Qing-Yu Zhang

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Scholarly Contributions

Chapters

• Zhang, Q., & Ding, X. (2018). Enzyme Regulation. In Comprehensive Toxicology, 3rd Edition(pp 9-29). Oxford: Academic Press.
• Zhang, Q., Li, L., & Van Winkle, L. S. (2018). Biochemical Function of the Respiratory Tract: Metabolism of Xenobiotics. In Comprehensive Toxicology, 3rd Edition(pp 171-179). Oxford: Academic Press.

Journals/Publications

• Chang, M., Gao, F., Gnawali, G., Xu, H., Dong, Y., Meng, X., Li, W., Wang, Z., Lopez, B., Carew, J. S., Nawrocki, S. T., Lu, J., Zhang, Q., & Wang, W. (2024). Selective Elimination of Senescent Cancer Cells by Galacto-Modified PROTACs. Journal of Medicinal Chemistry. doi:10.1021/acs.jmedchem.4c00152
• Ding, X., Han, J., Winkle, L. S., & Zhang, Q. (2022). Detection of Transgene Location in the CYP2A13/2B6/2F1-transgenic Mouse Model using Optical Genome Mapping Technology. Drug Metabolism and Disposition. doi:10.1124/dmd.122.001090
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  Most transgenic mouse models are generated through random integration of the transgene. The location of the transgene provides valuable information for assessing potential effects of the transgenesis on the host and for designing genotyping protocols that can amplify across the integration site, but it is challenging to identify. Here, we report the successful utility of optical genome mapping technology to identify the transgene insertion site in a CYP2A13/2B6/2F1-transgenic mouse model, which produces three human cytochrome P450 (P450) enzymes (CYP2A13, CYP2B6, and CYP2F1) that are encoded by neighboring genes on human chromosome 19. These enzymes metabolize many drugs, respiratory toxicants, and chemical carcinogens. Initial efforts to identify candidate insertion sites by whole genome sequencing was unsuccessful, apparently because the transgene is located in a region of the mouse genome that contains highly repetitive sequences. Subsequent utility of the optical genome mapping approach, which compares genome-wide marker distribution between the transgenic mouse genome and a reference mouse (GRCm38) or human (GRCh38) genome, localized the insertion site to mouse chromosome 14, between two marker positions at 4451324 base pair and 4485032 base pair. A transgene-mouse genome junction sequence was further identified through long-polymerase chain reaction amplification and DNA sequencing at GRCm38 Chr.14:4484726. The transgene insertion (∼2.4 megabase pair) contained 5–7 copies of the human transgenes, which replaced a 26.9–33.4 kilobase pair mouse genomic region, including exons 1–4 of Gm3182, a predicted and highly redundant gene. Finally, the sequencing results enabled the design of a new genotyping protocol that can distinguish between hemizygous and homozygous CYP2A13/2B6/2F1-transgenic mice. SIGNIFICANCE STATEMENT This study characterizes the genomic structure of, and provides a new genotyping method for, a transgenic mouse model that expresses three human P450 enzymes, CYP2A13, CYP2B6, and CYP2F1, that are important in xenobiotic metabolism and toxicity. The demonstrated success in applying the optical genome mapping technology for identification of transgene insertion sites should encourage others to do the same for other transgenic models generated through random integration, including most of the currently available human P450 transgenic mouse models.
• Ding, X., Ding, L., Fan, X., He, Y., Lian, W., Lin, G., Ma, J., Zhang, Q., & Zhang, Q. Y. (2021). Lung injury induced by pyrrolizidine alkaloids depends on metabolism by hepatic cytochrome P450s and blood transport of reactive metabolites.. Archives of Toxicology, 95(1), 103-116. doi:10.1007/s00204-020-02921-0
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  Pyrrolizidine alkaloids (PAs) are common phytotoxins with both hepatotoxicity and pneumotoxicity. Hepatic cytochrome P450 enzymes are known to bioactivate PAs into reactive metabolites, which can interact with proteins to form pyrrole-protein adducts and cause intrahepatic cytotoxicity. However, the metabolic and initiation biochemical mechanisms underlying PA-induced pneumotoxicity remain unclear. To investigate the in vivo metabolism basis for PA-induced lung injury, this study used mice with conditional deletion of the cytochrome P450 reductase (Cpr) gene and resultant tissue-selective ablation of microsomal P450 enzyme activities. After oral exposure to monocrotaline (MCT), a pneumotoxic PA widely used to establish animal lung injury models, liver-specific Cpr-null (LCN) mice, but not extrahepatic Cpr-low (xh-CL) mice, had significantly lower level of pyrrole-protein adducts in the serum, liver and lungs compared with wild-type (WT) mice. While MCT-exposed LCN mice had significantly higher blood concentration of intact MCT, compared to MCT-exposed WT or xh-CL mice. Consistent with the MCT in vivo bioactivation data, MCT-induced lung injury, represented by vasculature damage, in WT and xh-CL mice but not LCN mice. Furthermore, reactive metabolites of MCT were confirmed to exist in the blood efflux from the hepatic veins of MCT-exposed rats. Our results provide the first mode-of-action evidence that hepatic P450s are essential for the bioactivation of MCT, and blood circulating reactive metabolites of MCT to the lung causes pneumotoxicity. Collectively, this study presents the scientific basis for the application of MCT in animal lung injury models, and more importantly, warrants public awareness and further investigations of lung diseases associated with exposure to not only MCT but also different PAs.
• Ding, X., Ding, L., Fan, X., He, Y., Lin, G., Ma, J., & Zhang, Q. (2021). The key role of gut-liver axis in pyrrolizidine alkaloid-induced hepatotoxicity and enterotoxicity.. Acta pharmaceutica Sinica. B, 11(12), 3820-3835. doi:10.1016/j.apsb.2021.07.013
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  Pyrrolizidine alkaloids (PAs) are the most common phytotoxins with documented human hepatotoxicity. PAs require metabolic activation by cytochromes P450 to generate toxic intermediates which bind to proteins and form protein adducts, thereby causing cytotoxicity. This study investigated the role of the gut-liver axis in PA intoxication and the underlying mechanisms. We exposed mice to retrorsine (RTS), a representative PA, and for the first time found RTS-induced intestinal epithelium damage and disruption to intestinal barrier function. Using mice with tissue-selective ablation of P450 activity, we found that hepatic P450s, but not intestinal P450s, were essential for PA bioactivation. Besides, in RTS-exposed, bile duct-cannulated rats, we found the liver-derived reactive PA metabolites were transported by bile into the intestine to exert enterotoxicity. The impact of gut-derived pathogenic factors in RTS-induced hepatotoxicity was further studied in mice with dextran sulfate sodium (DSS)-induced chronic colitis. DSS treatment increased the hepatic endotoxin level and depleted hepatic reduced glutathione, thereby suppressing the PA detoxification pathway. Compared to RTS-exposed normal mice, the colitic mice displayed more severe RTS-induced hepatic vasculature damage, fibrosis, and steatosis. Overall, our findings provide the first mode-of-action evidence of PA-induced enterotoxicity and highlight the importance of gut barrier function in PA-induced liver injury.
• Begley, T. J., Dedon, P. C., Ding, X., Endres, L., Evke, S., Kovalchuk, N., Leonardi, A., Martin, S., Melendez, J. A., Nevins, S., Winkle, L. V., Yin, L., Zhang, Q. Y., & Zhang, Q. (2020). The epitranscriptomic writer ALKBH8 drives tolerance and protects mouse lungs from the environmental pollutant naphthalene.. Epigenetics, 15(10), 1121-1138. doi:10.1080/15592294.2020.1750213
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  The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a transfer RNA (tRNA) methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation of selenoproteins. Using Alkbh8 deficient (Alkbh8def) mice, we have investigated the importance of epitranscriptomic systems in the response to naphthalene, an abundant polycyclic aromatic hydrocarbon and environmental toxicant. We performed basal lung analysis and naphthalene exposure studies using wild type (WT), Alkbh8de f and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for naphthalene bioactivation. Under basal conditions, lungs from Alkbh8def mice have increased markers of oxidative stress and decreased thioredoxin reductase protein levels, and have reprogrammed gene expression to differentially regulate stress response transcripts. Alkbh8def mice are more sensitive to naphthalene induced death than WT, showing higher susceptibility to lung damage at the cellular and molecular levels. Further, WT mice develop a tolerance to naphthalene after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice. We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against naphthalene-induced lung dysfunction and promotes naphthalene tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.
• Ding, L., Li, L., Liu, S., Bao, X., Dickman, K. G., Sell, S. S., Mei, C., Zhang, Q. Y., Gu, J., & Ding, X. (2020). Proximal Tubular Vacuolization and Hypersensitivity to Drug-Induced Nephrotoxicity in Male Mice With Decreased Expression of the NADPH-Cytochrome P450 Reductase. Toxicological sciences : an official journal of the Society of Toxicology, 173(2), 362-372.
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  The effect of variations in the expression of cytochrome P450 reductase (CPR or POR) is determined in mice with decreased POR expression to identify potential vulnerabilities in people with low POR expression. There is an age-dependent appearance of increasing vacuolization in the proximal tubules of the renal cortex in 4- to 9-month-old male (but not female) Cpr-low (CL) mice. These mice have low POR expression in all cells of the body and upregulation of lysosome-associated membrane protein 1 expression in the renal cortex. Vacuolization is also seen in extrahepatic CL and extrarenal CL male mice, but not in mice with tissue-specific Por deletion in liver, intestinal epithelium, or kidney. The occurrence of vacuolization is accompanied by increases in serum blood-urea-nitrogen levels. Male CL mice are hypersensitive to cisplatin- and gentamicin-induced renal toxicity at 3 months of age, before proximal tubular (PT) vacuoles are detectable. At doses that do not cause renal toxicity in wild-type mice, both drugs cause substantial increases in serum blood-urea-nitrogen levels and PT vacuolization in male but not female CL mice. The hypersensitivity to drug-induced renal toxicity is accompanied by increases in circulating drug levels. These novel findings demonstrate deficiency of renal function in mice with globally reduced POR expression and suggest that low POR expression may be a risk factor for drug-induced nephrotoxicity in humans.
• Ding, X., Ding, L., Fan, X., Fu, P. P., He, Y., Lin, G., Song, Z., Zhang, Q., & Zhang, Q. Y. (2020). Investigation on Pneumotoxicity Induced by Pyrrolizidine AlkaloidsInvestigation on Pneumotoxicity Induced by Pyrrolizidine Alkaloids. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.08849
• Ding, X., Zhang, Q. Y., Winkle, L. V., Kovalchuk, N., Kovalchuk, N., Winkle, L. V., Zhang, Q. Y., & Ding, X. (2020). Contribution of Pulmonary CYP-mediated Bioactivation of Naphthalene to Airway Epithelial Injury in the Lung.. Toxicological sciences : an official journal of the Society of Toxicology, 177(2), 334-346. doi:10.1093/toxsci/kfaa114
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  Previous studies have established that cytochrome P450 enzymes (CYPs) in both liver and lung are capable of bioactivating naphthalene (NA), an omnipresent air pollutant and possible human carcinogen, in vitro and in vivo. The aim of this study was to examine the specific contribution of pulmonary CYPs in airway epithelial cells to NA-induced airway toxicity. We used a lung-Cpr-null mouse model, which undergoes doxycycline-induced, Cre-mediated deletion of the Cpr (a redox partner of all microsomal CYPs) gene specifically in airway epithelial cells. In 2-month-old lung-Cpr-null mice, Cpr deletion occurred in 75%-82% of epithelial cells of conducting airways. The extent of NA-induced acute lung toxicity (as indicated by total protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid collected at 24-h after initiation of a 4-h, nose-only, 10-ppm NA inhalation exposure) was substantially lower (by 37%-39%) in lung-Cpr-null mice, compared with control littermates. Moreover, the extent of cellular proliferation (as indicated by 5-bromo-2'-deoxyuridine incorporation) was noticeably lower in both proximal and distal airways (by 59% and 65%, respectively) of NA-treated lung-Cpr-null mice, compared with control littermates, at 2-day post-NA inhalation exposure. A similar genotype-related difference in the extent of postexposure cell proliferation was also observed in mice exposed to NA via intraperitoneal injection at 200 mg/kg. These results directly validate the hypothesis that microsomal CYP enzymes in airway epithelial cells play a large role in causing injury to airway epithelia following exposure to NA via either inhalation or intraperitoneal route.
• Fan, X., Ding, X., & Zhang, Q. Y. (2020). Hepatic and intestinal biotransformation gene expression and drug disposition in a dextran sulfate sodium-induced colitis mouse model. Acta pharmaceutica Sinica. B, 10(1), 123-135.
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  We examined the impact of gut inflammation on the expression of cytochrome P450 (P450) and other biotransformation genes in male mice using a dextran sulfate sodium (DSS)-induced colitis model. Several P450 isoforms, including CYP1A, CYP2B, CYP2C, and CYP3A, were down-regulated, accompanied by decreases in microsomal metabolism of diclofenac and nifedipine, in the liver and small intestine. The impact of the colitis on clearance of oral drugs varied for four different drugs tested: a small decrease for nifedipine, a relatively large decrease for lovastatin, but no change for pravastatin, and a large decrease in the absorption of cyclosporine A. To further assess the scope of influence of gut inflammation on gene expression, we performed genome-wide expression analysis using RNA-seq, which showed down-regulation of many CYPs, non-CYP phase-I enzymes, phase-II enzymes and transporters, and up-regulation of many other members of these gene families, in both liver and intestine of adult C57BL/6 mice, by DSS-induced colitis. Overall, our results indicate that gut inflammation suppresses the expression of many P450s and other biotransformation genes in the intestine and liver, and alters the pharmacokinetics for some but not all drugs, potentially affecting therapeutic efficacy or causing adverse effects in a drug-specific fashion.
• Zhang, Q. Y., Ding, X., Zhang, Q., Yin, L., Yang, W., Turesky, R. J., Sheng, J., Han, W., & Fan, X. (2020). Generation and characterization of a serum albumin‐humanized mouse model for biomarker discovery. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.04421
• Zhou, J., Zhang, Q. Y., Zhang, J., Xu, J., Trudeau, N., Tharappel, A. M., Samrat, S. K., Rugenstein, N., Li, Z., Li, H., Lang, Y., Kuo, L., Kramer, L. D., Koetzner, C. A., Hu, S., Fan, X., D'brant, L., Chen, H., & Butler, D. (2020). JMX0207, a Niclosamide Derivative with Improved Pharmacokinetics, Suppresses Zika Virus Infection Both In Vitro and In Vivo.. ACS infectious diseases, 6(10), 2616-2628. doi:10.1021/acsinfecdis.0c00217
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  Flaviviruses causes significant human disease. Recent outbreaks of the Zika virus highlight the need to develop effective therapies for this class of viruses. Previously we identified niclosamide as a broad-spectrum inhibitor for flaviviruses by targeting the interface between viral protease NS3 and its cofactor NS2B. Here, we screened a small library of niclosamide derivatives and identified a new analogue with improved pharmacokinetic properties. Compound JMX0207 showed improved efficacy in inhibition of the molecular interaction between NS3 and NS2B, better inhibition of viral protease function, and enhanced antiviral efficacy in the cell-based antiviral assay. The derivative also significantly reduced Zika virus infection on 3D mini-brain organoids derived from pluripotent neural stem cells. Intriguingly, the compound significantly reduced viremia in a Zika virus (ZIKV) animal model. In summary, a niclosamide derivative, JMX0207, was identified, which shows improved pharmacokinetics and efficacy against Zika virus both in vitro and in vivo.
• Ding, X., Zhang, Q. Y., Yang, W., Ding, L., Winkle, L. S., Han, W., Liu, P., Kovalchuk, N., Kovalchuk, N., Liu, P., Han, W., Winkle, L. S., Yang, W., Ding, X., Ding, L., & Zhang, Q. Y. (2019). Generation and characterization of a myeloid cell-specific Cyp19a1 knockout mouse model. The FASEB Journal, 33(S1). doi:10.1096/fasebj.2019.33.1_supplement.508.2
• Fan, X., Li, H., Ding, X., & Zhang, Q. Y. (2019). Contributions of Hepatic and Intestinal Metabolism to the Disposition of Niclosamide, a Repurposed Drug with Poor Bioavailability. Drug metabolism and disposition: the biological fate of chemicals, 47(7), 756-763.
