3 affects the route of drug excretion.
• Slitt, A. L., Cherrington, N. J., Maher, J. M., & Klaassen, C. D. (2003). Induction of multidrug resistance protein 3 in rat liver is associated with altered vectorial excretion of acetaminophen metabolites. Drug metabolism and disposition: the biological fate of chemicals, 31(9), 1176-86.
  More info

  Treatment with the microsomal enzyme inducer trans-stilbene oxide (TSO) can decrease biliary excretion of acetaminophen-glucuronide (AA-GLUC) and increase efflux of AA-GLUC into blood. The hepatic canalicular multidrug resistance protein (Mrp) 2 and sinusoidal protein Mrp3 transport AA-GLUC conjugates into bile and blood, respectively. Thus, TSO-induced alterations in the vectorial excretion of AA-GLUC may occur via increased hepatic Mrp3 levels. The goal of this study was to determine whether TSO, diallyl sulfide (DAS), and oltipraz (OLT) treatments can up-regulate Mrp3 protein expression, and whether treatment with DAS and OLT can correspondingly increase hepatovascular efflux of AA metabolites. Rats were administered phenobarbital, TSO, DAS, OLT, or vehicle for 4 days. Interestingly, all of the chemicals increased the plasma concentration and urinary excretion of AA-GLUC and decreased its biliary excretion. In control animals, approximately 77% and 23% of AA-GLUC was excreted into bile or urine, respectively, whereas with inducer-pretreated animals, 68% was excreted into urine. Correspondingly, all of the compounds increased hepatic Mrp3 mRNA levels by 13- to 37-fold and protein levels by 2- to 6-fold, respectively. In conclusion, these studies correlate increased Mrp3 protein levels in liver with increased hepatovascular excretion of AA-GLUC and suggest that induction of Mrp3 affects the route of drug excretion.
• Brady, J. M., Cherrington, N. J., Hartley, D. P., Buist, S. C., Li, N., & Klaassen, C. D. (2002). Tissue distribution and chemical induction of multiple drug resistance genes in rats. Drug metabolism and disposition: the biological fate of chemicals, 30(7), 838-44.
  More info

  Multiple drug resistance (mdr) genes encode P-glycoprotein, which is responsible for resistance to some cancer chemotherapeutic drugs and efflux of xenobiotics of cells. Thus, mdr can protect organs from xenobiotics. In rats, there are two mdr1 genes capable of xenobiotic transport, mdr1a and mdr1b. The purpose of this study was to determine the tissue distribution of rat mdr1a and mdr1b mRNA and whether microsomal enzyme inducers that increase phase I and II drug-metabolizing enzymes coordinately regulate mdr1a and/or mdr1b. The mRNA levels of mdr1a and mdr1b were determined using branched-DNA signal amplification technology. The highest level of expression of mdr1a mRNA was observed in the gastrointestinal tract, with levels increasing, respectively, from duodenum, jejunum, and ileum to large intestine. Expression levels of mdr1a mRNA in the cerebral cortex, cerebellum, kidney, lung, and liver were less than one-tenth of that in the ileum. The tissue distribution of mdr1b mRNA was similar to mdr1a with highest expression in the gastrointestinal tract but only about 3-fold higher than in most other tissues. The induction of mdr1a and mdr1b mRNA transcripts in liver, kidney, and ileum by treatment of rats with 18 chemicals representing aryl hydrocarbon receptor ligands, constitutive androstane receptor ligands, pregnane X receptor ligands, peroxisome proliferator-activated receptor ligands, electrophile-response-element activators, and CYP4502E1 inducers was assessed. Hepatic, renal, and intestinal expression of mdr1a and mdr1b mRNA were not significantly altered by treatment of rats with any of these classes of ligands. In conclusion, the primary expression of rat mdr1 genes is in the gastrointestinal tract where they are thought to function to decrease the absorption of some xenobiotics. Rat mdr1 gene expression is not readily increased by microsomal enzyme inducers in rats through coordinate mechanisms with phase I and II drug-metabolizing enzymes.
• Brady, J. M., Cherrington, N. J., Hartley, D. P., Buist, S. C., Ning, L. i., & Klaassen, C. D. (2002). Tissue distribution and chemical induction of multiple drug resistance genes in rats. Drug Metabolism and Disposition, 30(7), 838-844.
  More info

