Jay Gandolfi

Interests

Research

Mechanisms of toxicology, Environmental Health. and Toxic effects of drugs and chemicals

Teaching

Toxicology, Environmental Health, Chemical and Drug Disposition

Courses

Scholarly Contributions

Journals/Publications

• Bentley, J. B., Vaughan, R. W., Miller, M. S., Calkins, J. M., & Gandolfi, A. J. (2020). Serum inorganic fluoride levels in obese patients during and after enflurane anesthesia. Anesthesia and analgesia, 58(5), 409-12.
  More info

  Serum ionic fluoride levels in 24 markedly obese patients (127.6 +/- 6.0 kg) and seven nonobese control subjects (67.3 +/- 1.2 kg) were compared during and following enflurane anesthesia (less than 2.0 MAC hr). Peak serum fluoride levels were higher (28.0 +/- 1.9 vs 17.3 +/- 1.3 micrometers/L, p less than 0.01) and the rate at which fluoride levels increased was more rapid (slope 5.6 vs 2.5 micrometers/L/hr) in obese patients than in control patients. No clinical evidence of nephrotoxicity was found in either group. Vasopressin resistance tests were not performed, and thus it is inknown whether subclinical nephrotoxicity occurred in either study group. Possible reasons for increased enflurane metabolism in obesity are discussed. These possibilities include differences in fluoride ion kinetics, hepatic delivery and penetration of volatile anesthetics, and altered hepatic microsomal enzyme activity. Obesity rather than weight is an important determinant of anesthetic biotransformation.
• Chen, M., & Gandolfi, J. (2020). Characterization of the humoral immune response and hepatotoxicity after multiple halothane exposures in guinea pigs. Drug metabolism reviews, 29(1-2), 103-22.
• Gandolfi, A. J., & Buhler, D. R. (2020). Metabolism of hexachlorophene in the rabbit. Excretion, tissue distribution, and characterization of the urinary glucuronide conjugate. Journal of agricultural and food chemistry, 25(1), 21-5.
• Gandolfi, A. J., Sipes, I. G., & Brown, B. R. (2020). Detection of covalently bound halothane metabolites in the hypoxic rat model for halothane hepatotoxicity. Fundamental and applied toxicology : official journal of the Society of Toxicology, 1(3), 255-9.
  More info

  Using a non-radiometric technique, halothane metabolites have been shown to covalently bind to rat hepatic tissue with the production of a lesion. The conditions required for optimizing the lesion (hypoxia and biotransformation enzyme induction) also optimizes the binding of fluorinated halothane residues to hepatic tissue. The maximal binding of fluorinated halothane residues to the liver of rats precedes the development of the hepatic lesion. Female rats, which are resistant to the halothane initiated lesion, have one-third as much covalently bound halothane residue. Biotransformation inhibitors (SKF-525A, metyrapone), which inhibited lesion formation, also inhibit the covalent binding of halothane to hepatic tissue. Cystamine and cysteine, sulfhydryl agents which can inhibit hepatic lesion development when administered four hr after halothane exposure, also suppressed the amount of halothane metabolites covalently bound to hepatic tissue. Using this non-radioactive method for measuring the covalent binding of halothane to hepatic tissue, it appears that the bioactivation of halothane was a necessary event for the appearance of a halothane initiated hepatic lesion.
• Gandolfi, A. J., Wijeweera, J., & Brendel, K. (2020). Use of precision-cut liver slices as an in vitro tool for evaluating liver function. Toxicologic pathology, 24(1), 58-61.
  More info

  Precision-cut liver slices have been developed as an in vitro tool for assessing liver viability and function and for examining hepatotoxicants. Liver slices from a variety of species (including human) are prepared using mechanical slicers that produce reproducible slices of a uniform thickness, which allows optimum exchange of nutrients, waste, and gases. Slices are incubated in dynamic systems that allow the slices to be maintained viable in culture for 1-10 days. The viability of slices can be assessed by ion content (K+, Na+ ATPase status), intermediary metabolism, energy status (ATP), respiration, biosynthetic ability, and biotransformation activity. In addition, liver tissue slices allow the opportunity for extensive microscopic evaluation (light and electron) as well as newer technologies such as confocal microscopy. Assessment of the toxic potential of a chemical can be performed after a short-term or constant exposure by evaluating the viability parameters. Liver slices have been used extensively for rank-ordering the toxicity of chemicals as well as for examining the mechanisms of liver injury. Liver slices in culture also can be used for an examination of the induction of new enzymes such as cytochrome P-450 and the expression of stress proteins or peroxisomal enzymes. Finally, liver slices offer a system for evaluating whole or cryopreserved liver as well as regeneration of liver tissue after toxic insult. Liver slices have been shown to be a valid in vitro system for examining liver function and offer a bridge between in vivo and cell culture systems.
• Ghantous, H. N., Fernando, J., Gandolfi, A. J., & Brendel, K. (2020). Biotransformation of halothane in guinea pig liver slices. Drug metabolism and disposition: the biological fate of chemicals, 18(4), 514-8.
  More info

  The biotransformation of halothane was studied using liver slices. Precision-cut Hartley male guinea pig liver slices (1 cm diameter; 250-300 microns thick) were incubated in sealed roller vials containing supplemented Krebs-Henseleit buffer at 37 degrees C under different O2 tensions (2.5, 21, and 95%). After a 1-hr preincubation, halothane was vaporized in the vial producing a 1.9 mM medium concentration. Halothane metabolites (Br-, trifluoroacetic acid, F-) were measured at 2, 4, and 6 hr. Viability of the incubated slices was verified by determining intracellular K+ content and levels of cytochrome P-450, which were maintained under 95% O2 atmosphere but decreased with lower O2 tensions (2.5%). The highest fluoride production was 300 +/- 22 pmol/mg slice weight/6 hr at low O2 tension (2.5%). Defluorination decreased with increasing O2 tension to undetectable levels under 95% O2. Production of the oxidative metabolite, trifluoroacetic acid, was highest at 95% O2 (2.35 +/- 0.17 nmol/mg slice weight/6 hr). Trifluoroacetic acid production decreased with decreasing O2 tension. Br- production was the highest at 21% O2 (1.8 +/- 0.13 nmol/mg slice weight/6 hr). Production of Br- was not dependent on the O2 tension. The guinea pig slices are capable of biotransforming halothane (oxidative/reductive); therefore, this in vitro system appears suitable for studying the biotransformation of halothane.
• Hassall, C. D., Gandolfi, A. J., & Brendel, K. (2020). Effect of halogenated vinyl cysteine conjugates on renal tubular active transport. Toxicology, 26(3-4), 285-94.
  More info