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  Niclosamide, an antiparasitic, has been repositioned as a potential therapeutic drug for systemic diseases based on its antiviral, anticancer, and anti-infection properties. However, low bioavailability limits its in vivo efficacy. Our aim was to determine whether metabolic disposition by microsomal P450 enzymes in liver and intestine influences niclosamide's bioavailability in vivo, by comparing niclosamide metabolism in wild-type, liver-Cpr-null (LCN), and intestinal epithelium-Cpr-null (IECN) mice. In vitro stability of niclosamide in microsomal incubations was greater in the intestine than in liver in the presence of NADPH, but it was much greater in liver than in intestine in the presence of UDPGA. NADPH-dependent niclosamide metabolism and hydroxy-niclosamide formation were inhibited in hepatic microsomes of LCN mice, but not IECN mice, compared with wild-type mice. In intestinal microsomal reactions, hydroxy-niclosamide formation was not detected, but rates of niclosamide-glucuronide formation were ∼10-fold greater than in liver, in wild-type, LCN, and IECN mice. Apparent Km and values for microsomal niclosamide-glucuronide formation showed large differences between the two tissues, with the intestine having higher Km (0.47 M) and higher (15.8) than the liver (0.09 M and 0.75, respectively). In vivo studies in LCN mice confirmed the essential role of hepatic P450 in hydroxy-niclosamide formation; however, pharmacokinetic profiles of oral niclosamide were only minimally changed in LCN mice, compared with wild-type mice, and the changes seem to reflect the compensatory increase in hepatic UDP-glucuronosyltransferase activity. SIGNIFICANCE STATEMENT: These results suggest that efforts to increase the bioavailability of niclosamide by blocking its metabolism by P450 enzymes will unlikely be fruitful. In contrast, inhibition of niclosamide glucuronidation in both liver and intestine may prove effective for increasing niclosamide's bioavailability, thereby making it practical to repurpose this drug for treating systemic diseases.
• Hartog, M., Zhang, Q. Y., & Ding, X. (2019). Role of mouse cytochrome P450 enzymes of the CYP2ABFGS subfamilies in the induction of lung inflammation by cigarette smoke exposure. Toxicological sciences : an official journal of the Society of Toxicology.
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  Many constituents of tobacco smoke (TS) require bioactivation to exert toxic effects; however, few studies have examined the role of bioactivation enzymes in the adverse effects of TS exposure. This knowledge gap is a major source of uncertainty for risk assessment and chemoprevention efforts.
• Jia, K., Zhang, D., Jia, Q., & Zhang, Q. Y. (2019). Regulation of Fgf15 expression in the intestine by glucocorticoid receptor. Molecular medicine reports, 19(4), 2953-2959.
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  Fibroblast growth factor 15 (FGF15) was previously identified to be highly expressed in the ileum and functions as an endocrine factor to regulate bile acid synthesis in the liver. FGF15 targets its receptor fibroblast growth factor receptor 4 in the liver and serves important roles in energy metabolism, including bile acid homeostasis, glucose metabolism and protein synthesis. The expression of FGF15 is known to be regulated by the transcription factor farnesoid X receptor (FXR). In the present study, reverse transcription‑quantitative polymerase chain reaction was used for measuring Fgf15 expression from the animal and tissue culture experiments, and it was identified that dexamethasone, a drug widely used in anti‑inflammation therapy, and a classical inducer of glucocorticoid receptor (GR)‑ and pregnane X receptor (PXR)‑target genes, may downregulate Fgf15 expression in the ileum. GR was identified to be highly expressed in the ileum by western blot analysis. Furthermore, it was demonstrated that the downregulation of Fgf15 by dexamethasone is due to the repression of ileal FXR activity via GR; however, not PXR, in the ileum. The present results provide insight for a better understanding of the adverse effects associated with dexamethasone therapy.
• Kovalchuk, N., Zhang, Q. Y., Kelty, J., Van Winkle, L., & Ding, X. (2019). Toxicokinetic Interaction between Hepatic Disposition and Pulmonary Bioactivation of Inhaled Naphthalene Studied Using -Null and CYP2A13/2F1-Humanized Mice with Deficient Hepatic Cytochrome P450 Activity. Drug metabolism and disposition: the biological fate of chemicals, 47(12), 1469-1478.
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  Previous studies using -null (lacking all genes of the , , , , and subfamilies), CYP2A13/2F1-humanized, and liver--null (LCN) mice showed that although hepatic cytochrome P450 (P450) enzymes are essential for systemic clearance of inhaled naphthalene (a possible human carcinogen), both hepatic and extrahepatic P450 enzymes may contribute to naphthalene-induced lung toxicity via bioactivation. Herein, we aimed to further understand the toxicokinetics of inhaled naphthalene in order to provide a basis for predicting the effects of variations in rates of xenobiotic disposition on the extent of target tissue bioactivation. We assessed the impact of a hepatic deficit in naphthalene metabolism on the toxicokinetics of inhaled naphthalene using newly generated -null-and-LCN and CYP2A13/2F1-humanized-and-LCN mice. We determined plasma, lung, and liver levels of naphthalene and naphthalene-glutathione conjugate, a biomarker of naphthalene bioactivation, over time after naphthalene inhalation. We found that the loss of hepatic naphthalene metabolism severely decreased naphthalene systemic clearance and caused naphthalene to accumulate in the liver and other tissues. Naphthalene release from tissue, as evidenced by the continued increase in plasma naphthalene levels after termination of active inhalation exposure, was accompanied by prolonged bioactivation of naphthalene in the lung. In addition, transgenic expression of human CYP2A13/2F1 in the respiratory tract caused a reduction in plasma naphthalene levels (by 40%, relative to -null-and-LCN mice) and corresponding decreases in naphthalene-glutathione levels in the lung in mice with hepatic P450 deficiency, despite the increase in local naphthalene-bioactivating P450 activity. Thus, the bioavailability of naphthalene in the target tissue has a significant effect on the extent of naphthalene bioactivation in the lung. SIGNIFICANCE STATEMENT: In this study, we report several novel findings related to the toxicokinetics of inhaled naphthalene, the ability of which to cause lung carcinogenesis in humans is a current topic for risk assessment. We show the accumulation of naphthalene in the liver and lung in mice with compromised hepatic cytochrome P450 (P450) activity; the ability of tissue-stored naphthalene to redistribute to the circulation after termination of active inhalation exposure, prolonging exposure of target tissues to naphthalene; and the ability of non-CYP2ABFGS enzymes of the lung to bioactivate naphthalene. These results suggest potentially large effects of deficiencies in hepatic P450 activity on naphthalene tissue burden and bioactivation in human lungs.
• Bissig, K. D., Han, W., Barzi, M., Kovalchuk, N., Ding, L., Fan, X., Pankowicz, F. P., Zhang, Q. Y., & Ding, X. (2018). P450-Humanized and Human Liver Chimeric Mouse Models for Studying Xenobiotic Metabolism and Toxicity. Drug metabolism and disposition: the biological fate of chemicals, 46(11), 1734-1744.
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  Preclinical evaluation of drug candidates in experimental animal models is an essential step in drug development. Humanized mouse models have emerged as a promising alternative to traditional animal models. The purpose of this mini-review is to provide a brief survey of currently available mouse models for studying human xenobiotic metabolism. Here, we describe both genetic humanization and human liver chimeric mouse models, focusing on the advantages and limitations while outlining their key features and applications. Although this field of biomedical science is relatively young, these humanized mouse models have the potential to transform preclinical drug testing and eventually lead to a more cost-effective and rapid development of new therapies.
• Li, L., Zhang, Q. Y., & Ding, X. (2018). A CYP2B6-humanized mouse model and its potential applications. Drug metabolism and pharmacokinetics, 33(1), 2-8.
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  CYP2B6 is a human microsomal cytochrome P450 enzyme with broad substrate selectivity. CYP2B6 is the only functional member of the human CYP2B gene subfamily, which differs from the situation in rodents, such as mouse, where multiple functional Cyp2b genes are expressed. Recent studies with Cyp2b knockout or knockdown mouse models have yielded insights into the in vivo roles of mouse CYP2B enzymes in drug disposition and xenobiotic toxicity. A CYP2B6-humanized mouse model (CYP2A13/2B6/2F1-transgenic/Cyp2abfgs-null), which expresses human CYP2B6 in the liver, and human CYP2A13 and CYP2F1 in the respiratory tract, but not any of the mouse Cyp2b genes, has also been established. In the CYP2B6-humanized mouse, the CYP2B6 transgene is expressed primarily in the liver, where it was found to be active toward prototype CYP2B6 substrate drugs. The regulatory elements of the CYP2B6 transgene appear to be compatible with mouse nuclear receptors that mediate CYP2B induction. Therefore, the CYP2B6-humanized mouse is a valuable animal model for studying the impact of CYP2B6 expression or induction on drug metabolism, drug efficacy, drug-drug interaction, and drug/xenobiotic toxicity. In this mini-review, we provide a brief background on CYP2B6 and the Cyp2b-knockout and CYP2B6-humanized mice, and discuss the potential applications and limitations of the current models.
• Li, Z., Sakamuru, S., Huang, R., Brecher, M., Koetzner, C. A., Zhang, J., Chen, H., Qin, C. F., Zhang, Q. Y., Zhou, J., Kramer, L. D., Xia, M., & Li, H. (2018). Erythrosin B is a potent and broad-spectrum orthosteric inhibitor of the flavivirus NS2B-NS3 protease. Antiviral research, 150, 217-225.
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  Many flaviviruses, such as Zika virus (ZIKV), Dengue virus (DENV1-4) and yellow fever virus (YFV), are significant human pathogens. Infection with ZIKV, an emerging mosquito-borne flavivirus, is associated with increased risk of microcephaly in newborns and Guillain-Barré syndrome and other complications in adults. Currently, specific therapy does not exist for any flavivirus infections. In this study, we found that erythrosin B, an FDA-approved food additive, is a potent inhibitor for flaviviruses, including ZIKV and DENV2. Erythrosin B was found to inhibit the DENV2 and ZIKV NS2B-NS3 proteases with IC in low micromolar range, via a non-competitive mechanism. Erythrosin B can significantly reduce titers of representative flaviviruses, DENV2, ZIKV, YFV, JEV, and WNV, with micromolar potency and with excellent cytotoxicity profile. Erythrosin B can also inhibit ZIKV replication in ZIKV-relevant human placental and neural progenitor cells. As a pregnancy category B food additive, erythrosin B may represent a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.
• Ding, X., Zhang, Q. Y., & Zhang, Q. (2017). In vivo approaches to identification of metabolism-mediated toxicological mechanisms. Drug Metabolism and Pharmacokinetics, 32(1), S6. doi:10.1016/j.dmpk.2016.10.033
• Kovalchuk, N., Kelty, J., Li, L., Hartog, M., Zhang, Q. Y., Edwards, P., Van Winkle, L., & Ding, X. (2017). Impact of hepatic P450-mediated biotransformation on the disposition and respiratory tract toxicity of inhaled naphthalene. Toxicology and applied pharmacology, 329, 1-8.
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  We determined whether a decrease in hepatic microsomal cytochrome P450 activity would impact lung toxicity induced by inhalation exposure to naphthalene (NA), a ubiquitous environmental pollutant. The liver-Cpr-null (LCN) mouse showed decreases in microsomal metabolism of NA in liver, but not lung, compared to wild-type (WT) mouse. Plasma levels of NA and NA-glutathione conjugates (NA-GSH) were both higher in LCN than in WT mice after a 4-h nose-only NA inhalation exposure at 10ppm. Levels of NA were also higher in lung and liver of LCN, compared to WT, mice, following exposure to NA at 5 or 10ppm. Despite the large increase in circulating and lung tissue NA levels, the level of NA-GSH, a biomarker of NA bioactivation, was either not different, or only slightly higher, in lung and liver tissues of LCN mice, relative to that in WT mice. Furthermore, the extent of NA-induced acute airway injury, judging from high-resolution lung histopathology and morphometry at 20h following NA exposure, was not higher, but lower, in LCN than in WT mice. These results, while confirming the ability of extrahepatic organ to bioactivate inhaled NA and mediate NA's lung toxicity, suggest that liver P450-generated NA metabolites also have a significant, although relatively small, contribution to airway toxicity of inhaled NA. This hepatic contribution to the airway toxicity of inhaled NA may be an important risk factor for individuals with diminished bioactivation activity in the lung.
• Li, L., Bao, X., Zhang, Q. Y., Negishi, M., & Ding, X. (2017). Role of CYP2B in Phenobarbital-Induced Hepatocyte Proliferation in Mice. Drug metabolism and disposition: the biological fate of chemicals, 45(8), 977-981.
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  Phenobarbital (PB) promotes liver tumorigenesis in rodents, in part through activation of the constitutive androstane receptor (CAR) and the consequent changes in hepatic gene expression and increases in hepatocyte proliferation. A typical effect of CAR activation by PB is a marked induction of expression in the liver; the latter has been suspected to be vital for PB-induced hepatocellular proliferation. This hypothesis was tested here by using a -null (null) mouse model in which all genes are deleted. Adult male and female wild-type (WT) and null mice were treated intraperitoneally with PB at 50 mg/kg once daily for 5 successive days and tested on day 6. The liver-to-body weight ratio, an indicator of liver hypertrophy, was increased by 47% in male WT mice, but by only 22% in male -null mice, by the PB treatment. The fractions of bromodeoxyuridine-positive hepatocyte nuclei, assessed as a measure of the rate of hepatocyte proliferation, were also significantly lower in PB-treated male null mice compared with PB-treated male WT mice. However, whereas few proliferating hepatocytes were detected in saline-treated mice, many proliferating hepatocytes were still detected in PB-treated male null mice. In contrast, female WT mice were much less sensitive than male WT mice to PB-induced hepatocyte proliferation, and PB-treated female WT and PB-treated female null mice did not show significant difference in rates of hepatocyte proliferation. These results indicate that CYP2B induction plays a significant, but partial, role in PB-induced hepatocyte proliferation in male mice.
• Li, L., Carratt, S., Hartog, M., Kovalchik, N., Jia, K., Wang, Y., Zhang, Q. Y., Edwards, P., Winkle, L. V., & Ding, X. (2017). Human CYP2A13 and CYP2F1 Mediate Naphthalene Toxicity in the Lung and Nasal Mucosa of CYP2A13/2F1-Humanized Mice. Environmental health perspectives, 125(6), 067004.
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  The potential carcinogenicity of naphthalene (NA), a ubiquitous environmental pollutant, in human respiratory tract is a subject of intense debate. Chief among the uncertainties in risk assessment for NA is whether human lung CYP2A13 and CYP2F1 can mediate NA's respiratory tract toxicity.
• Ding, X., Zhang, Q. Y., Zhang, Q., & Xie, F. (2016). An update on the role of intestinal cytochrome P450 enzymes in drug disposition.. Acta pharmaceutica Sinica. B, 6(5), 374-383. doi:10.1016/j.apsb.2016.07.012
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  Oral administration is the most commonly used route for drug treatment. Intestinal cytochrome P450 (CYP)-mediated metabolism can eliminate a large proportion of some orally administered drugs before they reach systemic circulation, while leaving the passage of other drugs unimpeded. A better understanding of the ability of intestinal P450 enzymes to metabolize various clinical drugs in both humans and preclinical animal species, including the identification of the CYP enzymes expressed, their regulation, and the relative importance of intestinal metabolism compared to hepatic metabolism, is important for improving bioavailability of current drugs and new drugs in development. Here, we briefly review the expression of drug-metabolizing P450 enzymes in the small intestine of humans and several preclinical animal species, and provide an update of the various factors or events that regulate intestinal P450 expression, including a cross talk between the liver and the intestine. We further compare various clinical and preclinical approaches for assessing the impact of intestinal drug metabolism on bioavailability, and discuss the utility of the intestinal epithelium-specific NADPH-cytochrome P450 reductase-null (IECN) mouse as a useful model for studying in vivo roles of intestinal P450 in the disposition of orally administered drugs.