  PMID: 12065443;Abstract: Multiple drug resistance (mdr) genes encode P-glycoprotein, which is responsible for resistance to some cancer chemotherapeutic drugs and efflux of xenobiotics of cells. Thus, mdr can protect organs from xenobiotics. In rats, there are two mdr1 genes capable of xenobiotic transport, mdr1a and mdr1b. The purpose of this study was to determine the tissue distribution of rat mdr1a and mdr1b mRNA and whether microsomal enzyme inducers that increase phase I and II drug-metabolizing enzymes coordinately regulate mdr1a and/or mdr1b. The mRNA levels of mdr1a and mdr1b were determined using branched-DNA signal amplification technology. The highest level of expression of mdr1a mRNA was observed in the gastrointestinal tract, with levels increasing, respectively, from duodenum, jejunum, and ileum to large intestine. Expression levels of mdr1a mRNA in the cerebral cortex, cerebellum, kidney, lung, and liver were less than one-tenth of that in the ileum. The tissue distribution of mdr1b mRNA was similar to mdr1a with highest expression in the gastrointestinal tract but only about 3-fold higher than in most other tissues. The induction of mdr1a and mdr1b mRNA transcripts in liver, kidney, and ileum by treatment of rats with 18 chemicals representing aryl hydrocarbon receptor ligands, constitutive androstane receptor ligands, pregnane X receptor ligands, peroxisome proliferator-activated receptor ligands, electrophile-response-element activators, and CYP4502E1 inducers was assessed. Hepatic, renal, and intestinal expression of mdr1a and mdr1b mRNA were not significantly altered by treatment of rats with any of these classes of ligands. In conclusion, the primary expression of rat mdr1 genes is in the gastrointestinal tract where they are thought to function to decrease the absorption of some xenobiotics. Rat mdr1 gene expression is not readily increased by microsomal enzyme inducers in rats through coordinate mechanisms with phase I and II drug-metabolizing enzymes.
• Buist, S. C., Cherrington, N. J., Choudhuri, S., Hartley, D. P., & Klaassen, C. D. (2002). Gender-specific and developmental influences on the expression of rat organic anion transporters. The Journal of pharmacology and experimental therapeutics, 301(1), 145-51.
  More info

  Rat organic anion transporter 1 (Oat1), Oat2, and Oat3, members of the organic anion transporter family, transport some organic anions across cellular membranes. Previously, highest Oat1 and Oat3 mRNA expression was reported in kidney and Oat2 in liver. However, gender and developmental differences in Oat expression remain unknown. This study describes gender- and age-specific patterns of rat organic anion transporter expression in various tissues. Oat mRNA expression was evaluated in adult male and female Sprague-Dawley rat tissues, and developmental expression was also determined in kidneys of Sprague-Dawley rats ranging in age from days 0 through 45. Expression was quantified using branched-DNA signal amplification. Oat1 mRNA expression was primarily observed in kidney. Surprisingly, Oat2 mRNA expression was also highest in kidney rather than in liver. Moreover, considerably higher Oat2 levels were seen in female kidney as compared with male. Finally, Oat3 mRNA expression was highest in kidney of both genders, whereas a male-predominant pattern was observed in liver. At birth, all kidney Oat mRNA levels were low. Renal Oat1 expression gradually increased throughout development, approaching adult levels at 30 days of age, where at days 40 and 45 Oat1 levels were greater in males than females. Oat2 expression in kidney was minimal through day 30 but increased dramatically at day 35 in females only. Lastly, Oat3 mRNA expression in kidney matured earliest, rapidly increasing from birth through day 10. These data indicate that Oat mRNA expression is primarily localized to the kidney, and observed expression patterns may explain some previously recognized age- and gender-dependent toxicities associated with chemical exposure.
• C., S., Cherrington, N. J., Choudhuri, S., Hartley, D. P., & Klaassen, C. D. (2002). Gender-specific and developmental influences on the expression of rat organic anion transporters. Journal of Pharmacology and Experimental Therapeutics, 301(1), 145-151.
  More info

  PMID: 11907168;Abstract: Rat organic anion transporter 1 (Oat1), Oat2, and Oat3, members of the organic anion transporter family, transport some organic anions across cellular membranes. Previously, highest Oat1 and Oat3 mRNA expression was reported in kidney and Oat2 in liver. However, gender and developmental differences in Oat expression remain unknown. This study describes gender- and age-specific patterns of rat organic anion transporter expression in various tissues. Oat mRNA expression was evaluated in adult male and female Sprague-Dawley rat tissues, and developmental expression was also determined in kidneys of Sprague-Dawley rats ranging in age from days 0 through 45. Expression was quantified using branched-DNA signal amplification. Oat1 mRNA expression was primarily observed in kidney. Surprisingly, Oat2 mRNA expression was also highest in kidney rather than in liver. Moreover, considerably higher Oat2 levels were seen in female kidney as compared with male. Finally, Oat3 mRNA expression was highest in kidney of both genders, whereas a male-predominant pattern was observed in liver. At birth, all kidney Oat mRNA levels were low. Renal Oat1 expression gradually increased throughout development, approaching adult levels at 30 days of age, where at days 40 and 45 Oat1 levels were greater in males than females. Oat2 expression in kidney was minimal through day 30 but increased dramatically at day 35 in females only. Lastly, Oat3 mRNA expression in kidney matured earliest, rapidly increasing from birth through day 10. These data indicate that Oat mRNA expression is primarily localized to the kidney, and observed expression patterns may explain some previously recognized age- and gender-dependent toxicities associated with chemical exposure.
• Cherrington, N. J., Hartley, D. P., Li, N., Johnson, D. R., & Klaassen, C. D. (2002). Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats. The Journal of pharmacology and experimental therapeutics, 300(1), 97-104.
  More info