  Addition of halogenated vinyl cysteine conjugates to isolated rabbit kidney tubule suspensions resulted in a decrease in the active transport of para-aminohippuric acid (PAH) and tetraethylammonium bromide (TEA). At 10(-5) M vinyl cysteine conjugate, tubule to medium accumulation ratios (T/M) were similar to those of controls while at 10(-3) M the T/M values decreased to 1, indicating complete inhibition of active accumulation of PAH or TEA. The decreased active transport was not caused by inhibition of mitochondrial oxidation since incubations in the presence of 10(-3) M halogenated vinyl cysteine did not inhibit tubule O2 utilization or production of 14CO2 from [14C]glucose or [14C]succinate. A mechanism is proposed whereby toxicity may result from covalent binding of an active intermediate, produced by enzyme cleavage, to membrane associated nucleophilic groups thereby decreasing active transport.
• Jaffe, D. R., Hassall, C. D., Brendel, K., & Gandolfi, A. J. (2020). In vivo and in vitro nephrotoxicity of the cysteine conjugate of hexachlorobutadiene. Journal of toxicology and environmental health, 11(4-6), 857-67.
  More info

  Hexachlorobutadiene (HCBD), a renal toxin and carcinogen, is thought to require bioactivation to exert toxicity. The chemically synthesized cysteine conjugate of structurally similar halogenated hydrocarbons, trichloroethylene, chlorotrifluoroethylene, and chlorodifluoroethylene, have been shown to be nephrotoxic. Hence the cysteine conjugate of HCBD, S-pentachlorobuta-1,3-dienyl cysteine (PCBC), was assessed for potential nephrotoxicity. Active acid and base transport in isolated rabbit renal tubules was used to screen nephrotoxicity. A dose-dependent decrease in acid and base transport was observed after incubation with PCBC. At 10(-5) M PCBC transport was similar to that in controls, while at 10(-3) M PCBC completely inhibited active transport. In addition, in vivo exposure of Swiss-Webster male mice caused dose-dependent damage in the pars recta region of the proximal tubules (5-25 mg/kg ip). Genotoxicity in renal tissue was studied by using alkaline elution to detect DNA single-strand breaks and total cross-links. No DNA single-strand breaks were observed in isolated rabbit renal tubules after exposure to 10(-3) to 10(-5) M PCBC. However, at 10(-3) M PCBC there was some evidence of DNA cross-links. Therefore if cysteine conjugates of HCBD are formed in vivo, they could account for the toxicity observed with exposure to HCBD.
• Lind, R. C., & Gandolfi, A. J. (2020). Concentration-dependent inhibition of halothane biotransformation in the guinea pig. Drug metabolism and disposition: the biological fate of chemicals, 21(2), 386-9.
  More info

  Previous studies have indicated concentration-dependent inhibition of halothane's biotransformation by the hepatic cytochrome P-450 enzyme system. In order to investigate this phenomenon in the guinea pig model of acute halothane-associated hepatotoxicity, male outbred Hartley guinea pigs underwent 4 hr inhalation exposures to either subanesthetic (0.1%) or anesthetic (1.0%) concentrations of halothane with 40% O2. Plasma concentrations of the primary halothane metabolite, trifluoroacetic acid (TFA) were one-half as great immediately (0 hr) after the 1% exposure as they were with 0.1%. By 10 hr after exposure plasma TFA had increased significantly in both treatment groups. However, there was a much greater rate of increase with 1% halothane so that values were now more than 50% greater than with 0.1% halothane. Plasma TFA in the 1% halothane group remained significantly greater over the 96-hr time course of the experiment. Covalent binding of reactive halothane biotransformation intermediates to hepatic protein paralleled plasma TFA. At 0 hr, the degree of binding in the 1% halothane group was one-half as great as in the 0.1% group and by 10 hr after had increased to be nearly twice as great as the 0.1% group that had not increased between the time points. These data provide strong evidence for substrate-specific inhibition of halothane biotransformation with the majority of biotransformation occurring in the hours following exposure to an anesthetic (1%) concentration of the drug. These metabolic dynamics should be considered in studies of other organohalogens, including the new refrigerants that are structurally similar to halothane.
• Lind, R. C., & Gandolfi, A. J. (2020). Hepatoprotection by dimethyl sulfoxide. I. Protection when given twenty-four hours after chloroform or bromobenzene. Toxicologic pathology, 27(3), 342-7.
  More info

  Dimethyl sulfoxide (DMSO) has previously been reported to protect against hepatotoxicity resulting from chloroform (CHCl3) or bromobenzene (BB) when given 10 hr after the toxicant. The object of these studies was to further demonstrate the latent protective ability of DMSO by administering it at a much later time (24 hr) following toxicant exposure. In addition, a more detailed evaluation of the lesions was performed to better characterize the lesion progression and resolution. Male Sprague-Dawley rats received a hepatotoxic oral dose of either CHCl3 (1.0 ml/kg) or BB (0.5 ml/kg) and then received 2 ml/kg DMSO intraperitoneally 24 hr later. With both toxicants, limited centrilobular lesions were already present by the time DMSO was administered. Without treatment, liver injury rapidly progressed so that by 48 hr it occupied 40-50% of the liver, with accompanying large increases in plasma alanine aminotransferase (ALT) activity. Administration of DMSO greatly attenuated lesion development for both toxicants; the area injured was reduced by more than 4-fold, accompanied by a decrease in 48 hr ALT activity of 8-16-fold. The ability of DMSO to intervene in the development of liver injury at such a late time appears to be unique and may provide insight into therapies for acute xenobiotic-induced hepatitis.
• MacDonald, J. R., Lind, R. C., Sipes, I. G., & Gandolfi, A. J. (2020). Determination of hepatic tissue calcium levels by flame emission spectrophotometry. Journal of analytical toxicology, 8(4), 155-7.
  More info