• Sheng, J., Wang, Y., Turesky, R. J., Kluetzman, K., Zhang, Q. Y., & Ding, X. (2016). Novel Transgenic Mouse Model for Studying Human Serum Albumin as a Biomarker of Carcinogenic Exposure. Chemical research in toxicology, 29(5), 797-809.
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  Albumin is a commonly used serum protein for studying human exposure to xenobiotic compounds, including therapeutics and environmental pollutants. Often, the reactivity of albumin with xenobiotic compounds is studied ex vivo with human albumin or plasma/serum samples. Some studies have characterized the reactivity of albumin with chemicals in rodent models; however, differences between the orthologous peptide sequences of human and rodent albumins can result in the formation of different types of chemical-protein adducts with different interaction sites or peptide sequences. Our goal is to generate a human albumin transgenic mouse model that can be used to establish human protein biomarkers of exposure to hazardous xenobiotics for human risk assessment via animal studies. We have developed a human albumin transgenic mouse model and characterized the genotype and phenotype of the transgenic mice. The presence of the human albumin gene in the genome of the model mouse was confirmed by genomic PCR analysis, whereas liver-specific expression of the transgenic human albumin mRNA was validated by RT-PCR analysis. Further immunoblot and mass spectrometry analyses indicated that the transgenic human albumin protein is a full-length, mature protein, which is less abundant than the endogenous mouse albumin that coexists in the serum of the transgenic mouse. The transgenic protein was able to form ex vivo adducts with a genotoxic metabolite of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a procarcinogenic heterocyclic aromatic amine formed in cooked meat. This novel human albumin transgenic mouse model will facilitate the development and validation of albumin-carcinogen adducts as biomarkers of xenobiotic exposure and/or toxicity in humans.
• Ding, X., Zhang, Q. Y., Zhang, Q., Sells, S., Mei, C., Liu, S., Li, L., Gu, J., Ding, X., & Dickman, K. G. (2015). An Exploration of the Mechanisms Underlying Proximal Tubular Vacuolization in Mice with Low Expression of the NADPH-Cytochrome P450 Reductase. The FASEB Journal, 29.
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  Cytochrome P450 reductase (CPR or POR) is an obligatory redox partner for all microsomal P450 enzymes. A large, global decrease in POR expression, in a Cpr-low mouse model, was previously found to ...
• Liu, Z., Li, L., Wu, H., Hu, J., Ma, J., Zhang, Q. Y., & Ding, X. (2015). Characterization of CYP2B6 in a CYP2B6-humanized mouse model: inducibility in the liver by phenobarbital and dexamethasone and role in nicotine metabolism in vivo. Drug metabolism and disposition: the biological fate of chemicals, 43(2), 208-16.
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  The aim of this study was to further characterize the expression and function of human CYP2B6 in a recently generated CYP2A13/2B6/2F1-transgenic (TG) mouse model, in which CYP2B6 is expressed selectively in the liver. The inducibility of CYP2B6 by phenobarbital (PB) and dexamethasone (DEX), known inducers of CYP2B6 in human liver, was examined in the TG mice, as well as in TG/Cyp2abfgs-null (or "CYP2B6-humanized") mice. Hepatic expression of CYP2B6 mRNA and protein was greatly induced by PB or DEX treatment in both TG and TG/Cyp2abfgs-null mice. Function of the transgenic CYP2B6 was first studied using bupropion as a probe substrate. In PB-treated mice, the rates of hepatic microsomal hydroxybupropion formation (at 50 μM bupropion) were >4-fold higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice (for both male and female mice); the rate difference was accompanied by a 5-fold higher catalytic efficiency in the TG/Cyp2abfgs-null mice and was abolished by an antibody to CYP2B6. The ability of CYP2B6 to metabolize nicotine was then examined, both in vitro and in vivo. The rates of hepatic microsomal cotinine formation from nicotine were significantly higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice, pretreated with PB or DEX. Furthermore, systemic nicotine metabolism was faster in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice. Thus, the transgenic CYP2B6 was inducible and functional, and, in the absence of mouse CYP2A and CYP2B enzymes, it contributed to nicotine metabolism in vivo. The CYP2B6-humanized mouse will be valuable for studies on in vivo roles of hepatic CYP2B6 in xenobiotic metabolism and toxicity.
• Turesky, R. J., Konorev, D., Fan, X., Tang, Y., Yao, L., Ding, X., Xie, F., Zhu, Y., & Zhang, Q. Y. (2015). Effect of Cytochrome P450 Reductase Deficiency on 2-Amino-9H-pyrido[2,3-b]indole Metabolism and DNA Adduct Formation in Liver and Extrahepatic Tissues of Mice. Chemical research in toxicology, 28(12), 2400-10.
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  2-Amino-9H-pyrido[2,3-b]indole (AαC), a carcinogen formed during the combustion of tobacco and cooking of meat, undergoes cytochrome P450 (P450) metabolism to form the DNA adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). We evaluated the roles of P450 expressed in the liver and intestine to bioactivate AαC by employing male B6 wild-type (WT) mice, liver-specific P450 reductase (Cpr)-null (LCN) mice, and intestinal epithelium-specific Cpr-null (IECN) mice. Pharmacokinetic parameters were determined for AαC, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-OSO3H), and N(2)-(β-1-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N(2)-Glu) with animals dosed by gavage with AαC (13.6 mg/kg). The uptake of AαC was rapid with no difference in the plasma half-lives (t1/2) of AαC, AαC-3-OSO3H, and AαC-N(2)-Glu among mouse models. The maximal plasma concentrations (Cmax) and the areas under concentration-time curve (AUC0-24h) of AαC and AαC-N(2)-Glu were 4-24-fold higher in LCN than in WT mice, but they were not different between WT and IECN mice. These findings are consistent with the ablation of hepatic P450 activity in LCN mice. However, the Cmax and AUC0-24h of AαC-3-OSO3H in plasma were not substantially different among the mouse models. Similar pharmacokinetic parameters were obtained with WT and LCN mice treated with a lower AαC dose (1.36 mg kg(-1)). dG-C8-AαC was detected at similar levels in the livers of all three mouse models at the high AαC dose; levels of dG-C8-AαC in colon, bladder, and lung were greater in LCN than in WT mice and were the same in colon of IECN and WT mice. At the low AαC dose, dG-C8-AαC occurred at ∼ 40% lower levels in liver of LCN mouse than in WT mouse liver, but adduct levels remained higher in extrahepatic tissues of LCN mice. Therefore, hepatic P450 plays an important role in detoxication of AαC, but other hepatic or extrahepatic enzymes contribute to the bioactivation of AαC. P450s expressed in the intestine do not appreciably contribute to bioactivation of AαC in mice.
• Zhu, Y., Xie, F., Ding, L., Fan, X., Ding, X., & Zhang, Q. Y. (2015). Intestinal epithelium-specific knockout of the cytochrome P450 reductase gene exacerbates dextran sulfate sodium-induced colitis. The Journal of pharmacology and experimental therapeutics, 354(1), 10-7.
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  The potential involvement of intestinal microsomal cytochrome P450 (P450) enzymes in defending against colon inflammation and injury was studied in mice treated with dextran sulfate sodium (DSS) to induce colitis. Wild-type (WT) mice and mice with intestinal epithelium (IE)-specific deletion of the P450 reductase gene (IE-Cpr-null) were compared. IE-Cpr-null mice have little microsomal P450 activity in IE cells. DSS treatment (2.5% in drinking water for 6 days) caused more severe colon inflammation, as evidenced by the presence of higher levels of myeloperoxidase and proinflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-6, and IL-1β], and greater weight loss, colonic tissue damage, and colon shortening, in IE-Cpr-null mice than in WT mice. The IE-Cpr-null mice were deficient in colonic corticosterone (CC) synthesis, as indicated by the inability of ex vivo cultured colonic tissues from DSS-treated IE-Cpr-null mice (in contrast to DSS-treated WT mice) to show increased CC production, compared with vehicle-treated mice, and by the ability of added deoxycorticosterone (DOC), a precursor of CC biosynthesis via mitochondrial CYP11B1, to restore ex vivo CC production by colonic tissues from DSS-treated null mice. Intriguingly, null (but not WT) mice failed to show increased serum CC levels following DSS treatment. Nevertheless, cotreatment of DSS-exposed mice with DOC, which did not restore DSS-induced increase in serum CC, abolished the hypersensitivity of IE-Cpr-null mice to DSS-induced colon injury. Taken together, our results strongly support the notion that microsomal P450 enzymes in the intestine play an important role in protecting colon epithelium from DSS-induced inflammation and injury, possibly through increased local CC synthesis in response to DSS challenge.
• Ding, X., Ahlawat, S., D'hondt, R. E., Mantis, N. J., Xie, F., Zhang, Q. Y., Zhang, Q., & Zhu, Y. (2014). Mice deficient in intestinal epithelium cytochrome P450 reductase are prone to acute toxin-induced mucosal damage.. Scientific reports, 4(1), 5551. doi:10.1038/srep05551
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  Cytochrome P450 (P450) enzymes are a superfamily of heme-containing enzymes involved in the metabolism of various endogenous compounds, including retinoids, glucocorticoids, and eicosanoids, that are postulated to participate in the maintenance and/or development of inflammatory and immune reactions in the intestinal mucosa. To investigate the role of P450 enzymes in intestinal inflammation and immunity, we took advantage of IE-Cpr-null mice, which are deficient in intestinal epithelium of NADPH-cytochrome P450 reductase (CPR), the obligate redox partner of all microsomal P450 enzymes. We report that IE-Cpr-null mice, following an acute toxin challenge, had higher levels of pro-inflammatory chemokines and increased tissue damage compared to wild-type mice. IE-Cpr-null mice had normal Peyer's patch numbers and elicited normal secretory IgA (SIgA) responses. However, SIgA baseline levels in IE-Cpr-null mice were consistently elevated over WT littermates. While neither retinoic acid nor glucocorticoid levels in serum and intestinal homogenates were altered in IE-Cpr-null mice, basal levels of arachidonic acid metabolites (11,12-DiHETE and 14,15-DiHETE) with known anti-inflammatory property were significantly lower compared to WT controls. Overall, these findings reveal immunological and metabolic changes resulting from a genetic deficiency in CPR expression in the intestine, and support a role for microsomal P450 enzymes in mucosal homeostasis and immunity.
• Ding, X., Fang, C., Kovalchuk, N., Megaraj, V., Zhang, Q. Y., Zhang, Q., & Zhu, Y. (2014). Role of hepatic and intestinal p450 enzymes in the metabolic activation of the colon carcinogen azoxymethane in mice.. Chemical research in toxicology, 27(4), 656-62. doi:10.1021/tx4004769
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  P450-mediated bioactivation of azoxymethane (AOM), a colon carcinogen, leads to the formation of DNA adducts, of which O(6)-methylguanine (O(6)-mG) is the most mutagenic and contributes to colon tumorigenesis. To determine whether P450 enzymes of the liver and intestine both contribute to AOM bioactivation in vivo, we compared tissue levels of AOM-induced DNA adducts, microsomal AOM metabolic activities, and incidences of colonic aberrant crypt foci (ACF) among wild-type (WT), liver-specific P450 reductase (Cpr)-null (LCN), and intestinal epithelium-specific Cpr-null (IECN) mice. At 6 h following AOM treatment (at 14 mg/kg, s.c.), O(6)-mG and N(7)-mG levels were highest in the liver, followed by the colon, and then small intestine in WT mice. As expected, hepatic adduct levels were significantly lower (by >60%) in LCN mice but unchanged in IECN mice, whereas small-intestinal adduct levels were unchanged or increased in LCN mice but lower (by >50%) in IECN mice compared to that in WT mice. However, colonic adduct levels were unchanged in IECN mice compared to that in WT mice and increased in LCN mice (by 1.5-2.9-fold). The tissue-specific impact of the CPR loss in IECN and LCN mice on microsomal AOM metabolic activity was confirmed by rates of formation of formaldehyde and N(7)-mG in vitro. Furthermore, the incidence of ACF, a lesion preceding colon cancer, was similar in the three mouse strains. Thus, AOM-induced colonic DNA damage and ACF formation is not solely dependent on either hepatic or intestinal microsomal P450 enzymes. P450 enzymes in both the liver and intestine likely contribute to AOM-induced colon carcinogenesis.
• Ding, X., Fang, C., Zhang, Q. Y., Zhang, Q., & Zhu, Y. (2014). Regulation of intestinal cytochrome P450 expression by hepatic cytochrome P450: possible involvement of fibroblast growth factor 15 and impact on systemic drug exposure.. Molecular pharmacology, 85(1), 139-47. doi:10.1124/mol.113.088914
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  Tissue-specific deletion of the gene for NADPH-cytochrome P450 (P450) reductase (CPR), the essential electron donor to all microsomal P450 enzymes, in either liver or intestine, leads to upregulation of many P450 genes in the tissue with the Cpr deletion. Here, by studying the liver-specific Cpr-null (LCN) mouse, we examined whether an interorgan regulatory pathway exists, such that a loss of hepatic CPR would cause compensatory changes in intestinal P450 expression and capacity for first-pass metabolism of oral drugs. We show for the first time that intestinal expression of CYP2B, 2C, and 3A proteins was increased in LCN mice by 2- to 3-fold compared with wild-type (WT) mice, accompanied by significant increases in small intestinal microsomal lovastatin-hydroxylase activity and systemic clearance of oral lovastatin (at 5 mg/kg). Additional studies showed that the hepatic Cpr deletion, which caused large decreases in bile acid (BA) levels in the liver, intestine, plasma, and intestinal content, led to drastic decreases in the mRNA levels of intestinal fibroblast growth factor 15 (FGF15), a target gene of the BA receptor farnesoid X receptor. Furthermore, treatment of mice with FGF19 (the human counterpart of mouse FGF15) abolished the difference between WT and LCN mice in small intestinal (SI) CYP3A levels at 6 hours after the treatment. Our findings reveal a previously unrecognized direct role of intestinal FGF15/19 in the regulation of SI P450 expression and may have profound implications for the prediction of drug exposure in patients with compromised hepatic P450 function.
• Ding, X., Zhang, Q. Y., Zhang, Q., Liu, Z., Li, L., Kluetzman, K., Jia, K., & Hartog, M. (2014). Generation and characterization of a novel CYP2A13--transgenic mouse model.. Drug metabolism and disposition: the biological fate of chemicals, 42(8), 1341-8. doi:10.1124/dmd.114.059188
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  CYP2A13, CYP2B6, and CYP2F1 are neighboring cytochrome P450 genes on human chromosome 19, and the enzymes that they encode overlap in substrate specificity. A CYP2A13/2B6/2F1-transgenic mouse, in which CYP2A13 and 2F1 are both expressed in the respiratory tract and CYP2B6 is expressed in the liver, was recently generated. We generated a CYP2A13 (only) transgenic mouse so that the specific activity of CYP2A13 can be determined. The CYP2B6 and CYP2F1 genes in the CYP2A13/2B6/2F1 genomic clone were inactivated via genetic manipulations, and CYP2A13 was kept intact. A CYP2A13 (only) transgenic (2A13-TG) mouse was generated using the engineered construct and then characterized to confirm transgene integrity and determine copy numbers. The 2A13-TG mice were normal in gross morphology, development, and fertility. As in the CYP2A13/2B6/2F1-transgenic mouse, CYP2A13 expression in the 2A13-TG mouse was limited to the respiratory tract; in contrast, CYP2B6 and 2F1 proteins were not detected. Additional studies using the CYP2A13-humanized (2A13-TG/Cyp2abfgs-null) mouse produced by intercrossing between 2A13-TG and Cyp2abfgs-null mice confirmed that the transgenic CYP2A13 is active in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a lung procarcinogen. The 2A13-TG mouse should be valuable for assessing specific roles of human CYP2A13 in xenobiotic toxicity in the respiratory tract.