  Many phase I and II microsomal enzyme inducers share common mechanisms of transcriptional activation and thus share a similar battery of genes that are coordinately regulated. Many phase II metabolites are thought to be transported out of cells by multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3). The purpose of this study was to determine the organ distribution of these three transporters in rat, and whether they are coordinately regulated with phase I and II drug-metabolizing enzymes. Therefore, Mrp1, 2, and 3 mRNAs were quantified using branched DNA signal amplification in multiple tissues and in tissues from rats that were treated with 18 chemicals thought to induce drug-metabolizing enzymes by six different transcription activation mechanisms [aryl-hydrocarbon receptor ligands, constitutive androstane receptor (CAR) activators, pregnane-X-receptor ligands, peroxisome proliferator activator receptor ligands, electrophile response element (EpRE) activators, and CYP2E1 inducers]. It was found that Mrp1 was expressed at a high level in kidney, lung, intestine, and brain, with low expression in liver. Mrp2 was highly expressed in liver and duodenum, and Mrp3 was highly expressed throughout the intestine but very low in liver. Microsomal enzyme inducers did not markedly increase the expression of Mrp1 or Mrp2. However, Mrp3 expression was significantly increased by each of the CAR activators and an EpRE activator in liver. Mrp3 was not similarly induced in kidney and large intestine, demonstrating that the coordinate inducibility of Mrp3 is specific to the liver. We conclude that rat hepatic Mrp3 is induced by CAR activators, thus enhancing the vectoral excretion of some phase II metabolites from the liver to the blood.
• Cherrington, N. J., Hartley, D. P., Ning, L. i., Johnson, D. R., & Klaassen, C. D. (2002). Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats. Journal of Pharmacology and Experimental Therapeutics, 300(1), 97-104.
  More info

  PMID: 11752103;Abstract: Many phase I and II microsomal enzyme inducers share common mechanisms of transcriptional activation and thus share a similar battery of genes that are coordinately regulated. Many phase II metabolites are thought to be transported out of cells by multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3). The purpose of this study was to determine the organ distribution of these three transporters in rat, and whether they are coordinately regulated with phase I and II drug-metabolizing enzymes. Therefore, Mrp1, 2, and 3 mRNAs were quantified using branched DNA signal amplification in multiple tissues and in tissues from rats that were treated with 18 chemicals thought to induce drug-metabolizing enzymes by six different transcription activation mechanisms [aryl-hydrocarbon receptor ligands, constitutive androstane receptor (CAR) activators, pregnane-X-receptor ligands, peroxisome proliferator activator receptor ligands, electrophile response element (EpRE) activators, and CYP2E1 inducers]. It was found that Mrp1 was expressed at a high level in kidney, lung, intestine, and brain, with low expression in liver. Mrp2 was highly expressed in liver and duodenum, and Mrp3 was highly expressed throughout the intestine but very low in liver. Microsomal enzyme inducers did not markedly increase the expression of Mrp1 or Mrp2. However, Mrp3 expression was significantly increased by each of the CAR activators and an EpRE activator in liver. Mrp3 was not similarly induced in kidney and large intestine, demonstrating that the coordinate inducibility of Mrp3 is specific to the liver. We conclude that rat hepatic Mrp3 is induced by CAR activators, thus enhancing the vectoral excretion of some phase II metabolites from the liver to the blood.
• Li, N., Hartley, D. P., Cherrington, N. J., & Klaassen, C. D. (2002). Tissue expression, ontogeny, and inducibility of rat organic anion transporting polypeptide 4. The Journal of pharmacology and experimental therapeutics, 301(2), 551-60.
  More info