  Hepatotoxicants are known to cause significant increases in total liver calcium content. These increases are postulated to be the key irreversible event leading to necrosis of hepatic parenchymal cells. An investigation is presented of the use of flame emission spectrophotometry as an alternative analytical method to atomic absorption spectrophotometry in the assessment of hepatic calcium levels following hepatotoxicant challenge, using a proven method of tissue digestion. Flame emission and atomic absorption spectrophotometry were found to be comparable in sensitivity with detection limits in the ppm range and correlation coefficients of greater than 0.99 for the standard curves. Tissue sample analysis demonstrated comparable precision for the two methods. The liver calcium levels determined for an untreated control rat were 105 ppm with a coefficient of variation of 2.9% for flame emission spectrophotometry vs. 106 ppm with a coefficient of variation of 3.6% for atomic absorption spectrophotometry. Comparable precision was also found for the two methods when toxicant-damaged liver was analyzed. Flame emission spectrophotometry appears to be a useful alternative to atomic absorption spectrophotometry for tissue calcium analyses in experimental toxicologic studies.
• Michel-Ramirez, G., Recio-Vega, R., Lantz, R. C., Gandolfi, A. J., Olivas-Calderon, E., Chau, B. T., & Amistadi, M. K. (2020). Assessment of YAP gene polymorphisms and arsenic interaction in Mexican women with breast cancer. Journal of applied toxicology : JAT, 40(3), 342-351.
• Peraza, M. A., Cromey, D. W., Carolus, B., Carter, D. E., & Gandolfi, A. J. (2020). Morphological and functional alterations in human proximal tubular cell line induced by low level inorganic arsenic: evidence for targeting of mitochondria and initiated apoptosis. Journal of applied toxicology : JAT, 26(4), 356-67.
  More info

  The kidney is a known target organ for arsenic and is critical for both arsenic biotransformation and elimination. Previous studies have demonstrated that at high doses (ppm) inorganic arsenic is toxic to mitochondria primarily by affecting cellular respiration. However, the effect of inorganic arsenic on mitochondria after low level exposures is not known, particularly in the kidney. Thus the functional and morphological effects of low level inorganic arsenic were investigated in a human proximal tubular cell line, HK-2. Mitochondrial function was assessed at subcytotoxic concentrations of arsenite (< or = 10 microm) by examining the alteration of the mitochondrial membrane potential using MitoTracker Red, a mitochondrion selective dye. In a subset of cells, subcytotoxic arsenite led to mitochondrial membrane depolarization, which could subsequently lead to permeability transition and apoptosis. Subcytotoxic arsenite also induced translocation of phosphatidylserine, indicative of early-stage apoptosis. To confirm whether subcytotoxic arsenite induces cellular and/or mitochondrial morphological alterations consistent with initiated apoptosis, HK-2 cells were evaluated with transmission electron microscopy. Classic morphology of apoptosis was not observed with subcytotoxic arsenite exposures; however, evidence of necrotic changes in the cytoplasmic structure and mitochondrial morphology were apparent. Therefore, based on depolarization of mitochondria and the externalization of phosphatidylserine, HK-2 cells appear to initiate apoptosis following subcytotoxic arsenite insult, but morphological changes indicate that HK-2 cells fail to complete apoptosis and ultimately undergo necrosis. Therefore, subcytotoxic arsenite can be sufficiently toxic to mitochondria that they lose their ability to keep the cell on course for apoptotic cell death.
• Ramírez, D. M., Vea, L., Field, J. A., Baker, P. B., Gandolfi, A. J., & Maier, R. M. (2020). Transferable Training Modules: Building Environmental Education Opportunities With and for Mexican Community Health Workers (Promotores de Salud). Family & community health, 40(4), 306-315.
  More info

  Community health workers (promotores de salud) have the ability to empower communities to mitigate negative health outcomes. Current training efforts in environmental topics are lacking. This project addressed this gap by developing 4 transferable training modules on environmental health. By applying a series of surveys, interviews, and trainings, we evaluated their relevance. Partners provided favorable feedback for 3 of the 4 modules. It was also learned that the development method could be improved by engaging technically trained promotores de salud in the role of co-creators. This project has implications for environmental justice communities as it can lessen information disparities.
• Wood, C. L., Gandolfi, A. J., & Van Dyke, R. A. (2020). Lipid binding of a halothane metabolite. Relationship to lipid peroxidation in vitro. Drug metabolism and disposition: the biological fate of chemicals, 4(4), 305-13.
  More info

  Incubation of rat liver microsomes with 14C-halothane in a nitrogen atmosphere results in a high correlation between the formation of conjugated dienes and the binding of a halothane metabolite to phospholipids. Both the binding and the formation of the conjugated dienes required NADPH, were inhibited by carbon monoxide, and increased with duration of incubation and with protein concentration. Both [36Cl] halothane and [14C] halothane showed a similar pattern of binding to microsomal phospholipids suggesting that the chlorine atom is retained by the metabolite that binds. Neither high levels of conjugated dienes produced by halothane in microsomes incubated under nitrogen nor the binding of the halothane metabolite results in the destruction of cytochrome P-450.
• Gamboa-Loira, B., Hernández-Alcaraz, C., Gandolfi, A. J., Cebrián, M. E., Burguete-García, A., García-Martínez, A., & López-Carrillo, L. (2018). Arsenic methylation capacity in relation to nutrient intake and genetic polymorphisms in one-carbon metabolism. Environmental research, 164, 18-23.
  More info

  Nutrients and genetic polymorphisms participating in one-carbon metabolism may explain interindividual differences in inorganic arsenic (iAs) methylation capacity, which in turn may account for variations in susceptibility to iAs-induced diseases.
• Quiller, G., Mérida-Ortega, ., Rothenberg, S. J., Cebrián, M. E., Gandolfi, A. J., Franco-Marina, F., & López-Carrillo, L. (2018). Dietary flavonoids improve urinary arsenic elimination among Mexican women. Nutrition research (New York, N.Y.), 55, 65-71.
  More info

  Inorganic arsenic (iAs) exposure increases risk of several diseases, including cancer. Some nutrients such as flavonoids enhance glutathione activity, which in turn play a key role in iAs elimination. Our objective was to explore whether dietary non-soy flavonoids are associated with iAs metabolism. We hypothesized that the intake of flavonoids belonging to the following groups, flavan-3-ols, flavone, flavonol, flavanone, and anthocyanidin, is positively associated with urinary dimethylarsinic acid (DMA), which is the most soluble iAs metabolite excreted. We performed a cross-sectional study that included 1027 women living in an arsenic-contaminated area of northern Mexico. Flavonoid intake was estimated using a validated food frequency questionnaire. Concentration of urinary iAs and its metabolites (monomethylarsonic acid and DMA) were determined by high performance liquid chromatography ICP-MS. Results showed positive significant associations between DMA and the flavonoid groups flava-3-ols (β= 0.0112) and flavones (β= 0.0144), as well as the individual intake of apigenin (β= 0.0115), luteolin (β= 0.0138), and eriodictyol (β= 0.0026). Our findings suggest that certain non-soy flavonoids may improve iAs elimination; however, there is still very limited information available regarding the consumption of flavonoids and iAs metabolism.
• Michel-Ramirez, G., Recio-Vega, R., Ocampo-Gomez, G., Palacios-Sanchez, E., Delgado-Macias, M., Delgado-Gaona, M., Lantz, R. C., Gandolfi, J., & Gonzalez-Cortes, T. (2017). Association between YAP expression in neoplastic and non-neoplastic breast tissue with arsenic urinary levels. Journal of applied toxicology : JAT, 37(10), 1195-1202.
  More info