• Ding, X., Finn, R. D., Fluck, C. E., Gu, J., Henderson, C. J., Mclaughlin, L. A., Pandey, A. V., Porter, T. D., Riddick, D. S., Ronseaux, S., Wolf, C. R., Zhang, Q. Y., Zhang, Q., & Zou, L. (2013). NADPH-cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology.. Drug metabolism and disposition: the biological fate of chemicals, 41(1), 12-23. doi:10.1124/dmd.112.048991
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  This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b(5), squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b(5) are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b(5) on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.
• Ding, X., Zhang, Q. Y., Zhou, X., Zhang, Q., Yang, W., Wei, Y., Liu, F., Li, L., Kluetzman, K., & Dunbar, A. (2013). Generation and characterization of a novel Cyp2a(4/5)bgs-null mouse model.. Drug metabolism and disposition: the biological fate of chemicals, 41(1), 132-40. doi:10.1124/dmd.112.048736
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  Knockout mouse models targeting various cytochrome P450 (P450 or CYP) genes are valuable for determining P450's biologic functions, including roles in drug metabolism and chemical toxicity. In this study, a novel Cyp2a(4/5)bgs-null mouse model was generated, in which a 1.2-megabase pair genomic fragment containing nine Cyp genes in mouse chromosome 7 (including, sequentially, Cyp2a5, 2g1, 2b19, 2b23, 2a4, 2b9, 2b13, 2b10, and 2s1) are deleted, through Cre-mediated recombination in vivo. The resultant mouse strain was viable and fertile, without any developmental deficits or morphologic abnormalities. Deletion of the constitutive genes in the cluster was confirmed by polymerase chain reaction analysis of the genes and the mRNAs in tissues known to express each gene. The loss of this gene cluster led to significant decreases in microsomal activities toward testosterone hydroxylation in various tissues examined, including olfactory mucosa (OM), lung, liver, and brain. In addition, systemic clearance of pentobarbital was decreased in Cyp2a(4/5)bgs-null mice, as indicated by >60% increases in pentobarbital-induced sleeping time, compared with wild-type (WT) mice. This novel Cyp2a(4/5)bgs-null mouse model will be valuable for in vivo studies of drug metabolism and chemical toxicities in various tissues, including the liver, lung, brain, intestine, kidney, skin, and OM, where one or more of the targeted Cyp genes are known to be expressed in WT mice. The model will also be valuable for preparation of humanized mice that express human CYP2A6, CYP2A13, CYP2B6, or CYP2S1, and as a knockout mouse model for five non-P450 genes (Vmn1r184, Nalp9c, Nalp4a, Nalp9a, and Vmn1r185) that were also deleted.
• Ding, X., Zhang, Q. Y., Zhou, X., Zhang, Q., Zhang, P., Jia, K., & Fang, C. (2013). Dietary regulation of mouse intestinal P450 expression and drug metabolism.. Drug metabolism and disposition: the biological fate of chemicals, 41(2), 529-35. doi:10.1124/dmd.112.049403
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  The study was originally designed to test the hypothesis that the compensatory increase in intestinal P450 (cytochrome P450) expression in the intestinal epithelium-specific P450 reductase (CPR) knockout (IE-Cpr-null) mice was attributable to decreased metabolism of putative P450 inducers present in the diet. Thus, we determined the impact of a dietary change from regular rodent chow to a synthetic diet devoid of phytochemicals on the expression of P450 enzymes in the small intestine (SI) and liver of wild-type (WT) and IE-Cpr-null mice. The dietary change diminished expression of CYP1A, 2B, 2C, and 3A in SI and CYP2B, 2C, and 3A in liver of both WT and IE-Cpr-null mice. However, the compensatory increase in SI P450 expression still occurred in IE-Cpr-null, compared with WT, mice, on the synthetic diet. The diet change-induced decrease in P450 expression was accompanied by decreases in microsomal midazolam-hydroxylase activity in vitro and first-pass clearance of midazolam in vivo in WT mice. Further studies showed that the dietary change, but not Cpr deletion, caused large decreases in bile acid (BA) levels in plasma, liver, SI, and intestinal content and that treatment of WT mice on the synthetic diet with GW4064, a farnesoid-X-receptor agonist, restored the levels of CYP3A expression in both liver and SI to those seen in mice fed with regular chow. Taken together, these results highlight the vital role of diet in maintaining adequate expression of major drug-metabolizing P450s and their associated drug-metabolizing activities in the digestive tract and suggest potential involvement of BA signaling in the regulatory mechanisms.
• Ding, X., D'agostino, J., Fang, C., Jia, K., Spink, D. C., Zhang, Q. Y., Zhang, Q., Zhang, P., & Zhu, Y. (2012). Potential biological functions of cytochrome P450 reductase-dependent enzymes in small intestine: novel link to expression of major histocompatibility complex class II genes.. The Journal of biological chemistry, 287(21), 17777-17788. doi:10.1074/jbc.m112.354274
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  NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine.
• Ding, X., Zhang, Q. Y., Zhou, X., Zhang, X., Zhang, Q., Yao, Y., Wu, H., Weng, Y., Wei, Y., Liu, Z., Ling, G., Li, L., Kluetzman, K., & D'agostino, J. (2012). Generation and characterization of a CYP2A13/2B6/2F1-transgenic mouse model.. Drug metabolism and disposition: the biological fate of chemicals, 40(6), 1144-50. doi:10.1124/dmd.112.044826
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  CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (∼100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (∼0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10- and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (∼0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.
• Zhang, Q. Y., Zhang, Q., & Zhu, Y. (2012). Role of intestinal cytochrome p450 enzymes in diclofenac-induced toxicity in the small intestine.. The Journal of pharmacology and experimental therapeutics, 343(2), 362-70. doi:10.1124/jpet.112.198077
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  The aim of this study was to determine the role of small intestinal (SI) cytochrome P450 (P450) enzymes in the metabolic activation of diclofenac (DCF), a widely used nonsteroidal anti-inflammatory drug, and DCF-induced intestinal toxicity. DCF induces intestinal ulcers in humans and mice, but the underlying mechanisms, including the necessity for drug bioactivation in the target tissues and the sources and identities of reactive intermediates, are not fully understood. We found that the number of DCF-induced (at 50 mg/kg p.o.) intestinal ulcers was significantly smaller in an intestinal epithelium (IE)-specific P450 reductase (CPR) knockout (IE-Cpr-null) mouse model, which has little P450 activity in the IE, than in wild-type (WT) mice, determined at 14 h after DCF administration. The involvement of intestinal P450 enzymes was confirmed by large reductions (>80-90%) in the rates of in vitro formation, in SI microsomal reactions, of hydroxylated DCF metabolites and reactive intermediates, trapped as DCF-glutathione (GSH) conjugates, in the IE-Cpr-null, compared with WT mice. The SI levels of DCF-GSH conjugates (at 4 h after dosing) and DCF-protein adducts (at 14 h after dosing) were significantly lower in IE-Cpr-null than in WT mice. In additional experiments, we found that pretreatment of mice with grapefruit juice, which is known to inhibit SI P450 activity, ameliorated DCF-induced intestinal toxicity in WT mice. Our results not only strongly support the notion that SI P450 enzymes play an important role in DCF-induced intestinal toxicity, but also illustrate the possibility of preventing DCF-induced intestinal toxicity through dietary intervention.
• D'agostino, J., Zhang, Q. Y., Zhang, Q., & Zhu, Y. (2011). Role of intestinal cytochrome P450 (P450) in modulating the bioavailability of oral lovastatin: insights from studies on the intestinal epithelium-specific P450 reductase knockout mouse.. Drug metabolism and disposition: the biological fate of chemicals, 39(6), 939-43. doi:10.1124/dmd.110.037861
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  The extents to which small intestinal (SI) cytochrome P450 (P450) enzymes control the bioavailability of oral drugs are not well defined, particularly for drugs that are substrates for both P450 and the P-glycoprotein (P-gp). In this study, we have determined the role of SI P450 in the clearance of orally administered lovastatin (LVS), an anti-hypercholesterolemia drug, using an intestinal epithelium (IE)-specific P450 reductase knockout (IE-Cpr-null) mouse model. In the IE-Cpr-null mouse, which has little P450 activities in the IE, the oral bioavailability of LVS was substantially higher than that in wild-type (WT) mice (15 and 5%, respectively). In control experiments, the clearance rates were not different between the two strains, either for intraperitoneally dosed LVS, which bypasses SI metabolism, or for orally administered pravastatin, which is known to be poorly metabolized by P450. Thus, our results demonstrate a predominant role of SI P450 enzymes in the first-pass clearance of oral LVS. The absence of IE P450 activities in the IE-Cpr-null mice also facilitated the identification of the molecular targets for orally administered grapefruit juice (GFJ), which is known to inhibit LVS clearance in humans. We found that pretreatment of mice with oral GFJ enhanced the systemic exposure of LVS in WT, but not in IE-Cpr-null mice, a result suggesting that the main target of GFJ action in the small intestine is P450, but not P-gp.
• Ding, X., Zhang, Q. Y., Zhou, X., Zhang, Q., Xie, F., Wei, Y., Roberts, D. W., Laukaitis, C. M., Kluetzman, K., Karn, R. C., & Gu, J. (2011). A novel defensive mechanism against acetaminophen toxicity in the mouse lateral nasal gland: role of CYP2A5-mediated regulation of testosterone homeostasis and salivary androgen-binding protein expression.. Molecular pharmacology, 79(4), 710-23. doi:10.1124/mol.110.070045
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  To identify novel factors or mechanisms that are important for the resistance of tissues to chemical toxicity, we have determined the mechanisms underlying the previously observed increases in resistance to acetaminophen (APAP) toxicity in the lateral nasal gland (LNG) of the male Cyp2g1-null/Cyp2a5-low mouse. Initial studies established that Cyp2a5-null mice, but not a newly generated strain of Cyp2g1-null mice, were resistant to APAP toxicity in the LNG; therefore, subsequent studies were focused on the Cyp2a5-null mice. Compared with the wild-type (WT) male mouse, the Cyp2a5-null male mouse had intact capability to metabolize APAP to reactive intermediates in the LNG, as well as unaltered circulating levels of APAP, APAP-GSH, APAP-glucuronide, and APAP-sulfate. However, it displayed reduced tissue levels of APAP and APAP-GSH and increased tissue levels of testosterone and salivary androgen-binding protein (ABP) in the LNG. Furthermore, we found that ABP was able to compete with GSH and cellular proteins for adduction with reactive metabolites of APAP in vitro. The amounts of APAP-ABP adducts formed in vivo were greater, whereas the amounts of APAP adducts formed with other cellular proteins were substantially lower, in the LNG of APAP-treated male Cyp2a5-null mice compared with the LNG of APAP-treated male WT mice. We propose that through its critical role in testosterone metabolism, CYP2A5 regulates 1) the bioavailability of APAP and APAP-GSH (presumably through modulation of the rates of xenobiotic excretion from the LNG) and 2) the expression of ABP, which can quench reactive APAP metabolites and thereby spare critical cellular proteins from inactivation.
• Ding, X., Zhang, Q. Y., Zhou, X., Zhang, Q., Xie, F., Winkle, L. V., Wei, Y., Li, L., Kluetzman, K., & Hu, J. (2011). Generation and characterization of a Cyp2f2-null mouse and studies on the role of CYP2F2 in naphthalene-induced toxicity in the lung and nasal olfactory mucosa.. The Journal of pharmacology and experimental therapeutics, 339(1), 62-71. doi:10.1124/jpet.111.184671
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  The CYP2F enzymes, abundantly expressed in the respiratory tract, are active toward many xenobiotic compounds, including naphthalene (NA). However, the precise roles of these enzymes in tissue-selective chemical toxicity have been difficult to resolve. A Cyp2f2-null mouse was generated in this study by disrupting the Cyp2f2 fourth exon. Homozygous Cyp2f2-null mice, which had no CYP2F2 expression and showed no changes in the expression of other P450 genes examined, were viable and fertile and had no in utero lethality or developmental deficits. The loss of CYP2F2 expression led to substantial decreases in the in vitro catalytic efficiency of microsomal NA epoxygenases in lung (up to ~160-fold), liver (~3-fold), and nasal olfactory mucosa (OM; up to ~16-fold), and significant decreases in rates of systemic NA (300 mg/kg i.p.) clearance. The Cyp2f2-null mice were largely resistant to NA-induced cytotoxicity, when examined at 24 h after NA dosing (at 300 mg/kg i.p.), and to NA-induced depletion of total nonprotein sulfhydryl (NPSH), examined at 2 h after dosing, in the lungs. In contrast, the loss of CYP2F2 expression did not alleviate NA-induced NPSH depletion or tissue toxicity in the OM. Mouse CYP2F2 clearly plays an essential role in the bioactivation and toxicity of NA in the lung but not in the OM. The Cyp2f2-null mouse should be valuable for studies on the role of CYP2F2 in the metabolism and toxicity of numerous other xenobiotic compounds and for future production of a CYP2F1-humanized mouse.
• Ding, X., Zhang, Q. Y., Zhou, X., Zhang, Q., Yang, W., Wei, Y., Li, L., Kluetzman, K., & Fang, C. (2010). Generation of a mouse model with a reversible hypomorphic cytochrome P450 reductase gene: utility for tissue-specific rescue of the reductase expression, and insights from a resultant mouse model with global suppression of P450 reductase expression in extrahepatic tissues.. The Journal of pharmacology and experimental therapeutics, 334(1), 69-77. doi:10.1124/jpet.110.167411
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  A mouse model termed Cpr-low (CL) was recently generated, in which the expression of the cytochrome P450 reductase (Cpr) gene was globally down-regulated. The decreased CPR expression was accompanied by phenotypical changes, including reduced embryonic survival, decreases in circulating cholesterol, increases in hepatic P450 expression, and female infertility (accompanied by elevated serum testosterone and progesterone levels). In the present study, a complementary mouse model [named reversible-CL (r-CL)] was generated, in which the reduced CPR expression can be reversed in an organ-specific fashion. The neo cassette, which was inserted into the last Cpr intron in r-CL mice, can be deleted by Cre recombinase, thus returning the structure of the Cpr gene (and hence CPR expression) to normal in Cre-expressing cells. All previously identified phenotypes of the CL mice were preserved in the r-CL mice. As a first application of the r-CL model, we have generated an extrahepatic-CL (xh-CL) mouse for testing of the functions of CPR-dependent enzymes in all extrahepatic tissues. The xh-CL mice, generated by mating of r-CL mice with albumin-Cre mice, had normal CPR expression in hepatocytes but down-regulated CPR expression elsewhere. They were indistinguishable from wild-type mice in body and liver weights, circulating cholesterol levels, and hepatic microsomal P450 expression and activities; however, they still showed elevated serum testosterone and progesterone levels and sterility in females. Embryonic lethality was prevented in males, but apparently not in females, indicating a critical role for fetal hepatic CPR-dependent enzymes in embryonic development, at least in males.
• Ding, X., Zhang, Q. Y., Zhang, Q., Zhang, J., Kaminsky, L. S., Fang, C., & Dunbar, D. (2009). An intestinal epithelium-specific cytochrome P450 (P450) reductase-knockout mouse model: direct evidence for a role of intestinal p450s in first-pass clearance of oral nifedipine.. Drug metabolism and disposition: the biological fate of chemicals, 37(3), 651-7. doi:10.1124/dmd.108.025429
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  To determine the in vivo function of intestinal cytochrome P450 (P450) enzymes, we have generated an intestinal epithelium (IE)-specific P450 reductase gene (Cpr) knockout mouse model (designated IE-Cpr-null). In the IE-Cpr-null mouse, CPR expression was abolished in IE cells; however, CPR expression was not altered in other tissues examined. The loss of CPR expression in the small intestine (SI) led to increased expression of several P450 proteins examined, including CYP1A1, CYP2B, CYP2C, and CYP3A. It is interesting to note that the expression of CYP1A1 was also increased in the liver, kidney, and lung of the IE-Cpr-null mice compared with wild-type (WT) littermates, a result strongly supporting the notion that SI metabolism of putative dietary CYP1A1 inducers can influence the systemic bioavailability of these inducers. The rates of SI microsomal metabolism of nifedipine (NFP) in the IE-Cpr-null mice were approximately 10% of the rates in WT littermates, despite the compensatory expression of multiple P450 enzymes in the SI. Furthermore, the area under the concentration-time curve (AUC) values for blood NFP (dosed at 10 mg/kg) levels were 1.6-fold higher in IE-Cpr-null mice than in WT littermates when NFP was given orally; in contrast, the AUC values were comparable for the two strains when NFP was given intravenously. This result directly showed that P450-catalyzed NFP metabolism in the SI plays an important role in the first-pass clearance of oral NFP. Our findings indicate that the IE-Cpr-null mouse model can be used to study the in vivo function of intestinal P450 enzymes in the clearance of oral drugs and other xenobiotics.