  To date, organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is known as a liver-specific and sodium-independent transporter that mediates transport of a variety of compounds. The purpose of this study was to determine whether Oatp4 mRNA expression is specific to the liver compared with Oatp1, 2, 3, or 5. In addition, the effect of gender and age was determined by assessing the expression of Oatp4 mRNA during the postnatal development of rats. Furthermore, to determine whether Oatp4 gene expression is coordinately modulated by drug-metabolizing enzyme inducers, male rats were administered chemicals known to induce the expression of drug-metabolizing enzymes through six mechanisms: the aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, peroxisome proliferator-activated receptor, electrophile response element, or CYP2E1 inducers. The levels of Oatp1, 2, 3, 4, and 5 mRNA were measured using the branched DNA signal amplification technique. The tissue distribution of Oatp4 was almost exclusively expressed in liver in contrast to Oatp1, 2, 3, and 5. The hepatic expression of Oatp4 was low in newborn rats and increased gradually to the adult level with no significant difference between genders. The expression of Oatp4 was not consistently induced by any of the six groups of enzyme inducers. These findings continue to suggest that Oatp4 is expressed specifically in the liver. The preference of Oatp4 for endogenous compounds coupled with its refractory response to known drug-metabolizing enzyme inducers suggests that Oatp4 may be largely responsible for the homeostasis of endogenous rather than exogenous chemicals, including pharmaceuticals.
• Ning, L. i., Hartley, D. P., Cherrington, N. J., & Klaassen, C. D. (2002). Tissue expression, ontogeny, and inducibility of rat organic anion transporting polypeptide 4. Journal of Pharmacology and Experimental Therapeutics, 301(2), 551-560.
  More info

  PMID: 11961056;Abstract: To date, organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is known as a liver-specific and sodium-independent transporter that mediates transport of a variety of compounds. The purpose of this study was to determine whether Oatp4 mRNA expression is specific to the liver compared with Oatp1, 2, 3, or 5. In addition, the effect of gender and age was determined by assessing the expression of Oatp4 mRNA during the postnatal development of rats. Furthermore, to determine whether Oatp4 gene expression is coordinately modulated by drug-metabolizing enzyme inducers, male rats were administered chemicals known to induce the expression of drug-metabolizing enzymes through six mechanisms: the aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, peroxisome proliferator-activated receptor, electrophile response element, or CYP2E1 inducers. The levels of Oatp1, 2, 3, 4, and 5 mRNA were measured using the branched DNA signal amplification technique. The tissue distribution of Oatp4 was almost exclusively expressed in liver in contrast to Oatp1, 2, 3, and 5. The hepatic expression of Oatp4 was low in newborn rats and increased gradually to the adult level with no significant difference between genders. The expression of Oatp4 was not consistently induced by any of the six groups of enzyme inducers. These findings continue to suggest that Oatp4 is expressed specifically in the liver. The preference of Oatp4 for endogenous compounds coupled with its refractory response to known drug-metabolizing enzyme inducers suggests that Oatp4 may be largely responsible for the homeostasis of endogenous rather than exogenous chemicals, including pharmaceuticals.
• Park, J. D., Cherrington, N. J., & Klaassen, C. D. (2002). Intestinal absorption of cadmium is associated with divalent metal transporter 1 in rats. Toxicological Sciences, 68(2), 288-294.
  More info

  PMID: 12151624;Abstract: The intestinal absorption of cadmium (Cd) increases when the body iron (Fe) stores are depleted. The depletion of Fe upregulates the expression of divalent metal transporter 1 (DMT1), which is located at the apical membrane of enterocytes lining the small intestine. DMT1 has been shown to transport Fe and other divalent metal ions in vitro. However, it is not known whether DMT1 mediates the intestinal absorption of Cd. To investigate DMT1 involvement in Cd absorption, rats were fed a diet for 4 weeks either deficient in Fe (FeD diet, 2-6 mg Fe/kg) or supplemented with Fe (FeS diet, 120 mg Fe/kg), followed by a single oral administration of 109CdCl2. Body Fe status, hemoglobin, and tissue Cd concentration were determined at 48 h after Cd administration. Also, DMT1 mRNA levels were quantified in duodenum, kidney, and liver by the branched DNA signal amplification method. Animals fed the FeD diet exhibited a reduced body weight gain, depletion of body Fe, and Fe deficiency anemia. Tissue Cd concentration was significantly higher in FeD than in FeS diet-fed rats, especially in the duodenum. The amount of Cd retained in the body was 10-fold higher in rats fed the FeD diet than in those fed the FeS diet. DMT1 mRNA was highly expressed in duodenum and was 15-fold higher in the FeD diet group. The levels of DMT1 mRNA were significantly lower in kidney and liver than in duodenum, but were 30 and 40% higher, respectively, in rats fed the FeD diet than in rats fed the FeS diet. These findings suggest that functional DMT1 protein is likely upregulated in the small intestine at the mRNA level by body iron depletion and increases Cd uptake from the gastrointestinal tract with subsequent transfer of Cd to the circulation and body tissues. Furthermore, the data from this study may indicate that DMT1 is a nonspecific metal transporter, which can transport not only Fe, but probably the toxic metal as well.
• Park, J. D., Cherrington, N. J., & Klaassen, C. D. (2002). Intestinal absorption of cadmium is associated with divalent metal transporter 1 in rats. Toxicological sciences : an official journal of the Society of Toxicology, 68(2), 288-94.
  More info