  The Hippo pathway regulates cell proliferation and apoptosis and it has been noted that loss of critical components of this pathway can lead to uncontrolled cell growth. Yes-associated protein (YAP) is an important component of this Hippo pathway because YAP is the nuclear effector of the Hippo tumor suppressor pathway and it is crucial for the response to oxidative stress induced by cellular process and by different xenobiotics, including arsenic. It has been proposed that YAP dysregulation can contribute to a malignant cellular phenotype acting as both a tumor suppressor and an oncogene. The aim of the study was to assess and compare the expression of YAP in neoplastic and non-neoplastic breast tissue of women chronically exposed to arsenic through drinking water. YAP expression was assessed by immunohistochemistry in 120 breast biopsies from women with breast cancer and from women with other non-neoplastic breast pathologies. Arsenic concentration was quantified in urine. The results disclosed a significant lower percentage of cytoplasm YAP expression in cases and that YAP high-intensity staining in the cytoplasm but not in the nucleus decreases the risk for breast cancer. In conclusion, our overall data suggest that YAP may act as a tumor suppressor protein because their reduced expression in cases, which can induce an environment favorable for inhibition of apoptosis and promoting cellular proliferation by increasing genetic instability of cells, which might contribute to the pathogenesis of cancer. Copyright © 2017 John Wiley & Sons, Ltd.
• Oliva-González, C., Uresti-Rivera, E. E., Galicia-Cruz, O. G., Jasso-Robles, F. I., Gandolfi, A. J., & Escudero-Lourdes, C. (2017). The tumor suppressor phosphatase and tensin homolog protein (PTEN) is negatively regulated by NF-κb p50 homodimers and involves histone 3 methylation/deacetylation in UROtsa cells chronically exposed to monomethylarsonous acid. Toxicology letters, 280, 92-98.
  More info

  UROtsa cells have been accepted as a model to study carcinogenicity mechanisms of arsenic-associated human bladder cancer. In vitro continuous exposure to monomethylarsonous acid (MMA), leads UROtsa cells to commit to malignant transformation. In this process, NF-κβ-associated inflammatory response seems to play an important role since this transcription factor activates some minutes after cells are exposed in vitro to MMA and keeps activated during the cellular malignant transformation. It is known that a slight decrease in the protein phosphatase and tensin homologue (PTEN) gene expression is enough for some cells to become malignantly transformed. Interestingly, this tumor suppressor has been proven to be negatively regulated by NF-κβ through binding to its gene promoter. Based on these observations we propose that NF-κβ may be involved in arsenic associated carcinogenesis through the negative regulation of PTEN gene expression. Changes in PTEN expression and the binding of p50 NF-κβ subunit to PTEN promoter were evaluated in UROtsa cells exposed for 4, 12, 20, or 24 wk to 50nM MMA. Results showed that MMA induced a significant decrease in PTEN expression around 20 wk exposure to MMA,which correlated with increased binding of p50 subunit to the PTEN promoter. Consistent with these results, ChIP assays also showed a significant decrease in H3 acetylation (H3ac) but an increase in the repression marks H3k9me3 and H327me3 in PTEN promoter when compared with not treated cells. These results suggest that the activation of NF-κβ by MMA may participate in UROtsa cells malignant transformation through the negative regulation of PTEN expression involving p50 homodimers-mediated chromatin remodeling around the PTEN promoter.
• Escudero-Lourdes, C., Uresti-Rivera, E. E., Oliva-González, C., Torres-Ramos, M. A., Aguirre-Bañuelos, P., & Gandolfi, A. J. (2016). Cortical Astrocytes Acutely Exposed to the Monomethylarsonous Acid (MMA) Show Increased Pro-inflammatory Cytokines Gene Expression that is Consistent with APP and BACE-1: Over-expression. Neurochemical research, 41(10), 2559-2572.
  More info

  Long-term exposure to inorganic arsenic (iAs) through drinking water has been associated with cognitive impairment in children and adults; however, the related pathogenic mechanisms have not been completely described. Increased or chronic inflammation in the brain is linked to impaired cognition and neurodegeneration; iAs induces strong inflammatory responses in several cells, but this effect has been poorly evaluated in central nervous system (CNS) cells. Because astrocytes are the most abundant cells in the CNS and play a critical role in brain homeostasis, including regulation of the inflammatory response, any functional impairment in them can be deleterious for the brain. We propose that iAs could induce cognitive impairment through inflammatory response activation in astrocytes. In the present work, rat cortical astrocytes were acutely exposed in vitro to the monomethylated metabolite of iAs (MMA), which accumulates in glial cells without compromising cell viability. MMA LD in astrocytes was 10.52 μM, however, exposure to sub-toxic MMA concentrations (50-1000 nM) significantly increased IL-1β, IL-6, TNF-α, COX-2, and MIF-1 gene expression. These effects were consistent with amyloid precursor protein (APP) and β-secretase (BACE-1) increased gene expression, mainly for those MMA concentrations that also induced TNF-α over-expression. Other effects of MMA on cortical astrocytes included increased proliferative and metabolic activity. All tested MMA concentrations led to an inhibition of intracellular lactate dehydrogenase (LDH) activity. Results suggest that MMA induces important metabolic and functional changes in astrocytes that may affect brain homeostasis and that inflammation may play a major role in cognitive impairment-related pathogenicity in As-exposed populations.
• García-Rico, L., Meza-Figueroa, D., Gandolfi, A. J., Del Río-Salas, R., Romero, F. M., & Meza-Montenegro, M. M. (2016). Dust-Metal Sources in an Urbanized Arid Zone: Implications for Health-Risk Assessments. Archives of environmental contamination and toxicology, 70(3), 522-33.
  More info