• Ding, X., Zhang, Q. Y., Zhang, X., Zhang, Q., Wu, H., Wang, S., Liu, F., Ling, G., & D'agostino, J. (2008). Characterization of CYP2A13*2, a variant cytochrome P450 allele previously found to be associated with decreased incidences of lung adenocarcinoma in smokers.. Drug metabolism and disposition: the biological fate of chemicals, 36(11), 2316-23. doi:10.1124/dmd.108.022822
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  CYP2A13, a human cytochrome P450 enzyme expressed mainly in the respiratory tract, is believed to play an important role in the initiation of smoking-induced lung cancer. CYP2A13.1 has high efficiency in the metabolic activation of a major tobacco-specific carcinogenic nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). CYP2A13(*)2, a variant allele, was previously found to be associated with decreased incidence of lung adenocarcinoma in smokers. The aim of the present study was to determine whether the CYP2A13.2 protein has decreased enzyme activity and/or expression levels in the lung, compared with CYP2A13.1. CYP2A13.2 has two sequence variations from CYP2A13.1: R25Q and R257C. We compared the activities of heterologously expressed CYP2A13.1 and CYP2A13.2 toward several known CYP2A13.1 substrates: NNK, N-nitrosomethylphenylamine, N,N-dimethylaniline, 2'-methoxyacetophenone, and hexamethylphosphoramide. Our results indicated that CYP2A13.2 was 20 to 40% less active than CYP2A13.1 with the substrates tested. We also determined the levels of the CYP2A13(*)2 mRNA, relative to the level of the CYP2A13(*)1 mRNA, in the lung tissue from (*)1/(*)2 heterozygotes. We found that the CYP2A13(*)2 allele was associated with a level of allelic expression approximately 40% lower than that of the CYP2A13(*)1 allele. Sequence analysis of the promoter region of the CYP2A13(*)2 allele identified a 26-nucleotide deletion. Functional analysis of a 2-kilobase pair CYP2A13-luciferase promoter construct indicated that the 26-nucleotide deletion causes decreases in CYP2A13 promoter activity in the A549 human lung cell line. These findings suggest that the reported association of the CYP2A13(*)2 allele with decreased incidences of lung adenocarcinoma in smokers can be at least partly explained by a decrease in CYP2A13 function.
• Ding, X., Dunbar, D., Kaminsky, L. S., Zhang, Q. Y., Zhang, Q., & Zhang, J. (2007). Role of small intestinal cytochromes p450 in the bioavailability of oral nifedipine.. Drug metabolism and disposition: the biological fate of chemicals, 35(9), 1617-23. doi:10.1124/dmd.107.016543
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  To determine the effect of intestinal cytochrome P450 (P450) enzymes on the bioavailability of oral drugs, we have examined the metabolism of nifedipine, an antihypertensive drug and a model substrate of CYP3A4, in mouse models having deficient expression of the NADPH-cytochrome P450 reductase. Initial studies were performed on Cpr-low (CL) mice, which have substantial decreases in Cpr expression in all tissues examined, including the small intestine. In CL mice, area under the concentration-time curve (AUC) values for blood nifedipine after intraperitoneal and oral dosing were 1.8- and 4.0-fold, respectively, higher than in wild-type mice, despite increased expression of multiple P450 enzymes in both liver and intestine. The greater extent of the increase in the AUC value for oral than for intraperitoneal nifedipine suggested that intestinal P450s influence the bioavailability of oral nifedipine, a notion supported by results from further studies on LCN and CL-LCN mice. The LCN mice, which have liver-specific Cpr deletion, had 6.9-fold higher AUC values and 2.2-fold higher C(max) values for blood nifedipine than did wild-type mice after oral nifedipine, consistent with the critical role of hepatic P450s in systemic nifedipine clearance. However, in the CL-LCN mice, which have global decreases in Cpr expression in all tissues examined and Cpr deletion in the liver, AUC and C(max) values for oral nifedipine were, respectively, 2.2- and 1.8-fold higher than in LCN mice, confirming the fact that P450-catalyzed metabolism in the small intestine, the portal-of-entry organ for oral drugs, plays an important role in the first-pass clearance of oral nifedipine.
• Ding, X., Zhang, Q. Y., Zhang, X., Zhang, Q., Wu, H., Weymarn, L. V., Murphy, S. E., & D'agostino, J. (2007). CYP2A13: variable expression and role in human lung microsomal metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.. The Journal of pharmacology and experimental therapeutics, 323(2), 570-8. doi:10.1124/jpet.107.127068
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  CYP2A13 is the most efficient cytochrome P450 enzyme in the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen. The aims of this study were to determine the levels of CYP2A13 protein in human lung microsomes and to ascertain whether CYP2A13 plays any role in lung microsomal NNK metabolic activation. The expression of CYP2A6 and CYP2A13 was examined using a high-resolution immunoblotting method, following immunopurification with an anti-CYP2A5 antibody. We found that, of 116 human lung microsomal samples analyzed, approximately 90% had detectable CYP2A6, whereas only 12% had detectable CYP2A13 with a detection limit of approximately 2 fmol of CYP2A/mg protein. For the majority of microsomal samples analyzed, the level of CYP2A13 was found to be lower than the level of CYP2A6; overall, the highest level of CYP2A13 found ( approximately 20 fmol/mg protein) was approximately 10-fold lower than the highest level of CYP2A6 detected. Quantitative RNA-polymerase chain reaction analysis confirmed that the highly variable expression of the CYP2A proteins was consistent with variations in the levels of the corresponding CYP2A mRNAs in the same tissue samples. It is noteworthy that the level of CYP2A13, but not CYP2A6, was correlated with lung microsomal NNK metabolic activation activity. Furthermore, the addition of 8-methoxypsoralen, a CYP2A inhibitor, led to greater inhibition of NNK metabolic activation in microsomes containing relatively high levels of CYP2A13 than in samples containing no detectable CYP2A13. Taken together, these data indicate that human lung microsomal CYP2A13 is active in NNK metabolic activation. Therefore, individuals having relatively high levels of CYP2A13 expression will likely have an increased risk of developing smoking-related lung cancer.
• Ding, X., Behr, M. J., Cui, H., Fang, C., Gu, J., Kaminsky, L. S., Kluetzman, K., Swiatek, P. J., Weng, Y., Wu, L., Yang, W., Zhang, Q. Y., & Zhang, Q. (2005). Transgenic mice with a hypomorphic NADPH-cytochrome P450 reductase gene: effects on development, reproduction, and microsomal cytochrome P450.. The Journal of pharmacology and experimental therapeutics, 312(1), 35-43. doi:10.1124/jpet.104.073353
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  A mouse model with a hypomorphic NADPH-cytochrome P450 reductase (Cpr) gene (designated Cpr(low) allele) was generated and characterized in this study. The Cpr gene in these mice was disrupted by the insertion of a neo gene in intron 15, which led to 74 to 95% decreases in CPR expression in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver, and heart. In the liver, a pattern of pericentral distribution of CPR protein was preserved in the Cpr(low/low) mice, despite an overall reduction in CPR expression. Genotype distribution in F2 pups indicated limited embryonic lethality associated with the Cpr(low) allele, a finding that confirms the role of CPR-dependent enzymes in development. Adult male homozygotes had decreased body weight and decreased heart, lung, and kidney weights, whereas homozygous Cpr(low) females, which had increased serum testosterone and progesterone and decreased copulatory activities, were infertile. Furthermore, adult Cpr(low/low) mice had decreased plasma cholesterol, and some mice developed mild centrilobular hepatic lipidosis. In addition, despite apparently compensatory increases in total microsomal cytochrome P450 content in the liver and kidney, the decreases in CPR expression were accompanied by reductions in systemic clearance of pentobarbital, as well as in hepatic microsomal metabolism of acetaminophen and testosterone. These phenotypes illustrate the potential impact of a globally decreased CPR activity in human adults, and this novel knock-in mouse model provides a unique opportunity for further explorations of the in vivo roles of CPR and CPR-dependent enzymes.
• Ding, X., Gu, J., Hecht, S. S., Murphy, S. E., Wong, H. L., Zhang, X., Zhang, Q. Y., & Zhang, Q. (2005). Metabolic activation of the tobacco carcinogen 4-(methylnitrosamino)-(3-pyridyl)-1-butanone by cytochrome P450 2A13 in human fetal nasal microsomes.. Chemical research in toxicology, 18(6), 913-8. doi:10.1021/tx0500777
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  Among human P450s studied to date, P450 2A13 is the most efficient catalyst of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-hydroxylation. This reaction is a key bioactivation pathway in NNK-induced carcinogenesis. P450 2A13 mRNA has been detected in human tissues, but it is unknown whether the enzyme is functional in vivo. Therefore, we studied NNK alpha-hydroxylation in human fetal nasal mucosal microsomes, which have been shown to contain high levels of P450 2A protein, presumed to be a mixture of P450 2A6 and 2A13. The microsomes efficiently catalyzed NNK alpha-hydroxylation at the methylene and methyl carbons, as well as carbonyl reduction. Antibodies against mouse P450 2A5 inhibited alpha-hydroxylation by these microsomes greater than 90%. K(m) and V(max) values for alpha-methylene hydroxylation were 6.5 +/- 1.1 muM and 3.0 +/- 0.1 pmol/min/mg; for alpha-methyl hydroxylation, they were 6.7 +/- 0.8 microM and 0.85 +/- 0.03 pmol/min/mg. The K(m) values agree closely with those for NNK metabolism by P450 2A13. Using a new technique, we separated P450 2A13 from P450 2A6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Quantitative immunoblot analysis indicated that the level of P450 2A13 in the pooled fetal nasal microsome sample used for kinetic analysis was approximately 1.6 pmol/mg protein. In the same sample, P450 2A6 was not detected (detection limit, 67 fmol/mg protein). These kinetic, immunoinhibition, and immunoblot data confirm that P450 2A13 is a functional enzyme and the catalyst of NNK alpha-hydroxylation in human fetal nasal mucosa. The results are also the first to demonstrate high efficiency NNK alpha-hydroxylation in a human tissue.
• Ding, X., Zhang, Q. Y., Zhang, Q., Zhang, L., Yang, W., Hinson, J. A., Gu, J., Cui, H., & Behr, M. J. (2005). In vivo mechanisms of tissue-selective drug toxicity: effects of liver-specific knockout of the NADPH-cytochrome P450 reductase gene on acetaminophen toxicity in kidney, lung, and nasal mucosa.. Molecular pharmacology, 67(3), 623-30. doi:10.1124/mol.104.007898
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  Acetaminophen overdose causes toxicity in liver and extrahepatic tissues. Although it is well established that cytochrome P450 enzymes play a critical role in the metabolic activation of acetaminophen, it is not yet clear whether acetaminophen toxicity in extrahepatic tissues is a consequence of hepatic biotransformation. The aim of this study was to determine whether extrahepatic acetaminophen toxicity is altered in a mouse model that has liver-specific deletion of the NADPH-cytochrome P450 reductase (Cpr) gene. Liver-specific Cpr-null (Null) mice were resistant to acetaminophen hepatotoxicity, and they showed faster acetaminophen clearance than did wild-type mice at a toxic acetaminophen dose (400 mg/kg i.p.). However, when circulating acetaminophen levels were made equivalent in the two strains, the severity of extrahepatic acetaminophen toxicity was decreased in the Null relative to that in the wild-type mice in the lung, kidney, and lateral nasal glands, although not in the nasal olfactory and respiratory mucosa. In the lung and liver, the decreased acetaminophen toxicity was accompanied by substantial decreases in the formation of acetaminophen-protein adducts in the Null mice; adducts were not detected in other tissues examined. These results indicate that acetaminophen toxicity in the nasal mucosa is not dependent on hepatic microsomal P450-catalyzed metabolic activation and that acetaminophen toxicity in the lung, kidney, and lateral nasal glands is at least partly caused by liver-derived acetaminophen metabolites.
• Ding, X., Zhang, Q. Y., Zhang, X., Zhang, Q., Weng, Y., Su, T., Schilling, B., Liu, D., Ling, G., Gu, J., & Chen, Y. (2005). Expression of cytochrome p450 and other biotransformation genes in fetal and adult human nasal mucosa.. Drug metabolism and disposition: the biological fate of chemicals, 33(10), 1423-8. doi:10.1124/dmd.105.005769
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  Despite recent progress in the identification and characterization of numerous nasal biotransformation enzymes in laboratory animals, the expression of biotransformation genes in human nasal mucosa remains difficult to study. Given the potential role of nasal biotransformation enzymes in the metabolism of airborne chemicals, including fragrance compounds and therapeutic agents, as well as the potential interspecies differences between laboratory animals and humans, it would be highly desirable to identify those biotransformation genes that are expressed in human nasal mucosa. In this study, a global gene expression analysis was performed to compare biotransformation enzymes expressed in human fetal and adult nasal mucosa to those expressed in liver. The identities of a list of biotransformation genes with apparently nasal mucosa-selective expression were subsequently confirmed by RNA-polymerase chain reaction (PCR) and DNA sequencing of the PCR products. Further quantitative RNA-PCR experiments indicated that, in the fetus, aldehyde dehydrogenase 6 (ALDH6), CYP1B1, CYP2F1, CYP4B1, and UDP glucuronosyltransferase 2A1 are expressed preferentially in the nasal mucosa and that ALDH7, flavin-containing monooxygenase 1, and glutathione S-transferase P1 are at least as abundant in the nasal mucosa as in the liver. The nasal mucosal expression of CYP2E1 was also detected. These findings provide a basis for further explorations of the metabolic capacity of the human nasal mucosa for xenobiotic compounds.
• Ding, X., Zhang, Q. Y., Zhuo, X., Zhang, X., Zhang, Q., Yang, W., Swiatek, P. J., Su, T., Gu, J., D'agostino, J., & Cui, H. (2005). Generation and characterization of a transgenic mouse model with hepatic expression of human CYP2A6.. Biochemical and biophysical research communications, 338(1), 318-24. doi:10.1016/j.bbrc.2005.08.086
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  The aim of this study was to prepare and characterize a transgenic mouse model in which CYP2A6, a human P450 enzyme, is expressed specifically in the liver. CYP2A6, which is mainly expressed in human liver, is active toward many xenobiotics. Our transgene construct contained the mouse transthyretin promoter/enhancer, a full-length CYP2A6 cDNA, and a downstream neomycin-resistance gene for positive selection in embryonic stem cells. Hepatic expression of the CYP2A6 transgene was demonstrated by immunoblotting, whereas tissue specificity of CYP2A6 expression was confirmed by RNA-PCR. The transgenic mouse was further characterized after being backcrossed to the B6 strain for six generations. Hepatic microsomes from homozygous transgenic mice had activities significantly higher than those of B6 mice toward coumarin. The in vivo activity of transgenic CYP2A6 was also determined. Systemic clearance of coumarin was significantly higher in the transgenic mice than in B6 controls, consistent with the predicted role of CYP2A6 as the major coumarin hydroxylase in human liver. The CYP2A6-transgenic mouse model should be valuable for studying the in vivo function of this polymorphic human enzyme in drug metabolism and chemical toxicity.