  The intestinal absorption of cadmium (Cd) increases when the body iron (Fe) stores are depleted. The depletion of Fe upregulates the expression of divalent metal transporter 1 (DMT1), which is located at the apical membrane of enterocytes lining the small intestine. DMT1 has been shown to transport Fe and other divalent metal ions in vitro. However, it is not known whether DMT1 mediates the intestinal absorption of Cd. To investigate DMT1 involvement in Cd absorption, rats were fed a diet for 4 weeks either deficient in Fe (FeD diet, 2-6 mg Fe/kg) or supplemented with Fe (FeS diet, 120 mg Fe/kg), followed by a single oral administration of 109 CdCl2. Body Fe status, hemoglobin, and tissue Cd concentration were determined at 48 h after Cd administration. Also, DMT1 mRNA levels were quantified in duodenum, kidney, and liver by the branched DNA signal amplification method. Animals fed the FeD diet exhibited a reduced body weight gain, depletion of body Fe, and Fe deficiency anemia. Tissue Cd concentration was significantly higher in FeD than in FeS diet-fed rats, especially in the duodenum. The amount of Cd retained in the body was 10-fold higher in rats fed the FeD diet than in those fed the FeS diet. DMT1 mRNA was highly expressed in duodenum and was 15-fold higher in the FeD diet group. The levels of DMT1 mRNA were significantly lower in kidney and liver than in duodenum, but were 30 and 40% higher, respectively, in rats fed the FeD diet than in rats fed the FeS diet. These findings suggest that functional DMT1 protein is likely upregulated in the small intestine at the mRNA level by body iron depletion and increases Cd uptake from the gastrointestinal tract with subsequent transfer of Cd to the circulation and body tissues. Furthermore, the data from this study may indicate that DMT1 is a nonspecific metal transporter, which can transport not only Fe, but probably the toxic metal as well.
• Slitt, A. L., Cherrington, N. J., Hartley, D. P., Leazer, T. M., & Klaassen, C. D. (2002). Tissue distribution and renal developmental changes in rat organic cation transporter mRNA levels. Drug Metabolism and Disposition, 30(2), 212-219.
  More info

  PMID: 11792693;Abstract: Organic cation transporters (OCTs) are responsible for excretion of cationic substances into urine. Tissue OCT expression may be important for the disposition and excretion of xenobiotics. Therefore, OCT1, OCT2, OCT3, OCTN1, and OCTN2 mRNA levels were measured in adult rat tissues and rat kidney tissue at various stages of development from day 0 to 45. OCT1 mRNA expression was highest in kidney and spleen, moderate in skin, and low in the gastrointestinal tract, brain, lung, thymus, muscle, and prostate. OCT2 mRNA levels were highest in kidney, with low expression in other tissues, and with renal OCT2 levels being approximately 4 times higher in males than that in females. In gonadectomized males, OCT2 mRNA levels were attenuated to female levels, suggesting a role for testosterone in OCT2 expression. OCT3 was moderately expressed in kidney and was highest in blood vessel, skin, and thymus. OCTN1 was expressed in most of the tissues examined, with relatively higher expression in kidney and ileum and lower levels in thymus. Lastly, OCTN2 was expressed abundantly in kidney and ileum, moderately in large intestine, dorsal prostate, bladder, duodenum, and cerebellum, and minimally in thymus, spleen, and cerebral cortex. Renal OCT1, OCTN1, and OCTN2 mRNA levels increased gradually from postnatal day 0 through day 45 in both genders. Renal OCT2 levels remained the same in males and females through day 25 and then dramatically increased only in male kidney after day 30. In summary, OCT mRNA was detected primarily in kidney, and the high level of renal OCT expression may explain why the kidney is a target organ for xenobiotics with cationic properties.
• Slitt, A. L., Cherrington, N. J., Hartley, D. P., Leazer, T. M., & Klaassen, C. D. (2002). Tissue distribution and renal developmental changes in rat organic cation transporter mRNA levels. Drug metabolism and disposition: the biological fate of chemicals, 30(2), 212-9.
  More info