  The available information concerning metal pollution in different dust sources and the health effects in children remains limited in Mexico. This study focuses on Hermosillo, which is an urbanized area located in the Sonoran Desert in which soil resuspension and dust emission processes are common. The metal content of arsenic (As), chromium (Cr), manganese (Mn), and lead (Pb) were determined in three dust sources (playgrounds, roofs, and roads), each representing different exposure media (EM) for these elements. The metal levels in dust were found in the order of Mn > Cr > Pb > As with the highest metal content found in road dust. Despite the similar average metal distributions, principal component analysis shows a clear separation of the three EM with playground dust related to Cr and Mn and road dust to As and Pb. However, the geoaccumulation index results indicate that dust samples are uncontaminated to moderately polluted, except for Pb in road dust, which is considerably high. In addition, the enrichment factor suggests an anthropogenic origin for all of the studied metals except for Mn. In this context, the hazard index (HI) for noncarcinogenic risk is >1 in this population and thus represents a potential health risk. The spatial distribution for each metal on EM and the HI related to the marginality index could represent a more accurate decision-making tool in risk assessment studies.
• López-Carrillo, L., Gamboa-Loira, B., Becerra, W., Hernández-Alcaraz, C., Hernández-Ramírez, R. U., Gandolfi, A. J., Franco-Marina, F., & Cebrián, M. E. (2016). Dietary micronutrient intake and its relationship with arsenic metabolism in Mexican women. Environmental research, 151, 445-450.
  More info

  Concentrations of inorganic arsenic (iAs) metabolites in urine present intra- and interindividual variations, which are determined not only by the magnitude of exposure to iAs, but also by differences in genetic, environmental and dietary factors.
• Carignan, C. C., Cottingham, K. L., Jackson, B. P., Farzan, S. F., Gandolfi, A. J., Punshon, T., Folt, C. L., & Karagas, M. R. (2015). Estimated exposure to arsenic in breastfed and formula-fed infants in a United States cohort. Environmental health perspectives, 123(5), 500-6.
  More info

  Previous studies indicate that concentrations of arsenic in breast milk are relatively low even in areas with high drinking-water arsenic. However, it is uncertain whether breastfeeding leads to reduced infant exposure to arsenic in regions with lower arsenic concentrations.
• Olivas-Calderón, E., Recio-Vega, R., Gandolfi, A. J., Lantz, R. C., González-Cortes, T., Gonzalez-De Alba, C., Froines, J. R., & Espinosa-Fematt, J. A. (2015). Lung inflammation biomarkers and lung function in children chronically exposed to arsenic. Toxicology and applied pharmacology, 287(2), 161-167.
  More info

  Evidence suggests that exposure to arsenic in drinking water during early childhood or in utero has been associated with an increase in respiratory symptoms or diseases in the adulthood, however only a few studies have been carried out during those sensitive windows of exposure. Recently our group demonstrated that the exposure to arsenic during early childhood or in utero in children was associated with impairment in the lung function and suggested that this adverse effect could be due to a chronic inflammation response to the metalloid. Therefore, we designed this cross-sectional study in a cohort of children associating lung inflammatory biomarkers and lung function with urinary As levels. A total of 275 healthy children were partitioned into four study groups according with their arsenic urinary levels. Inflammation biomarkers were measured in sputum by ELISA and the lung function was evaluated by spirometry. Fifty eight percent of the studied children were found to have a restrictive spirometric pattern. In the two highest exposed groups, the soluble receptor for advanced glycation end products' (sRAGE) sputum level was significantly lower and matrix metalloproteinase-9 (MMP-9) concentration was higher. When the biomarkers were correlated to the urinary arsenic species, negative associations were found between dimethylarsinic (DMA), monomethylarsonic percentage (%MMA) and dimethylarsinic percentage (%DMA) with sRAGE and positive associations between %DMA with MMP-9 and with the MMP-9/tissue inhibitor of metalloproteinase (TIMP-1) ratio. In conclusion, chronic arsenic exposure of children negatively correlates with sRAGE, and positively correlated with MMP-9 and MMP-9/TIMP-1 levels, and increases the frequency of an abnormal spirometric pattern. Arsenic-induced alterations in inflammatory biomarkers may contribute to the development of restrictive lung diseases.
• Ramirez-Andreotta, M. D., Brusseau, M. L., Artiola, J., Maier, R. M., & Gandolfi, A. J. (2015). Building a co-created citizen science program with gardeners neighboring a superfund site: The Gardenroots case study. International public health journal, 7(1).
  More info

  A research project that is only expert-driven may ignore the role of local knowledge in research, give low priority to the development of a comprehensive communication strategy to engage the community, and may not deliver the results of the study to the community in an effective way.
• Recio-Vega, R., Gonzalez-Cortes, T., Olivas-Calderon, E., Lantz, R. C., Gandolfi, A. J., & Gonzalez-De Alba, C. (2015). In utero and early childhood exposure to arsenic decreases lung function in children. Journal of applied toxicology : JAT, 35(4), 358-66.
  More info

  The lung is a target organ for adverse health outcomes following exposure to As. Several studies have reported a high prevalence of respiratory symptoms and diseases in subjects highly exposed to As through drinking water; however, most studies to date has been performed in exposed adults, with little information on respiratory effects in children. The objective of the study was to evaluate the association between urinary levels of As and its metabolites with lung function in children exposed in utero and in early childhood to high As levels through drinking water. A total of 358 healthy children were included in our study. Individual exposure was assessed based on urinary concentration of inorganic As. Lung function was assessed by spirometry. Participants were exposed since pregnancy until early childhood to an average water As concentration of 152.13 µg l⁻¹. The mean urinary As level registered in the studied subjects was 141.2 µg l⁻¹ and only 16.7% had a urinary concentration below the national concern level. Forced vital capacity was significantly decreased in the studied population and it was negatively associated with the percentage of inorganic As. More than 57% of the subjects had a restrictive spirometric pattern. The urinary As level was higher in those children with restrictive lung patterns when compared with the levels registered in subjects with normal spirometric patterns. Exposure to As through drinking water during in utero and early life was associated with a decrease in forced vital capacity and with a restrictive spirometric pattern in the children evaluated.
• Burchiel, S. W., Lauer, F. T., Beswick, E. J., Gandolfi, A. J., Parvez, F., Liu, K. J., & Hudson, L. G. (2014). Differential susceptibility of human peripheral blood T cells to suppression by environmental levels of sodium arsenite and monomethylarsonous acid. PloS one, 9(10), e109192.
  More info