• Ding, X., Behr, M. J., Collins, D. N., Cui, H., Gu, J., Swiatek, P. J., Xie, Y., Zhang, Q. Y., Zhang, Q., & Zhuo, X. (2004). Targeted disruption of the olfactory mucosa-specific Cyp2g1 gene: impact on acetaminophen toxicity in the lateral nasal gland, and tissue-selective effects on Cyp2a5 expression.. The Journal of pharmacology and experimental therapeutics, 308(2), 719-28. doi:10.1124/jpet.103.060301
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  CYP2G1 is a cytochrome P450 monooxygenase expressed uniquely in the olfactory mucosa (OM). We have generated Cyp2g1-null mice to identify the roles of CYP2G1 in the biology and the tissue-specific toxicity of xenobiotic compounds in the nose. Homozygous Cyp2g1-null mice are viable and fertile; they show no evidence of embryonic lethality, morphological abnormality, or developmental deficits; and they seem to have normal olfactory ability. However, OM microsomes from Cyp2g1-null mice were found to have significantly lower activities than microsomes from wild-type mice in the metabolism of testosterone and progesterone (approximately 60% decrease) and in the metabolic activation of coumarin (>70% decrease). Unexpectedly, a significant reduction in the expression of the Cyp2a5 gene was found in the liver, the lateral nasal gland (LNG), and, to a lesser extent, the kidney of adult Cyp2g1-null mice. The loss of CYP2G1 expression, and the associated decrease in the hepatic expression of CYP2A5, did not decrease systemic clearance, extent of hepatotoxicity, or OM toxicity of acetaminophen (AP). However, the LNG was protected from AP (at 400 mg/kg) toxicity in the Cyp2g1-null mice. Paradoxically, the LNG did not have detectable CYP2G1, and the decrease in LNG CYP2A5 expression in the Cyp2g1-null mice was not accompanied by decreases in microsomal AP metabolism. We hypothesize that OM CYP2G1 (through a paracrine pathway) or LNG CYP2A5 may indirectly influence resistance of the LNG to chemical toxicity, possibly by regulating gene expression in the LNG through steroid hormones or other endogenous P450 substrates and their metabolites.
• Ding, X., Behr, M. J., Cui, H., Gu, J., Weng, Y., Wu, L., Yang, W., Zhang, Q. Y., Zhang, Q., & Zhang, L. (2003). Liver-specific deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase.. The Journal of biological chemistry, 278(28), 25895-901. doi:10.1074/jbc.m303125200
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  A mouse model with liver-specific deletion of the NADPH-cytochrome P450 reductase (Cpr) gene (designated Alb-Cre/Cprlox mice) was generated and characterized in this study. Hepatic microsomal CPR expression was significantly reduced at 3 weeks and was barely detectable at 2 months of age in the Alb-Cre+/-/Cprlox+/+ (homozygous) mice, with corresponding decreases in liver microsomal cytochrome P450 (CYP) and heme oxygenase (HO) activities, in pentobarbital clearance, and in total plasma cholesterol level. Nevertheless, the homozygous mice are fertile and are normal in gross appearance and growth rate. However, at 2 months, although not at 3 weeks, the homozygotes had significant increases in liver weight, accompanied by hepatic lipidosis and other pathologic changes. Intriguingly, total microsomal CYP content was increased in the homozygotes about 2-fold at 3 weeks and about 3-fold at 2 months of age; at 2 months, there were varying degrees of induction in protein (1-5-fold) and mRNA expression (0-67-fold) for all CYPs examined. There was also an induction of HO-1 protein (nearly 9-fold) but no induction of HO-2. These data indicate the absence of significant alternative redox partners for liver microsomal CYP and HO, provide in vivo evidence for the significance of hepatic CPR-dependent enzymes in cholesterol homeostasis and systemic drug clearance, and reveal novel regulatory pathways of CYP expression associated with altered cellular homeostasis. The Alb-Cre/Cprlox mouse represents a unique model for studying the in vivo function of hepatic HO and microsomal CYP-dependent pathways in the biotransformation of endogenous and xenobiotic compounds.
• Ding, X., Caggana, M., Chen, Y., Liu, Y., Ren, X., Zhang, X., Zhang, Q. Y., & Zhang, Q. (2003). Single nucleotide polymorphisms of the human cyp2a13 gene: evidence for a null allele.. Drug metabolism and disposition: the biological fate of chemicals, 31(9), 1081-5. doi:10.1124/dmd.31.9.1081
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  Human CYP2A13 is believed to be important in the metabolic activation of tobacco-specific nitrosamines in the respiratory tract; therefore, genetic polymorphisms of the CYP2A13 gene may be associated with interindividual differences in the risks of tobacco-related tumorigenesis. Our earlier studies identified a frequent single nucleotide polymorphism in CYP2A13 exon 5, Arg257Cys, which led to an approximate 50% decrease in metabolic activities. In the present study, three additional coding region mutations (Arg25Gln, Arg101Stop, and Asp158Glu) and several mutations in the introns and flanking regions were identified in a Chinese patient population. Of particular interest is the Arg101Stop mutation, which was due to a C > T change in exon 2. Thus, individuals homozygous for this nonsense mutation would not have a functional CYP2A13 protein and, therefore, might have reduced sensitivity to xenobiotic toxicity resulting from CYP2A13-mediated metabolic activation in the respiratory tract. The frequencies of the coding region mutations were further examined using random samples of white, black, Hispanic, and Asian newborns from New York. The frequency of the Arg25Gln mutation in Asian newborns (9.6%) was very similar to that found in the Chinese population (10.9%). On the other hand, the Arg101Stop mutation was not detected in 136 newborn samples examined (23 white, 21 black, 19 Hispanic, and 73 Asian), suggesting that this mutation may be unique for the Chinese patient population. Haplotype analysis indicated that the Arg25Gln and Arg257Cys mutations are parts of a common haplotype. However, an additional haplotype that consists of the 25Gln but not the 257Cys allele was also identified.
• Ding, X., Zhang, Q. Y., Liu, Y. Q., Zhang, Q., Su, T., Shi, L., Ren, X., Liu, Y., Liu, D., Gu, J., & Chen, Y. (2003). Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa.. Biochemical pharmacology, 66(7), 1245-51. doi:10.1016/s0006-2952(03)00476-3
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  The aim of the present study was to further characterize the expression of the CYP2A genes in human nasal mucosa. Fetal nasal tissues at 12-26 weeks of gestational age and surgical biopsy tissues from various regions of nasal cavity of adult patients were studied to determine whether CYP2A proteins can be detected by immunoblot in adults, whether higher levels of CYP2A proteins are present in adult than in fetal nasal mucosal microsomes, and whether CYP2A13 mRNA is more abundant than CYP2A6 mRNA in fetal nasal mucosa. In adults, immunoblot analysis detected CYP2A proteins in microsomes of the olfactory region from 8 of 10 individuals, but in none of the nasal microsomes of the respiratory region from 47 patients. Quantitative immunoblot analysis confirmed that CYP2A proteins are selectively expressed in the olfactory region in both adult and fetal tissues. Interestingly, the levels of CYP2A proteins in nasal microsomes were generally higher in fetuses than in adults. In the fetus, the level of CYP2A13 mRNA was much higher than that of CYP2A6 mRNA, as has been previously found in adult nasal mucosa. Immunohistochemical studies confirmed that, in the fetus, the CYP2A proteins are expressed in the supporting cells in the olfactory epithelium and in the Bowman's glands in the lamina propria. The prenatal expression of the CYP2A proteins in the olfactory mucosa suggests potential risks of developmental toxicity from maternally derived xenobiotics, since both CYP2A6 and CYP2A13 are known to be efficient in the metabolic activation of tobacco-specific nitrosamines and other respiratory toxicants.
• Ding, X., Zhang, Q. Y., Zhuo, X., Zhang, Q., Xie, Q., Wu, L., Weng, Y., Swiatek, P., Kluetzman, K., Gu, J., & Behr, M. J. (2003). Conditional knockout of the mouse NADPH-cytochrome p450 reductase gene.. Genesis (New York, N.Y. : 2000), 36(4), 177-81. doi:10.1002/gene.10214
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  NADPH-cytochrome P450 reductase (CPR or POR) is the obligatory electron donor for all microsomal cytochrome P450 (CYP or P450)-catalyzed monooxygenase reactions. Disruption of the mouse Cpr gene has been reported to cause prenatal developmental defects and embryonic lethality. In this study, we generated a mouse model with a floxed Cpr allele (termed Cpr(lox)). Homozygous Cpr(lox) mice are fertile and without any histological abnormality or any change in CPR expression. The floxed Cpr allele was subsequently deleted efficiently by crossing Cpr(lox) mice with transgenic mice having liver-specific Cre expression (Alb-Cre); the result was a decrease in the level of CPR protein in liver microsomes. The Cpr(lox) strain will be valuable for conditional Cpr gene deletion and subsequent determination of the impact of CPR loss on the metabolism of endogenous and xenobiotic compounds, as well as on postnatal development and other biological functions.
• Kaminsky, L. S., Zhang, Q. Y., & Zhang, Q. (2003). The small intestine as a xenobiotic-metabolizing organ.. Drug metabolism and disposition: the biological fate of chemicals, 31(12), 1520-5. doi:10.1124/dmd.31.12.1520
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  The mammalian small intestine serves principally as the site for absorption of nutrients, water, and both beneficial and potentially harmful xenobiotics. However, it has become apparent over the past 20 years, and most notably during the past 10 years, that an array of metabolic machinery is also
• Zhang, Q. Y., Zhang, Q., Kaminsky, L. S., & Dunbar, D. (2003). Characterization of mouse small intestinal cytochrome P450 expression.. Drug metabolism and disposition: the biological fate of chemicals, 31(11), 1346-51. doi:10.1124/dmd.31.11.1346
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  The expression of biotransformation enzymes in mouse small intestine is poorly characterized, which limits the utility of transgenic or knockout mouse models for first-pass drug metabolism studies. In response, we have systematically examined the composition and inducibility of cytochrome P450 (P450) protein and mRNA in mouse small intestinal epithelial cells (enterocytes). RNA-PCR was conducted to confirm the expression and identity of CYP1A1, 1B1, 2B10, 2B19, 2B20, 2C29, 2C38, 2C40, 2E1, 3A11, 3A13, 3A16, 3A25, and 3A44 in the enterocytes of untreated mice, but CYP1A2, 2A4/5, 2A12, 2C37, 2C39, and 2F2 were not detected. The inducibility of CYP2B, 2C, and 3A subfamily forms was determined by real-time quantitative RNA-PCR. All five CYP3A forms were induced, in a range from 1.7- to 4.5-fold, by dexamethasone (DEX). Phenobarbital (PB) induced CYP2B9, CYP2B10, and CYP2B20 mRNAs and suppressed CYP2B19 mRNA levels. PB also induced CYP2C29 and CYP2C40, but not CYP2C38 mRNA. At the protein level, CYP1A1, CYP1B1, CYP2B, CYP2C, CYP2E1, and CYP3A were detected in enterocytes from untreated mice by immunoblot analysis. CYP1A1 was inducible by beta-naphthoflavone (BNF), CYP2B and CYP2C by PB, and CYP3A by DEX. CYP2B, 2C, and 3A proteins were all expressed at high levels proximally, and decreased distally. The inducibility of CYP1A1 followed a similar pattern. Intestinal P450 expression was compared between C57BL/6 (B6) and 129/sv (129) mice, strains commonly used in the preparation of transgenic and knockout mouse models. There was no significant strain difference in constitutive levels or induction patterns for CYP2B, 2C, and 3A protein. However, CYP1A1 was induced to a high level by BNF in B6 mice, but was not induced in the 129 mice.
• Ding, X., Su, T., Swiatek, P., Zhang, Q. Y., Zhang, Q., & Zhang, J. (2002). Expression of the rat CYP2A3 gene in transgenic mice.. Drug metabolism and disposition: the biological fate of chemicals, 30(5), 548-52. doi:10.1124/dmd.30.5.548
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  Rat CYP2A3 and mouse CYP2A5 are predominantly expressed in the olfactory mucosa. CYP2A3 is also expressed in the lung at a low level, whereas CYP2A5 is expressed in several additional tissues. To better understand the transcriptional regulation of the CYP2A genes, transgenic mice were generated with a full-length CYP2A3 gene fragment containing 3.4 kilobases of the 5'-flanking region. CYP2A3 mRNA was detected in the brain and olfactory bulb in four transgenic mouse lines, in the olfactory mucosa in three lines, and in kidney, liver, lung, and small intestine in two lines. Thus, the expression of the CYP2A3 transgene mimicked the tissue distribution pattern of mouse CYP2A5 rather than that of rat CYP2A3. Furthermore, the levels of CYP2A3 mRNA were very low in all lines examined, suggesting that more distal regulatory regions may be involved in the abundant expression of the CYP2A genes in the olfactory mucosa.
• Ding, X., Zhang, Q. Y., Zhang, X., Zhang, Q., Su, T., Li, H., Gu, J., & Caggana, M. (2002). Genetic polymorphisms of the human CYP2A13 gene: identification of single-nucleotide polymorphisms and functional characterization of an Arg257Cys variant.. The Journal of pharmacology and experimental therapeutics, 302(2), 416-23. doi:10.1124/jpet.302.2.416
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  Human cytochrome P450 2A13 (CYP2A13), which is highly efficient in the metabolic activation of a major tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), may play important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the respiratory tract. The aim of this study was to identify any genetic polymorphisms of the CYP2A13 gene, which may alter the metabolic capacities of the enzyme. Polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was used to identify single-nucleotide polymorphisms (SNPs) in all of the exons and at the exon-intron boundaries, and PCR-restriction fragment length polymorphism analysis and DNA sequencing were used to determine the frequencies of the newly identified variant alleles in the four major ethnic groups. Blood spot DNA from more than 100 individuals was used for these analyses. Seven variant alleles were found, but only one SNP was detected in the coding region, in exon 5, leading to an Arg257Cys amino acid change. The frequencies of the Arg257Cys allele in white, black, Hispanic, and Asian individuals are 1.9%, 14.4%, 5.8%, and 7.7%, respectively. Functional analysis of the variant protein was performed following its heterologous expression. The Arg257Cys variant was 37 to 56% less active than the wild-type Arg-257 protein toward all substrates tested. With NNK, Cys-257 had higher K(m) and lower V(max) values than did Arg-257, with a >2-fold decrease in catalytic efficiency. The Arg257Cys mutation could provide some protection against xenobiotic toxicity in the respiratory tract to individuals who are homozygous for the Cys-257 allele.
• Zhang, Q. Y., Bessette, E. E., Fasco, M. J., Kaminsky, L. S., Liu, N., Pause, R., Vakharia, D. D., & Zhang, Q. Y. (2001). Polycyclic aromatic hydrocarbon/metal mixtures: effect on PAH induction of CYP1A1 in human HEPG2 cells.. Drug metabolism and disposition: the biological fate of chemicals, 29(7), 999-1006.
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  Environmental polycyclic aromatic hydrocarbons (PAHs) and metals coexist, and such mixtures could affect the carcinogenicity of PAHs, possibly by modification of PAH induction of the PAH-bioactivating CYP1A. The effect on PAH-mediated CYP1A induction of arsenic, lead, mercury, or cadmium (ranked as the most hazardous environmental metals by the Environmental Protection Agency and the Agency for Toxic Substances and Disease Registry) has thus been investigated. Induction of CYP1A1 by benzo[a]pyrene (BAP), benzo[b]fluoranthene (BBF), dibenzo[a,h]anthracene (DBAHA), benzo[a]anthracene (BAA), or benzo[k]fluoranthene (BKF) was probed by ethoxyresorufin-O-deethylase activity (EROD) in 96-well plates of human HepG2 cells, by immunoblot analysis, and by reverse transcription-polymerase chain reaction. Cells rapidly took up PAHs (BAP, BKF) from medium; by 24 h only 14% remained in the medium, and no detectable PAH bound to well walls. Induction efficiency (relative to dimethyl sulfoxide controls) was in the order BKF (16-fold) > DBAHA (14-fold) > BAA (4-fold) > BAP (3-fold) > BBF (1-fold), all at 5 microM PAH. The metals did not markedly affect cell viability at concentrations of arsenic, 5 microM; lead, 50 microM; mercury, 5 microM; and cadmium, 5 microM. At 5 microM PAH concentration, all of the metals decreased levels of PAH-induced CYP1A1 activities (direct inhibition of EROD activity was excluded) by variable extents and in a PAH-dependent manner. With BAP as inducer decreases in induction were arsenic, 57%; cadmium, 82%; mercury, 4%; and lead, 20%. The decreases were not a consequence of transcriptional down-regulation. One possible conclusion is that these metals could diminish PAH carcinogenic potential by decreasing PAH-mediated induction of their bioactivation by CYP1A1.