  Organic cation transporters (OCTs) are responsible for excretion of cationic substances into urine. Tissue OCT expression may be important for the disposition and excretion of xenobiotics. Therefore, OCT1, OCT2, OCT3, OCTN1, and OCTN2 mRNA levels were measured in adult rat tissues and rat kidney tissue at various stages of development from day 0 to 45. OCT1 mRNA expression was highest in kidney and spleen, moderate in skin, and low in the gastrointestinal tract, brain, lung, thymus, muscle, and prostate. OCT2 mRNA levels were highest in kidney, with low expression in other tissues, and with renal OCT2 levels being approximately 4 times higher in males than that in females. In gonadectomized males, OCT2 mRNA levels were attenuated to female levels, suggesting a role for testosterone in OCT2 expression. OCT3 was moderately expressed in kidney and was highest in blood vessel, skin, and thymus. OCTN1 was expressed in most of the tissues examined, with relatively higher expression in kidney and ileum and lower levels in thymus. Lastly, OCTN2 was expressed abundantly in kidney and ileum, moderately in large intestine, dorsal prostate, bladder, duodenum, and cerebellum, and minimally in thymus, spleen, and cerebral cortex. Renal OCT1, OCTN1, and OCTN2 mRNA levels increased gradually from postnatal day 0 through day 45 in both genders. Renal OCT2 levels remained the same in males and females through day 25 and then dramatically increased only in male kidney after day 30. In summary, OCT mRNA was detected primarily in kidney, and the high level of renal OCT expression may explain why the kidney is a target organ for xenobiotics with cationic properties.
• Takiguchi, M., Cherrington, N. J., Hartley, D. P., Klaassen, C. D., & Waalkes, M. P. (2001). Cyproterone acetate induces a cellular tolerance to cadmium in rat liver epithelial cells involving reduced cadmium accumulation. Toxicology, 165(1), 13-25.
  More info

  PMID: 11551428;Abstract: Several reports indicate that some steroids, in particular sex steroid hormones, can modify cadmium toxicity. We recently reported that cyproterone acetate (CA), a synthetic steroidal antiandrogen that is closely related in structure to progesterone, affects cadmium toxicity in mice. In the present study, we investigated the effect of CA on cadmium toxicity in a rat liver epithelial cell line (TRL 1215) in vitro. Cells were exposed to various concentrations of CA (0,1,10, or 50 μM) for 24 h and subsequently exposed to cadmium (0,50, or 100 μM; as CdCl2) for an additional 24 h. CA pretreatment resulted in a clear decrease in the sensitivity to cadmium. Additional time course study showed CA pretreatment provided protection against cadmium toxicity but only when given for 6 or more hours prior to cadmium exposure. Cellular cadmium accumulation was markedly reduced (60% decrease) in cells pretreated for 6 or more hours with CA. In the presence of protein synthesis inhibitors the protective effect of CA toward cadmium toxicity was abolished. However, in the presence of the GSH synthesis inhibitor, L-buthionine (S,R)-sulfoximide (BSO), the protective effect of CA toward cadmium toxicity remained. CA alone increased metallothionein (MT) levels 2.4-fold, while cadmium (50 μM) alone resulted in a 8.9-fold increase over control. However, cadmium-induced MT synthesis was markedly decreased by CA pretreatment probably because of reduced cadmium accumulation. Analysis of various metal transporters by bDNA signal amplification assay revealed that the ZnT-1 transporter gene, which encodes for a membrane protein associated with zinc efflux, was expressed three-fold more in CA treated cells than control. These data show that CA pretreatment provides protection against cadmium toxicity in vitro and indicate that this protection is due to a decreased accumulation of cadmium rather than through activation of MT synthesis. This decrease of cellular cadmium accumulation appears to be related to events that require protein synthesis and may be due to activation of the genes associated with zinc efflux.
• Cherrington, N. J., Cao, Y., Cherrington, J. W., Rose, R. L., & Hodgson, E. (1998). Physiological factors affecting protein expression of flavin-containing monooxygenases 1, 3 and 5. Xenobiotica; the fate of foreign compounds in biological systems, 28(7), 673-82.
  More info