• Zhang, Q. Y., Dunbar, D., Kaminsky, L. S., Liu, N., Vakharia, D. D., & Zhang, Q. Y. (2001). Induction of CYP1A by benzo[k]fluoranthene in human hepatocytes: CYP1A1 or CYP1A2?. Archives of biochemistry and biophysics, 389(1), 130-4. doi:10.1006/abbi.2001.2323
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  While fresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by, e.g., polycyclic aromatic hydrocarbons is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis makes resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris-HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat anti-human CYP1A1/CYP1A2 and rabbit anti-human CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocyte cultures induced with 5 microM benzo[k]fluoranthene (BKF) for 24 h. In six of the cultures only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocyte cultures treated individually with BKF and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay (Drug Metab. Disp. (1995) 23, 1339-1345) was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism-such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures.
• Zhang, Q. Y., Dunbar, D., Kaminsky, L. S., Obach, R. S., & Zhang, Q. Y. (2001). Metabolic characterization of the major human small intestinal cytochrome p450s.. Drug metabolism and disposition: the biological fate of chemicals, 29(3), 347-52.
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  Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. CYP3A4 is the major form of CYP expressed in enterocytes and CYP2C is also expressed at a significant level. In this study, we further characterized the expression of CYP3A4 and CYP2C in human enterocytes and their interindividual variations by examining the metabolic activities from 10 individuals. CYP3A4 in human jejunum microsomes, as determined by 6beta-testosterone hydroxylase activity, varied from 0.36 to 2.46 nmol/min/mg. The apparent average K(m) and V(max) values from two representative individuals were 54 microM and 3.2 nmol/min/mg, respectively. CYP2C9 and CYP2C19 in human jejunum microsomes, as determined by diclofenac 4'-hydroxylase and mephenytoin 4'-hydroxylase activities, varied over an 18-fold range (7.3-129 pmol/min/mg) and 17-fold range (0.8-13.1 pmol/min/mg), respectively. The mean apparent K(m) for diclofenac 4'-hydroxylase was 9.9 microM , whereas the apparent mean K(m) for S-mephenytoin 4'-hydroxylase was 79.3 microM . The mean intrinsic clearance (V(max)/K(m)) was approximately 130-fold greater for diclofenac 4'-hydroxylase than for mephenytoin 4'-hydroxylase. The metabolic activities of CYP2C9 and CYP2C19 were confirmed by inhibition by sulfaphenazole for CYP2C9 and ticlopidine for CYP2C19. In addition, CYP2C9 activities did not correlate with CYP3A4 activities, while CYP2C19 activities had a significant but poor correlation with those of CYP3A4. Thus the major CYP activities in human enterocytes have large interindividual variabilities that are not strongly related.
• Zhang, Q. Y., Zhang, Q. Y., Vakharia, D. D., Pause, R., Liu, N., Kaminsky, L. S., Fasco, M. J., & Bessette, E. (2001). Effect of metals on polycyclic aromatic hydrocarbon induction of CYP1A1 and CYP1A2 in human hepatocyte cultures.. Toxicology and applied pharmacology, 170(2), 93-103. doi:10.1006/taap.2000.9087
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  Environmental cocontamination by polycyclic aromatic hydrocarbons (PAHs) and metals could affect the carcinogenic consequences of PAH exposure by modifying PAH induction of PAH-bioactivating CYP1A. The effect of As, Pb, Hg, or Cd (ranked as the most hazardous environmental metals by EPA and ATSDR) on CYP1A1 and 1A2 induction by benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), dibenzo[a,h]anthracene (DBahA), benzo[a]anthracene (BaA), and benzo[k]fluoranthene (BkF) has thus been investigated in fresh human hepatocyte cultures. Induction was probed by ethoxyresorufin-O-deethylase activity, by immunoblots, and by RT-PCR. Uptake of PAHs into the hepatocytes varied according to PAH and liver donor: 84% of 5 microM BaA and 25-40% of 5 microM DBahA was taken up in 24 h. Hepatocytes retained viability up to 1 microM Cd and 5 microM Pb, Hg, or As and 5 microM PAHs. PAH induction of CYP1A in hepatocytes was variable, some cultures expressed CYP1A1 and others CYP1A1 and 1A2, and to variable extents. Induction efficiency (relative to DMSO controls) at 2.5 microM PAH concentration was in the order BkF (7.6-fold) > DBahA (6.1 fold) > BaP (5.7 fold) > BbF (3.9-fold) > BaA (2.5-fold). All four metals (1-5 microM) decreased CYP1A1/1A2 induction by some of the PAHs with dose-, metal-, and PAH-dependency. Arsenic (5 microM) decreased induction by 47% for BaP, 68% for BaA, 45% for BbF, 79% for BkF, and 53% for DBahA. Induced CYP1A2 protein was much more extensively decreased than 1A1 protein, and CYP1A2 mRNA and, to variable extents, CYP1A1 mRNA were decreased by As. Thus the metals in PAH/metal mixtures could diminish PAH carcinogenicity by decreasing induction of their bioactivation by CYP1A1/1A2.
• Ding, X., Bao, Z., Hong, J. Y., Smith, T. J., Su, T., & Zhang, Q. Y. (2000). Human cytochrome P450 CYP2A13: predominant expression in the respiratory tract and its high efficiency metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.. Cancer research, 60(18), 5074-9.
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  The human CYP2A subfamily comprises three genes, CYP2A6, CYP2A7, and CYP2A13. CYP2A6 is active toward many carcinogens and is the major coumarin 7-hydroxylase and nicotine C-oxidase in the liver, whereas CYP2A7 is not functional. The function of CYP2A13 has not been characterized. In this study, a CYP2A13 cDNA was prepared by RNA-PCR from human nasal mucosa and was translated using a baculovirus expression system. In a reconstituted system, the expressed CYP2A13 was more active than CYP2A6 in the metabolic activation of hexamethylphosphoramide, N,N-dimethylaniline, 2'-methoxyacetophenone, and N-nitrosomethylphenylamine but was much less active than CYP2A6 in coumarin 7-hydroxylation. Of particular interest, CYP2A13 was highly active in the metabolic activation of a major tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with a catalytic efficiency much greater than that of other human cytochrome P450 isoforms examined previously. The tissue distribution of CYP2A13 was determined with isoform-specific RNA-PCR. CYP2A13 mRNA was detected in liver and a number of extrahepatic tissues, including nasal mucosa, lung, trachea, brain, mammary gland, prostate, testis, and uterus, but not in heart, kidney, bone marrow, colon, small intestine, spleen, stomach, thymus, or skeletal muscle. Quantitative PCR analysis further revealed that CYP2A13 mRNA is expressed at the highest level in the nasal mucosa, followed by the lung and the trachea. Together, these findings suggest that CYP2A13 plays important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the human respiratory tract.
• Zhang, Q. Y., Ding, X., Chen, Y., Gu, J., Su, T., & Zhang, Q. Y. (2000). Expression of biotransformation enzymes in human fetal olfactory mucosa: potential roles in developmental toxicity.. Toxicology and applied pharmacology, 165(2), 158-62. doi:10.1006/taap.2000.8923
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  High levels of cytochrome P450 are present in the olfactory mucosa (OM) in mammalian animals and contribute to the known tissue-selective toxicity of numerous chemical compounds. Olfactory toxicity in the perinatal period may have a greater impact on behavior, growth, and development than in adults. To establish a molecular basis for determining the risk of developmental toxicity in OM, the expression of several cytochrome P450 enzymes, as well as NADPH-cytochrome P450 reductase and microsomal epoxide hydrolase, was examined in hepatic and nasal microsomes prepared from human fetal tissues at gestational day 91-125. The relative microsomal concentrations of these biotransformation enzymes were determined on immunoblots. Expression of CYP2A, CYP2J2, the reductase, and epoxide hydrolase was detected in both OM and liver. The microsomal levels of these enzymes were generally lower in OM than in liver of the same fetuses, except for the CYP2A-related proteins, which were expressed in OM at much higher levels. OM expression of CYP2A6, CYP2A13, CYP2B6, and CYP2J2 mRNAs was detected using RNA-PCR. These results document, for the first time, prenatal expression of xenobiotic-bioactivating cytochrome P450 enzymes in human OM and suggest that the human fetal OM may be a preferred target tissue for the toxicity of maternally derived chemical compounds that are activated by the CYP2A enzymes.
• Zhang, Q. Y., Ding, X., Gu, J., Kaminsky, L. S., Su, T., Xie, Q., Zhang, Y., & Zhang, Q. Y. (2000). Induction of mouse CYP2J by pyrazole in the eye, kidney, liver, lung, olfactory mucosa, and small intestine, but not in the heart.. Drug metabolism and disposition: the biological fate of chemicals, 28(11), 1311-6.
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  We have recently shown that rat CYP2J4 is inducible by pyrazole in liver, small intestine, and olfactory mucosa. The aim of the present study was to determine whether mouse CYP2Js are also inducible by pyrazole, which was known to induce CYP2A5 in mouse liver and kidney, but not in lung or olfactory mucosa. CYP2J proteins were detected in mouse liver, lung, kidney, heart, eye, olfactory mucosa, and small intestine by immunoblot analysis with an anti-CYP2J4 antibody. The microsomal level of the CYP2J4-related P450s in various mouse tissues ranked in the order of small intestine > olfactory mucosa > liver > kidney > or = heart > lung > eye. Induction of the CYP2J proteins was observed in the eye, liver, lung, kidney, olfactory mucosa, and small intestine, but not in the heart, after daily i.p. injection of pyrazole at 120 or 200 mg/kg for 3 days. CYP2J proteins were induced similarly in C57BL/6 and DBA/2 mice. CYP2A5 was detected in the small intestine in addition to liver and olfactory mucosa; however, treatment with pyrazole induced CYP2A5 in the liver, but not in the olfactory mucosa or the small intestine. Induction of CYP2J mRNAs was also observed by RNA blot analysis with a CYP2J4 cDNA probe. RNA-polymerase chain reaction analysis showed that, in both untreated and pyrazole-treated mice, CYP2J5 was expressed in the kidney and liver, but not in the other tissues examined, whereas CYP2J6 was detected in all tissues examined. The different tissue selectivities in CYP2A5 and CYP2J induction by pyrazole suggest involvement of different regulatory mechanisms.
• Zhang, Q. Y., Ding, X., Guo, J., Zhang, Q. Y., Zhang, J., & Zhou, Y. (2000). Identification and functional characterization of a conserved, nuclear factor 1-like element in the proximal promoter region of CYP1A2 gene specifically expressed in the liver and olfactory mucosa.. The Journal of biological chemistry, 275(12), 8895-902. doi:10.1074/jbc.275.12.8895
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  CYP1A2 is a major cytochrome P-450 isoform in the liver and the olfactory mucosa but is essentially not expressed in other tissues. A nuclear factor 1 (NF-1) -like element was identified in the proximal promoter region of rat, mouse, rabbit, and human CYP1A2 genes through data base analysis. In vitro DNase I footprinting with a -211 to +81 probe from the rat CYP1A2 gene and nuclear extracts from rat liver and olfactory mucosa revealed a single protected region corresponding to the NF-1-like element at -129 to -111. Protein binding to this NF-1-like element was tissue-selective and was confirmed by in vivo footprinting in native chromatin from rat liver. Multiple DNA-binding complexes were detected in gel-shift assays using the CYP1A2 NF-1-like element and nuclear extracts from liver and olfactory mucosa, all of which were supershifted in the presence of an anti-NF1 antibody. The NF-1-like element was essential for transcriptional activity of the CYP1A2 gene in an in vitro transcription assay using nuclear extracts from the two tissues. Thus, members of the NF-1 family of transcription factors may play an important role in the tissue-selective expression of the CYP1A2 gene in the liver and olfactory mucosa.
• Zhang, Q. Y., Zhang, Q. Y., Kaminsky, L. S., & Dunbar, D. (2000). Human cytochrome P-450 metabolism of retinals to retinoic acids.. Drug metabolism and disposition: the biological fate of chemicals, 28(3), 292-7.
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  Retinoic acids have important pleiotropic biological effects and thus the potential for human cytochrome P-450s (CYPs) to mediate retinoic acid synthesis was investigated. We examined the retinoic acid synthetic activity of human cDNA-expressed CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A4+ cytochrome b(5) (b(5)), 3A5, and 4A11, expressed individually in insect cells together with NADPH-P-450 reductase. Only CYP1A1, 1A2, 1B1, and 3A4+b(5) converted all-trans-retinal (20 microM) to all-trans-retinoic acid with turnover numbers of 0.53, 0.18, 0.20, and 0.41 nmol/min/nmol P-450, respectively. With 9-cis-retinal as substrate, CYP1A2 exhibited a turnover number of 1.58 nmol/min/nmol P-450 whereas CYP1A1, 2C19, and 3A4+b(5) had turnover numbers of 0.40, 0.27, and 0.41 nmol/min/nmol P-450, respectively. For CYP3A4 activities with both retinals, b(5) was required. Kinetic analyses revealed that CYP1A1, 1A2, and 3A4+b(5) with all-trans-retinal had apparent K(m) values of 55, 356, and 255 microM, and V(max) values of 2.0, 8.3, and 6.3 nmol/min/nmol P-450, respectively, and with 9-cis-retinal had K(m) values of 77, 91, and 368 microM, and V(max) values of 2.7, 9.7, and 7.6 nmol/min/nmol P-450, respectively. The 9-cis retinoic acid synthetic activity of a group of 12 human liver microsomes correlated only with the CYP1A2 activity (r = 0.96), implicating CYP1A2 in human liver microsomal metabolism of 9-cis- retinal to 9-cis-retinoic acid. These studies have indicated that human CYPs are capable of catalyzing retinal to retinoic acid metabolism, but the physiological relevance of this metabolism is still unclear.
• Zhang, Q. Y., & Zhang, Q. Y. (1999). Retinoic acid biosynthetic activity and retinoid receptors in the olfactory mucosa of adult mice.. Biochemical and biophysical research communications, 256(2), 346-51. doi:10.1006/bbrc.1999.0320
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  Retinoids play important roles in the ontogenic development of the olfactory system in mammals, but their function in adult olfactory mucosa has not been explored. In the present study, the olfactory mucosal expression of nuclear retinoid receptors was examined in adult mice. Several retinoic acid receptor isotypes were identified in olfactory mucosa from adult C57BL/6 mice by RNA-PCR and DNA sequence analysis, including RARbeta, RXRalpha, RXRbeta, and RXRgamma. In addition, a previously unidentified mouse RXRbeta isoform containing a 12-nucleotide insertion in exon 7 was detected. Furthermore, in vitro metabolic studies demonstrated that olfactory mucosal cytosolic and microsomal preparations are active in the biosynthesis of retinoic acids from all-trans- and 9-cis-retinal. These results indicate that components of the retinoid signal transduction system are expressed in adult olfactory mucosa and may play important roles in gene regulation in this unique tissue where olfactory neuronal cells are continuously replaced.
• Zhang, Q. Y., Ding, X., Zhang, Q. Y., Su, T., Spink, D. C., Moss, R. L., Gu, J., & Dudley, C. A. (1999). Cytochrome P450 and steroid hydroxylase activity in mouse olfactory and vomeronasal mucosa.. Biochemical and biophysical research communications, 266(1), 262-7. doi:10.1006/bbrc.1999.1807
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  The aims of this study are to identify the sex steroid-metabolizing cytochrome P450 enzymes of the vomeronasal organ (VNO) and to determine the activities of VNO microsomes to metabolize estradiol, progesterone, and testosterone. Several P450 isoforms, including CYP1A2, CYP2A, CYP2B, CYP2C, CYP2G1, and CYP3A, NADPH P450-reductase, and microsomal epoxide hydrolase were detected in mouse VNO, although their expression levels were much lower than those in the main olfactory epithelium. VNO microsomes were active toward the three steroid hormones, producing metabolite profiles similar to those seen with olfactory mucosal microsomes. Thus, the mammalian VNO, a steroid hormone target tissue, contains multiple steroid-metabolizing P450 isoforms and is capable of metabolic disposition of the three major sex steroid hormones. These findings support the proposed roles of olfactory mucosal and VNO microsomal P450 enzymes in maintaining cellular hormonal homeostasis and other perireceptor processes associated with olfactory chemosensory function.