  1. The mouse and rat exhibit substantial differences in the gender expression of flavin-containing monooxygenase (FMO) forms. Hepatic FMO1 is gender-dependent in both species, selective to the male in rat, female in mouse. Human FMO1 is nearly undetectable. FMO3 in mouse is gender-specific to the female, but gender-independent in rat and man. FMO5 is gender-independent for mouse, rat and man. 2. Gender differences in substrate metabolism do not reflect overall FMO or isoform differences. Methimazole, imipramine and thiobenzamide are much better substrates for FMO1 than for FMO3 or FMO5. 3. Activities of microsomal samples toward these substrates reflect the relative abundance of FMO1. Hepatic samples show a 3-fold greater activity toward methimazole in the female mouse and male rat. Human microsomal samples show minimal activity. 4. Developmentally, FMO1 and FMO5 are expressed in foetuses as early as gestation days 15 and 17 and equally between genders until puberty. FMO3 is not found until 2 weeks post-partum and is found equally in the male and female until 6 weeks post-partum when it becomes undetectable in the male. 5. An event takes place after birth but before puberty that confers the ability to produce FMO3. The developmental pattern observed for mouse FMO3 is similar to human FMO3.
• Cherrington, N. J., Cao, Y., Cherrington, J. W., Rose, R. L., & Hodgson, E. (1998). Physiological factors affecting protein expression of flavin-containing monooxygenases 1, 3 and 5. Xenobiotica, 28(7), 673-682.
  More info

  PMID: 9711811;Abstract: 1. The mouse and rat exhibit substantial differences in the gender expression of flavin-containing monooxygenase (FMO) forms. Hepatic FMO1 is gender-dependent in both species, selective to the male in rat, female in mouse. Human FMO1 is nearly undetectable. FMO3 in mouse is gender-specific to the female, but gender-independent in rat and man. FMO5 is gender-independent for mouse, rat and man. 2. Gender differences in substrate metabolism do not reflect overall FMO or isoform differences. Methimazole, imipramine and thiobenzamide are much better substrates for FMO1 than for FMO3 or FMO5. 3. Activities of microsomal samples toward these substrates reflect the relative abundance of FMO1. Hepatic samples show a 3-fold greater activity toward methimazole in the female mouse and male rat. Human microsomal samples show minimal activity. 4. Developmentally, FMO1 and FMO5 are expressed in foetuses as early as gestation days 15 and 17 and equally between genders until puberty. FMO3 is not found until 2 weeks post-partum and is found equally in the male and female until 6 weeks post-partum when it becomes undetectable in the male. 5. An event takes place after birth but before puberty that confers the ability to produce FMO3. The developmental pattern observed for mouse FMO3 is similar to human FMO3.
• Cherrington, N. J., Falls, J. G., Rose, R. L., Clements, K. M., Philpot, R. M., Levi, P. E., & Hodgson, E. (1998). Molecular Cloning, Sequence, and Expression of Mouse Flavin-Containing Monooxygenases 1 and 5 (FMO1 and FMO5). Journal of Biochemical and Molecular Toxicology, 12(4), 205-212.
  More info

  PMID: 9580872;Abstract: Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse. The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5′-flanking region, and 413 in the 3′-flanking region. The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5′-flanking region, and 757 in the 3′-flanking region. The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83-94% identity to other FMO1 homologs. FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83-84% identity to other FMO5 orthologs. Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5. Mouse FMO1 and FMO5 were expressed in E. coli and show similar mobility to the native proteins as determined by SDS-PAGE. The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward n-octylamine. In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate. © 1998 John Wiley & Sons, Inc.
• Cherrington, N. J., Falls, J. G., Rose, R. L., Clements, K. M., Philpot, R. M., Levi, P. E., & Hodgson, E. (1998). Molecular cloning, sequence, and expression of mouse flavin-containing monooxygenases 1 and 5 (FMO1 and FMO5). Journal of biochemical and molecular toxicology, 12(4), 205-12.
  More info

  Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse. The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5'-flanking region, and 413 in the 3'-flanking region. The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5'-flanking region, and 757 in the 3'-flanking region. The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83-94% identity to other FMO1 homologs. FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83-84% identity to other FMO5 orthologs. Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5. Mouse FMO1 and FMO5 were expressed in E. coli and show similar mobility to the native proteins as determined by SDS-PAGE. The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward noctylamine. In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate.
• Hodgson, E., Cherrington, N. J., Coleman, S., Siming, L., Falls, J. G., Yao, C., Goldstein, J. E., Rose, R. L., Kuhr, R. J., & Motoyama, N. (1998). Flavin-containing monooxygenase and cytochrome P450 mediated metabolism of pesticides: from mouse to human.. Revs. Toxicology, 2 (1-4), 231-44.
• Falls, J. G., Cherrington, N. J., Clements, K. M., Philpot, R. M., Levi, P. E., Rose, R. L., & Hodgson, E. (1997). Molecular cloning, sequencing, and expression in Escherichia coli of mouse flavin-containing monooxygenase 3 (FMO3): Comparison with the human isoform. Archives of Biochemistry and Biophysics, 347(1), 9-18.
  More info

  PMID: 9344459;Abstract: The sequence of mouse flavin-containing monooxygenase 3 (FMO3) was obtained from several clones isolated from a mouse liver cDNA library. The nucleotide sequence of mouse FMO3 was 2020 bases in length containing 37 bases in the 5' flanking region, 1602 in the coding region, and 381 in the 3' flanking region. The derived protein sequence consisted of 634 amino acids including the putative flavin adenine dinucleotide and NADP+ pyrophosphate binding sites (characteristic of mammalian FMOs) starting at positions 9 and 191, respectively. The mouse FMO3 protein sequence was 79 and 82% identical to the human and rabbit FMO3 sequences, respectively. Mouse FMO3 was expressed in Escherichia coli and compared to E. coli expressed human FMO3. The FMO3 proteins migrated with the same mobility (~58 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The expressed FMO3 enzymes (mouse and human forms) were sensitive to heat and reacted in a similar manner toward metal ions and detergent. Catalytic activities of mouse and human FMO3 were high toward the substrate methimazole; however, in the presence of trimethylamine and thioacetamide, FMO-dependent methimazole oxidation by both enzymes was reduced by greater than 85%. Other substrates which inhibited methimazole oxidation were thiourea and thiobenzamide and to a lesser degree N,N-dimethylaniline. When probed with mouse FMO3 cDNA, FMO3 transcripts were detected in hepatic mRNA samples from female mice, but not in samples from males. FMO3 was detected in mRNA samples from male and female mouse lung, but FMO3 message was not detected in mouse kidney sample from either gender. Results of immunoblotting confirmed the tissue- and gender-dependent expression of mouse FMO3.
• Falls, J. G., Cherrington, N. J., Clements, K. M., Philpot, R. M., Levi, P. E., Rose, R. L., & Hodgson, E. (1997). Molecular cloning, sequencing, and expression in Escherichia coli of mouse flavin-containing monooxygenase 3 (FMO3): comparison with the human isoform. Archives of biochemistry and biophysics, 347(1), 9-18.
  More info

  The sequence of mouse flavin-containing monooxygenase 3 (FMO3) was obtained from several clones isolated from a mouse liver cDNA library. The nucleotide sequence of mouse FMO3 was 2020 bases in length containing 37 bases in the 5' flanking region, 1602 in the coding region, and 381 in the 3' flanking region. The derived protein sequence consisted of 534 amino acids including the putative flavin adenine dinucleotide and NADP+ pyrophosphate binding sites (characteristic of mammalian FMOs) starting at positions 9 and 191, respectively. The mouse FMO3 protein sequence was 79 and 82% identical to the human and rabbit FMO3 sequences, respectively. Mouse FMO3 was expressed in Escherichia coli and compared to E. coli expressed human FMO3. The FMO3 proteins migrated with the same mobility ( approximately 58 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The expressed FMO3 enzymes (mouse and human forms) were sensitive to heat and reacted in a similar manner toward metal ions and detergent. Catalytic activities of mouse and human FMO3 were high toward the substrate methimazole; however, in the presence of trimethylamine and thioacetamide, FMO-dependent methimazole oxidation by both enzymes was reduced by greater than 85%. Other substrates which inhibited methimazole oxidation were thiourea and thiobenzamide and to a lesser degree N,N-dimethylaniline. When probed with mouse FMO3 cDNA, FMO3 transcripts were detected in hepatic mRNA samples from female mice, but not in samples from males. FMO3 was detected in mRNA samples from male and female mouse lung, but FMO3 message was not detected in mouse kidney sample from either gender. Results of immunoblotting confirmed the tissue- and gender-dependent expression of mouse FMO3.

Presentations

• Cherrington, N. J. (2015, February). Genes and Big Jeans: Precision Medicine and the Sources of Variable Drug Transport and Exposure. Genetics Grand Rounds. University of Arizona.
• Cherrington, N. J. (2015, November). Obesity and Risk of Adverse Drug Reactions. Professional Development. Tucson, AZ: Canyon Ranch.
• Cherrington, N. J. (2015, October). Genotype or Phenotype: Precision Medicine and the Sources of Variable Drug Transport and Exposure. Meet the Experts: The Transporter Conference. Boston, Massachusetts: Solvo.
• Cherrington, N. J. (2015, September). ADME in Metabolic Syndrome: Increased Risk of Drug-Induced Toxicity. Deleware Valley Drug Metabolism Discussion Group Keynote Address. Philadelphia, PA: Deleware Valley Drug Metabolism Discussion Group.