• Zhang, Q. Y., Ding, X., Zhuo, X., Zhang, Q. Y., Spink, D. C., Kaminsky, L. S., & Gu, J. (1999). Biotransformation of coumarin by rodent and human cytochromes P-450: metabolic basis of tissue-selective toxicity in olfactory mucosa of rats and mice.. The Journal of pharmacology and experimental therapeutics, 288(2), 463-71.
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  Coumarin was previously found to cause tissue-selective toxicity in the olfactory mucosa (OM) of rats and mice, with rats being the more sensitive species. The aim of this study was to explore the role of target tissue biotransformation in OM-selective toxicity and the metabolic basis of the species differences in coumarin toxicity. At least six coumarin metabolites were detected in OM microsomal reactions, with o-hydroxyphenylacetaldehyde (o-HPA) being the most abundant. Formation of o-HPA was inhibited by reduced glutathione, confirming its origin from a reactive intermediate. There were significant differences in the rates and metabolite profiles of coumarin metabolism in the livers of Wistar rats and C57BL/6 mice. The rates of metabolic activation of coumarin, as indicated by the formation of o-HPA, were comparable in OM microsomes of the two species but about 25- and 3-fold higher in OM than in liver microsomes of rats and mice, respectively. Thus, target tissue activation seems to play an important role in the tissue-selective toxicity, whereas differences in the rates of hepatic metabolism may be responsible for the species difference in olfactory toxicity. Purified, heterologously expressed mouse CYP2A5 and CYP2G1 produced 7-hydroxycoumarin and o-HPA as the predominant products, respectively. Kinetic analysis and immunoinhibition studies indicated that the OM-specific CYP2G1 plays the major role in metabolic activation of coumarin. Furthermore, of 13 human cytochrome P-450s (P-450s) examined, five (CYP1A1, CYP1A2, CYP2B6, CYP2E1, and CYP3A4) were active in the metabolic activation of coumarin, suggesting a potential risk of coumarin toxicity in humans.
• Zhang, Q. Y., Zhang, Q. Y., Zeisloft, S., Yang, J., Ostrowska, A., Kaminsky, L. S., & Dunbar, D. (1999). Characterization of human small intestinal cytochromes P-450.. Drug metabolism and disposition: the biological fate of chemicals, 27(7), 804-9.
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  Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P-450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. The CYP composition of enterocytes could thus affect the potential toxicity or therapeutic efficacy of xenobiotics by modifying systemic uptake. We have characterized human enterocyte CYP composition to enable assessment of its functional roles. An isolation method for enterocytes from human small intestine was developed using EDTA buffer-mediated elution. Villous enterocytes were isolated in high yield, separated from crypt cells. Reverse transcriptase-polymerase chain reaction of total RNA from enterocytes revealed that CYP1A1, 1B1, 2C, 2D6, 2E1, 3A4, and 3A5 mRNA were expressed, but only CYP2C and 3A4 were detectable by Western immunoblotting in enterocyte microsomes from 10 human small intestines, whereas CYP1A1 was weakly detectable in two of eight intestines tested. Microsomal protein content decreased markedly along the small intestine from the duodenum to the ileum, whereas total CYP content and CYP3A4 erythromycin N-demethylase activity increased slightly in progressing from the duodenum to the jejunum and then decreased markedly toward the ileum. Levels of CYP3A4 and 2C protein did not decrease in concert as a function of length along the intestine distally. Maximal CYP content for the 10 intestines varied from 0.06 to 0.18 nmol/mg microsomal protein and maximal CYP3A4 erythromycin N-demethylase activity varied from 0.30 to 0.76 nmol/min/mg microsomal protein. In conclusion, CYP3A4 is the major form of CYP expressed in human small intestine enterocytes, CYP3A5 expression was not detected, CYP2C and, in some intestines, CYP1A1 were expressed. The highest metabolic activity occurred in the proximal intestine.
• Zhang, Q. Y., Ding, X., Genter, M. B., Gu, J., Lipinskas, T. W., Nebert, D. W., Negishi, M., & Zhang, Q. Y. (1998). Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes.. The Journal of pharmacology and experimental therapeutics, 285(3), 1287-95.
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  The metabolic activation of two known olfactory mucosal (OM) toxicants, acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined with mouse liver and OM microsomes and purified, heterologously expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both isoforms were active in metabolizing DCBN and AP to metabolites that formed protein adducts. The formation of DCBN- or AP-protein adducts and other AP metabolites, including 3-hydroxy-AP and, in the presence of glutathione, AP-glutathione conjugate, was also detected in OM microsomal reactions and to a much greater extent than in liver microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major roles in mouse OM microsomal metabolic activation of DCBN and AP. Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450 isoforms in OM microsomes. OM microsomal AP and DCBN metabolic activation was inhibited by 5- and 8-methoxsalen, which inhibit both CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM bioactivation of AP and DCBN were ruled out by comparing activities of acetone-treated mice or Cyp1a2(-/-) mice with those of control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known OM-selective toxicity of AP and DCBN. Further analysis of the kinetics of AP and DCBN metabolism by the purified P450s suggested that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas their relative roles in DCBN metabolism may be dose dependent, with CYP2G1 playing more important roles at low substrate concentrations.
• Zhang, Q. Y., Ding, X., Zhang, Q. Y., Raner, G. M., Kaminsky, L. S., Dunbar, D., & Coon, M. J. (1998). Characterization of the cytochrome P450 CYP2J4: expression in rat small intestine and role in retinoic acid biotransformation from retinal.. Archives of biochemistry and biophysics, 353(2), 257-64. doi:10.1006/abbi.1998.0654
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  The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. Apparent K(m) values for the formation of retinoic acids were 54 and 49 microM, respectively, and apparent Vmax values were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestine in vivo.
• Hua, Z., Zhang, Q. Y., Ding, X., Zhang, Q. Y., Su, T., Lipinskas, T. W., & Hua, Z. (1997). cDNA cloning, heterologous expression, and characterization of mouse CYP2G1, an olfactory-specific steroid hydroxylase.. Archives of biochemistry and biophysics, 340(2), 208-14. doi:10.1006/abbi.1997.9899
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  CYP2G1 is expressed specifically in the olfactory mucosa in rabbits and rats. In the present study, a full-length cDNA for mouse CYP2G1 was obtained using a PCR approach with RNA preparations from the olfactory mucosa of C57BL/6 mice. Sequence comparisons indicated that mouse CYP2G1 is highly homologous in deduced amino acid sequence to rabbit (82.4% identity) and rat CYP2G1 (94.9% identity). RNA blot and immunoblot analyses indicated that mouse CYP2G1 is expressed only in the olfactory mucosa. The coding region of the mouse CYP2G1 cDNA was cloned into a baculoviral expression vector for heterologous production of the enzyme in cultured insect cells. Heterologously expressed mouse CYP2G1 was active in a reconstituted system toward testosterone and progesterone, producing all the major metabolites detected in olfactory microsomal reactions, including 15 alpha-, 15 beta-, and 2 beta-hydroxytestosterone from testosterone and two unidentified metabolites from progesterone. Kinetic analysis indicated that mouse CYP2G1 has relatively high affinities toward the steroid substrates, with K(m) values in the micromolar range for both testosterone and progesterone. At a substrate concentration of 10 microM, microsomes of olfactory mucosa had much higher turnover numbers toward testosterone and progesterone than hepatic microsomes, consistent with the olfactory-specific expression of a high-affinity sex steroid hydroxylase. These findings will facilitate further molecular genetics studies on the biological function of CYP2G1 in a mouse model.
• Zhang, Q. Y., Ding, X., Zhang, Q. Y., & Kaminsky, L. S. (1997). CDNA cloning, heterologous expression, and characterization of rat intestinal CYP2J4.. Archives of biochemistry and biophysics, 340(2), 270-8. doi:10.1006/abbi.1997.9922
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  The small intestine is the major portal of entry of ingested xenobiotics. Previous studies from this and other laboratories indicated that at least 6 of the 33 xenobiotic metabolizing forms of P450 currently identified are expressed in rat small intestinal epithelial cells. In the present study, a previously unidentified rat P450, designated CYP2J4, was identified in rat small intestine using PCR. The full-length CYP2J4 cDNA contains an open reading frame for a protein of 501 residues and is 72.5 and 75.8% identical to rabbit CYP2J1 and human CYP2J2, respectively, in deduced amino acid sequences. The coding region of CYP2J4 cDNA has been cloned into a baculoviral expression vector (pVL1392) and expressed in cultured Spodoptera frugiperta (SF9) cells. The heterologously expressed CYP2J4 protein displayed a typical p450 CO-difference spectrum, with maximum absorbance at 449 nm. When purified to near electrophoretic homogeneity, it was active toward arachidonic acid in a reconstituted system with NADPH-P450 reductase and phospholipid, producing both hydroxyeicosatetraenoic and epoxyeicosatrienoic acids. RNA blot analysis with CYP2J4 cDNA as a probe detected two mRNA species, about 2.0 and 2.4 kb, respectively, in RNA preparations from liver, intestine, olfactory mucosa, kidney, heart, and lung. The 2.0-kb mRNA species was abundant in liver, small intestine, and olfactory mucosa, whereas the 2.4-kb mRNA species was predominant only in the olfactory mucosa. Immunoblot analysis of microsomal fractions from different rat tissues with a polyclonal anti-peptide antibody to CYP2J4 detected a protein with the same electrophoretic mobility as purified CYP2J4 most abundantly in small intestine and to a lesser extent in liver and other immunoreactive proteins with slightly higher electrophoretic mobility than purified CYP2J4 in a number of tissues, including small intestine, liver, kidney, lung, and olfactory mucosa. The predominant distribution of CYP2J4, which has activity toward arachidonic acid, is provocative, but its physiological function is as yet unknown.
• Zhang, Q. Y., Dunbar, D., Fasco, M. J., Kaminsky, L. S., Wikoff, J., & Zhang, Q. Y. (1997). Regulation of cytochrome P4501A1 expression in rat small intestine.. Drug metabolism and disposition: the biological fate of chemicals, 25(1), 21-6.
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  The predominant inducible cytochrome P450 (CYP) in rat small intestine is CYP1A1, which, when induced to elevated levels by xenobiotics or dietary constituents, has the potential to metabolize and consequently reduce the systemic uptake of low concentrations of orally ingested, bioactivatable polycyclic aromatic hydrocarbons and heterocyclic aromatic amines. We investigated the regulation of small intestinal CYP1A1 in an effort to develop its anticancer potential. The time courses of hepatic and intestinal CYP1A1 induction by beta-naphthoflavone (BNF) were compared quantitatively at the protein and mRNA levels by immunoblot and competitive RNA-polymerase chain reaction analyses. CYP1A1 mRNA levels in both organs increased sharply and were maximal at approximately 6 hr and returned to near basal levels by 12 hr after BNF treatment. In contrast, hepatic CYP1A2 mRNA levels increased much more gradually. Small intestinal CYP1A2 mRNA concentrations were insufficient to support translation of detectable protein. Maximal levels of intestinal and hepatic CYP1A1 protein occurred between 12 and 24 hr, and 24 and 48 hr, respectively, after BNF. Intestinal CYP1A1 protein was detectable earlier and for a shorter duration than hepatic CYP1A1. CYP1A1 induction was first detected in crypt cells 3 hr before the appearance of activity in villous cells, and maximal levels of activity were reached in crypt cells 12 to 18 hr before maximal and 1.5-fold (per mg protein) higher responses in villous cells-induction thus occurs in both villous and crypt cells. Previously detected decreases in CYP1A1 inducibility from duodenum to ileum correlated with decreases in immunoblot determined-Ah receptor levels. Intestinal CYP1A1 induction does not involve the glucocorticoid receptor in contrast to hepatic induction. These studies have revealed several novel features of small intestinal CYP1A1 regulation.
• Zhang, Q. Y., Dunbar, D., He, W., Kaminsky, L. S., & Zhang, Q. Y. (1997). Induction of CYP1A1 by beta-naphthoflavone in IEC-18 rat intestinal epithelial cells and potentiation of induction by dibutyryl cAMP.. Biochemical and biophysical research communications, 233(3), 623-6. doi:10.1006/bbrc.1997.6508
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  We have examined the inducibility of CYP1A1 by beta-naphthoflavone (BNF) in a rat intestinal epithelial cell line, IEC-18, and the associated interaction between cAMP and BNF. CYP1A1 was not constitutively expressed in IEC-18 cells. Upon treatment with BNF, CYP1A1 RNA, protein, and microsomal 7-ethoxyresorufin O-deethylase activity were detected. Treatment with dibutyryl cAMP resulted in a 2-fold increase in the extent of induction at both RNA and protein levels, with corresponding increases in CYP1A1 enzymatic activity. These results support the involvement of protein kinase A in Ah receptor-mediated induction of CYP1A1 and provide an in vitro model for further studies on the mechanisms underlying regional and cellular differences in the regulation of CYP1A1 gene expression in the small intestine.
• Zhang, Q. Y., Zhang, Q. Y., Wikoff, J., Kaminsky, L. S., & Dunbar, D. (1996). Characterization of rat small intestinal cytochrome P450 composition and inducibility.. Drug metabolism and disposition: the biological fate of chemicals, 24(3), 322-8.
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  The composition and inducibility of cytochrome P450 (P450) in rat small intestinal epithelial cells were investigated with the use of RNA-polymerase chain reaction and immunoblot techniques. The complement of intestinal P450s is more restricted than hepatic forms. P450s 1A1, 2B1, and 3A1 were detected in enterocytes of untreated rats and were inducible by beta-naphthoflavone (BNF), phenobarbital, and pregnenolone-16alpha-carbonitrile or dexamethasone, respectively. In addition, P450s 2C6 and 2C11 were both constitutively expressed at low levels. In contrast, several P450 forms, which are found in the liver, were not detected in enterocytes of untreated or induced rats, including P450s 2A1, 2B2, 2E1, 3A2, and 4A1. P4501A2 mRNA was detected only in BNF-induced rat small intestine and at levels that did not result in its detectable translation. The most prominent inducible form in rat small intestine is P4501A1. Its inducibility diminishes markedly along the length of the small intestine from the duodenum to the ileum. Furthermore, the induction of P4501A1 in enterocytes was affected by the route of administration of the inducing agent. Thus, intestinal P4501A1 was more sensitive to orally administered BNF, whereas induction of hepatic P4501A1 was more sensitive to intraperitoneal administered BNF. Overall, the results demonstrate the differential regulation of P450s between the liver and small intestine, and provide a basis for further studies in assessing the potential of intestinal P450s to protect against orally ingested polycyclic aromatic hydrocarbon carcinogens.

Reviews

• Xie, F., Ding, X., & Zhang, Q. Y. (2016. An update on the role of intestinal cytochrome P450 enzymes in drug disposition(pp 374-383).
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  Oral administration is the most commonly used route for drug treatment. Intestinal cytochrome P450 (CYP)-mediated metabolism can eliminate a large proportion of some orally administered drugs before they reach systemic circulation, while leaving the passage of other drugs unimpeded. A better understanding of the ability of intestinal P450 enzymes to metabolize various clinical drugs in both humans and preclinical animal species, including the identification of the CYP enzymes expressed, their regulation, and the relative importance of intestinal metabolism compared to hepatic metabolism, is important for improving bioavailability of current drugs and new drugs in development. Here, we briefly review the expression of drug-metabolizing P450 enzymes in the small intestine of humans and several preclinical animal species, and provide an update of the various factors or events that regulate intestinal P450 expression, including a cross talk between the liver and the intestine. We further compare various clinical and preclinical approaches for assessing the impact of intestinal drug metabolism on bioavailability, and discuss the utility of the intestinal epithelium-specific NADPH-cytochrome P450 reductase-null (IECN) mouse as a useful model for studying roles of intestinal P450 in the disposition of orally administered drugs.