  Human exposure to arsenic in drinking water is known to contribute to many different health outcomes such as cancer, diabetes, and cardiopulmonary disease. Several epidemiological studies suggest that T cell function is also altered by drinking water arsenic exposure. However, it is unclear how individual responses differ to various levels of exposure to arsenic. Our laboratory has recently identified differential responses of human peripheral blood mononuclear cell (HPMBC) T cells as measured by polyclonal T cell activation by mitogens during sodium arsenite exposure. T cells from certain healthy individuals exposed to various concentrations (1-100 nM) of arsenite in vitro showed a dose-dependent suppression at these extremely low concentrations (∼ 0.1-10 ppb) of arsenite, whereas other individuals were not suppressed at low concentrations. In a series of more than 30 normal donors, two individuals were found to be sensitive to low concentration (10 nM equivalent ∼ 1 ppb drinking water exposure) to sodium arsenite-induced inhibition of T cell proliferation produced by phytohemagglutinin (PHA) and anti-CD3/anti-CD28. In an arsenite-susceptible individual, arsenite suppressed the activation of Th1 (Tbet) cells, and decreased the percentage of cells in the double positive Th17 (RORγt) and Treg (FoxP3) population. While the majority of normal blood donors tested were not susceptible to inhibition of proliferation at the 1-100 nM concentrations of As(+3), it was found that all donors were sensitive to suppression by 100 nM monomethylarsonous acid (MMA+3), a key metabolite of arsenite. Thus, our studies demonstrate for the first time that low ppb-equivalent concentrations of As(+3) are immunosuppressive to HPBMC T cells in some individuals, but that most donor HPBMC are sensitive to suppression by MMA(+3) at environmentally relevant exposure levels.
• Ezeh, P. C., Lauer, F. T., MacKenzie, D., McClain, S., Liu, K. J., Hudson, L. G., Gandolfi, A. J., & Burchiel, S. W. (2014). Arsenite selectively inhibits mouse bone marrow lymphoid progenitor cell development in vivo and in vitro and suppresses humoral immunity in vivo. PloS one, 9(4), e93920.
  More info

  It is known that exposure to As(+3) via drinking water causes a disruption of the immune system and significantly compromises the immune response to infection. The purpose of these studies was to assess the effects of As(+3) on bone marrow progenitor cell colony formation and the humoral immune response to a T-dependent antigen response (TDAR) in vivo. In a 30 day drinking water study, mice were exposed to 19, 75, or 300 ppb As(+3). There was a decrease in bone marrow cell recovery, but not spleen cell recovery at 300 ppb As(+3). In the bone marrow, As(+3) altered neither the expression of CD34+ and CD38+ cells, markers of early hematopoietic stem cells, nor CD45-/CD105+, markers of mesenchymal stem cells. Spleen cell surface marker CD45 expression on B cells (CD19+), T cells (CD3+), T helper cells (CD4+) and cytotoxic T cells (CD8+), natural killer (NK+), and macrophages (Mac 1+) were not altered by the 30 day in vivo As(+3) exposure. Functional assays of CFU-B colony formation showed significant selective suppression (p
• López-Carrillo, L., Hernández-Ramírez, R. U., Gandolfi, A. J., Ornelas-Aguirre, J. M., Torres-Sánchez, L., & Cebrian, M. E. (2014). Arsenic methylation capacity is associated with breast cancer in northern Mexico. Toxicology and applied pharmacology, 280(1), 53-9.
  More info

  Exposure to environmental contaminants, dietary factors and lifestyles may explain worldwide different breast cancer (BC) incidence. Inorganic arsenic (iAs) in the drinking water is a concern in many regions, such as northern Mexico. Studies in several countries have associated the proportion of urinary monomethylarsenic (%MMA) with increased risks for many As-related diseases, including cancer. To investigate the potential relationships between the risk of BC and the capacity to methylate iAs, a hospital-based case-control study (1016 cases/1028 controls) was performed in northern Mexico. Women were directly interviewed about their reproductive histories. The profile of As metabolites in urine was determined by HPLC-ICP-MS and methylation capacity was assessed by metabolite percentages and indexes. Total urinary As, excluding arsenobetaine (TAs-AsB), ranged from 0.26 to 303.29μg/L. Most women (86%) had TAs-AsB levels below As biological exposure index (35μg/L). Women with higher %MMA and/or primary methylation index (PMI) had an increased BC risk (%MMA ORQ5vs.Q1=2.63; 95%CI 1.89,3.66; p for trend
• Medeiros, M., Le, T. M., Troup, D., Novak, P., & Gandolfi, A. J. (2014). Expression Of Selected Pathway-Marker Genes In Human Urothelial Cells Exposed Chronically To A Non-Cytotoxic Concentration Of Monomethylarsonous Acid. Toxicology reports, 1, 421-434.
  More info

  Bladder cancer has been associated with chronic arsenic exposure. Monomethylarsonous acid [MMA(III)] is a metabolite of inorganic arsenic and has been shown to transform an immortalized urothelial cell line (UROtsa) at concentrations 20-fold less than arsenite. MMA(III) was used as a model arsenical to examine the mechanisms of arsenical-induced transformation of urothelium. A previous microarray analysis revealed only minor changes in gene expression at one and two months of chronic exposure to MMA(III), contrasting with substantial changes observed at three months of exposure. To address the lack of information between two and three months of exposure (the critical period of transformation), the expression of select pathway marker genes was measured by PCR array analysis on a weekly basis. Cell proliferation rate, anchorage-independent growth, and tumorigenicity in SCID mice were also assessed to determine the early, persistent phenotypic changes and their association with the changes in expression of these selected marker genes. A very similar pattern of alterations in these genes was observed when compared to the microarray results, and suggested that early perturbations in cell signaling cascades, immunological pathways, cytokine expression, and MAPK pathway are particularly important in driving malignant transformation. These results showed a strong association between the acquired phenotypic changes that occurred as early as one to two months of chronic MMA(III) exposure, and the observed gene expression pattern that is indicative of the earliest stages in carcinogenesis.
• Ramirez-Andreotta, M. D., Brusseau, M. L., Artiola, J. F., Maier, R. M., & Gandolfi, A. J. (2014). Environmental Research Translation: enhancing interactions with communities at contaminated sites. The Science of the total environment, 497-498, 651-664.
  More info

  The characterization and remediation of contaminated sites are complex endeavors fraught with numerous challenges. One particular challenge that is receiving increased attention is the development and encouragement of full participation by communities and community members affected by a given site in all facets of decision-making. Many disciplines have been grappling with the challenges associated with environmental and risk communication, public participation in environmental data generation, and decision-making and increasing community capacity. The concepts and methods developed by these disciplines are reviewed, with a focus on their relevance to the specific dynamics associated with environmental contamination sites. The contributions of these disciplines are then synthesized and integrated to help develop Environmental Research Translation (ERT), a proposed framework for environmental scientists to promote interaction and communication among involved parties at contaminated sites. This holistic approach is rooted in public participation approaches to science, which includes: a transdisciplinary team, effective collaboration, information transfer, public participation in environmental projects, and a cultural model of risk communication. Although there are challenges associated with the implementation of ERT, it is anticipated that application of this proposed translational science method could promote more robust community participation at contaminated sites.
• Zhou, X., Sun, X., Mobarak, C., Gandolfi, A. J., Burchiel, S. W., Hudson, L. G., & Liu, K. J. (2014). Differential binding of monomethylarsonous acid compared to arsenite and arsenic trioxide with zinc finger peptides and proteins. Chemical research in toxicology, 27(4), 690-8.
  More info

  Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV-vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis.
• Díaz-Villaseñor, A., Cruz, L., Cebrián, A., Hernández-Ramírez, R. U., Hiriart, M., García-Vargas, G., Bassol, S., Sordo, M., Gandolfi, A. J., Klimecki, W. T., López-Carillo, L., Cebrián, M. E., & Ostrosky-Wegman, P. (2013). Arsenic exposure and calpain-10 polymorphisms impair the function of pancreatic beta-cells in humans: a pilot study of risk factors for T2DM. PloS one, 8(1), e51642.
  More info

  The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function.
• Farzan, S. F., Korrick, S., Li, Z., Enelow, R., Gandolfi, A. J., Madan, J., Nadeau, K., & Karagas, M. R. (2013). In utero arsenic exposure and infant infection in a United States cohort: a prospective study. Environmental research, 126, 24-30.
  More info

  Arsenic (As), a ubiquitous environmental toxicant, has recently been linked to disrupted immune function and enhanced infection susceptibility in highly exposed populations. In drinking water, as levels above the EPA maximum contaminant level occur in our US study area and are a particular health concern for pregnant women and infants. As a part of the New Hampshire Birth Cohort Study, we investigated whether in utero exposure to As affects risk of infant infections. We prospectively obtained information on 4-month-old infants (n=214) using a parental telephone survey on infant infections and symptoms, including respiratory infections, diarrhea and specific illnesses, as well as the duration and severity of infections. Using logistic regression and Poisson models, we evaluated the association between maternal urinary As during pregnancy and infection risks adjusted for potentially confounding factors. Maternal urinary As concentrations were related to total number of infections requiring a physician visit (relative risk (RR) per one-fold increase in As in urine=1.5; 95% confidence interval (CI)=1.0, 2.1) or prescription medication (RR=1.6; 95% CI=1.1, 2.4), as well as lower respiratory infections treated with prescription medication (RR=3.3; 95% CI=1.2, 9.0). Associations were observed with respiratory symptoms (RR=4.0; 95% CI=1.0, 15.8), upper respiratory infections (RR=1.6; 95% CI=1.0, 2.5), and colds treated with prescription medication (RR=2.3; 95% CI=1.0, 5.2). Our results provide initial evidence that in utero As exposure may be related to infant infection and infection severity and provide insight into the early life impacts of fetal As exposure.
• Gilbert-Diamond, D., Li, Z., Perry, A. E., Spencer, S. K., Gandolfi, A. J., & Karagas, M. R. (2013). A population-based case-control study of urinary arsenic species and squamous cell carcinoma in New Hampshire, USA. Environmental health perspectives, 121(10), 1154-60.
  More info

  Chronic high arsenic exposure is associated with squamous cell carcinoma (SCC) of the skin, and inorganic arsenic (iAs) metabolites may play an important role in this association. However, little is known about the carcinogenicity of arsenic at levels commonly observed in the United States.
• Meza-Montenegro, M. M., Valenzuela-Quintanar, A. I., Balderas-Cortés, J. J., Yañez-Estrada, L., Gutiérrez-Coronado, M. L., Cuevas-Robles, A., & Gandolfi, A. J. (2013). Exposure assessment of organochlorine pesticides, arsenic, and lead in children from the major agricultural areas in Sonora, Mexico. Archives of environmental contamination and toxicology, 64(3), 519-27.
  More info

  There is a lack of information of exposure to organochlorine pesticides (OCP) and some metals, such as lead (Pb) and arsenic (As), both of which were used as arsenicals pesticides, in children living in the major agricultural areas of Mexico. The objective of this study was to assess the exposure of children to different OCP, As, and Pb in the Yaqui and Mayo valleys of Sonora to generate population baseline levels of these toxins. A cross-sectional study was undertaken in 165 children (age 6-12 years old) from 10 communities from both valleys during 2009. Blood samples were analyzed for OCP and Pb and first morning void urine for inorganic As (InAs). All of the blood samples had detectable levels of dichlorodiphenyltrichloroethylene (p,p'-DDE) ranging from 0.25 to 10.3 μg/L. However lindane, dichlorodiphenyltrichloroethane (p,p'-DDT), aldrin, and endosulfan were detected in far less of the population (36.4, 23.6, 9.1, and 3 %, respectively). Methoxychlor and endrin were not found in any sample. The average value of Pb in this population was 3.2 μg Pb/dL (range 0.17-9.0) with 8.5 % of the samples having levels 50 μg/L were observed in 12.7 % of the samples. Our results show that is important to start a risk-reduction program to decrease exposure to these toxins in Mexican communities. In addition, the results can be used to establish the baseline levels of exposure to these toxins in this agricultural region and may be used as a reference point for regulatory agencies.
• Canet, M. J., Hardwick, R. N., Lake, A. D., Kopplin, M. J., Scheffer, G. L., Klimecki, W. T., Gandolfi, A. J., & Cherrington, N. J. (2012). Altered arsenic disposition in experimental nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 40(9), 1817-24.
  More info

  Nonalcoholic fatty liver disease (NAFLD) is represented by a spectrum of liver pathologies ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Liver damage sustained in the progressive stages of NAFLD may alter the ability of the liver to properly metabolize and eliminate xenobiotics. The purpose of the current study was to determine whether NAFLD alters the disposition of the environmental toxicant arsenic. C57BL/6 mice were fed either a high-fat or a methionine-choline-deficient diet to model simple steatosis and NASH, respectively. At the conclusion of the dietary regimen, all mice were given a single oral dose of either sodium arsenate or arsenic trioxide. Mice with NASH excreted significantly higher levels of total arsenic in urine (24 h) compared with controls. Total arsenic in the liver and kidneys of NASH mice was not altered; however, NASH liver retained significantly higher levels of the monomethyl arsenic metabolite, whereas dimethyl arsenic was retained significantly less in the kidneys of NASH mice. NASH mice had significantly higher levels of the more toxic trivalent form in their urine, whereas the pentavalent form was preferentially retained in the liver of NASH mice. Moreover, hepatic protein expression of the arsenic biotransformation enzyme arsenic (3+ oxidation state) methyltransferase was not altered in NASH animals, whereas protein expression of the membrane transporter multidrug resistance-associated protein 1 was increased, implicating cellular transport rather than biotransformation as a possible mechanism. These results suggest that NASH alters the disposition of arsenical species, which may have significant implications on the overall toxicity associated with arsenic in NASH.
• Carranza-Rosales, P., Santiago-Mauricio, M. G., Guzmán-Delgado, N. E., Vargas-Villarreal, J., Lozano-Garza, G., Viveros-Valdez, E., Ortiz-López, R., Morán-Martínez, J., & Gandolfi, A. J. (2012). Induction of virulence factors, apoptosis, and cytokines in precision-cut hamster liver slices infected with Entamoeba histolytica. Experimental parasitology, 132(4), 424-33.
  More info

  Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24 h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis.
• Escudero-Lourdes, C., Wu, T., Camarillo, J. M., & Gandolfi, A. J. (2012). Interleukin-8 (IL-8) over-production and autocrine cell activation are key factors in monomethylarsonous acid [MMA(III)]-induced malignant transformation of urothelial cells. Toxicology and applied pharmacology, 258(1), 10-8.
  More info

  The association between chronic human exposure to arsenicals and bladder cancer development is well recognized; however, the underlying molecular mechanisms have not been fully determined. We propose that inflammatory responses can play a pathogenic role in arsenic-related bladder carcinogenesis. In previous studies, it was demonstrated that chronic exposure to 50 nM monomethylarsenous acid [MMA(III)] leads to malignant transformation of an immortalized model of urothelial cells (UROtsa), with only 3 mo of exposure necessary to trigger the transformation-related changes. In the three-month window of exposure, the cells over-expressed pro-inflammatory cytokines (IL-1β, IL-6 and IL-8), consistent with the sustained activation of NFKβ and AP1/c-jun, ERK2, and STAT3. IL-8 was over-expressed within hours after exposure to MMA(III), and sustained over-expression was observed during chronic exposure. In this study, we profiled IL-8 expression in UROtsa cells exposed to 50 nM MMA(III) for 1 to 5 mo. IL-8 expression was increased mainly in cells after 3 mo MMA(III) exposure, and its production was also found increased in tumors derived from these cells after heterotransplantation in SCID mice. UROtsa cells do express both receptors, CXCR1 and CXCR2, suggesting that autocrine cell activation could be important in cell transformation. Supporting this observation and consistent with IL-8 over-expression, CXCR1 internalization was significantly increased after three months of exposure to MMA(III). The expression of MMP-9, cyclin D1, bcl-2, and VGEF was significantly increased in cells exposed to MMA(III) for 3 mo, but these mitogen-activated kinases were significantly decreased after IL-8 gene silencing, together with a decrease in cell proliferation rate and in anchorage-independent colony formation. These results suggest a relevant role of IL-8 in MMA(III)-induced UROtsa cell transformation.
• Fei, D. L., Sanchez-Mejias, A., Wang, Z., Flaveny, C., Long, J., Singh, S., Rodriguez-Blanco, J., Tokhunts, R., Giambelli, C., Briegel, K. J., Schulz, W. A., Gandolfi, A. J., Karagas, M., Zimmers, T. A., Jorda, M., Bejarano, P., Capobianco, A. J., & Robbins, D. J. (2012). Hedgehog signaling regulates bladder cancer growth and tumorigenicity. Cancer research, 72(17), 4449-58.
  More info

  The role of Hedgehog (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in urothelial carcinoma cell lines was HH ligand dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, on the basis of the difference in intrinsic HH dependence of urothelial carcinoma cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer.
• Medeiros, M., Zheng, X., Novak, P., Wnek, S. M., Chyan, V., Escudero-Lourdes, C., & Gandolfi, A. J. (2012). Global gene expression changes in human urothelial cells exposed to low-level monomethylarsonous acid. Toxicology, 291(1-3), 102-12.
  More info

  Bladder cancer has been associated with chronic arsenic exposure. Monomethylarsonous acid [MMA(III)] is a metabolite of inorganic arsenic and has been shown to transform an immortalized urothelial cell line (UROtsa) at concentrations 20-fold less than arsenite. MMA(III) was used as a model arsenical to examine the mechanisms of arsenical-induced transformation of urothelium. A microarray analysis was performed to assess the transcriptional changes in UROtsa during the critical window of chronic 50nM MMA(III) exposure that leads to transformation at 3 months of exposure. The analysis revealed only minor changes in gene expression at 1 and 2 months of exposure, contrasting with substantial changes observed at 3 months of exposure. The gene expression changes at 3 months were analyzed showing distinct alterations in biological processes and pathways such as a response to oxidative stress, enhanced cell proliferation, anti-apoptosis, MAPK signaling, as well as inflammation. Twelve genes selected as markers of these particular biological processes were used to validate the microarray and these genes showed a time-dependent changes at 1 and 2 months of exposure, with the most substantial changes occurring at 3 months of exposure. These results indicate that there is a strong association between the acquired phenotypic changes that occur with chronic MMA(III) exposure and the observed gene expression patterns that are indicative of a malignant transformation. Although the substantial changes that occur at 3 months of exposure may be a consequence of transformation, there are common occurrences of altered biological processes between the first 2 months of exposure and the third, which may be pivotal in driving transformation.
• Meza-Montenegro, M. M., Gandolfi, A. J., Santana-Alcántar, M. E., Klimecki, W. T., Aguilar-Apodaca, M. G., Del Río-Salas, R., De la O-Villanueva, M., Gómez-Alvarez, A., Mendivil-Quijada, H., Valencia, M., & Meza-Figueroa, D. (2012). Metals in residential soils and cumulative risk assessment in Yaqui and Mayo agricultural valleys, northern Mexico. The Science of the total environment, 433, 472-81.
  More info

  This investigation examines the extent of soil metal pollution associated with the Green Revolution, relative to agricultural activities and associated risks to health in the most important agricultural region of Mexico. Metal contents in bulk soil samples are commonly used to assess contamination, and metal accumulations in soils are usually assumed to increase with decreasing particle size. This study profiled the spatial distribution of metals (Ni, Cr, Pb, Cu, Fe, Cd, V, Hg, Co, P, Se, and Mn) in bulk soil and fine-grained fractions (soil-derived dust) from 22 towns and cities. The contamination of soil was assessed through the use of a geoaccumulation index (Igeo) and pollution index (PI). The results of this study indicated that a number of towns and cities are moderately to highly polluted by soil containing Be, Co, Hg, P, S, V, Zn, Se, Cr, and Pb in both size fractions (coarse and fine). Hazard index in fine fraction (HI(children)=2.1) shows that risk assessment based on Co, Mn, V, and Ni spatially related to power plants, have the potential to pose health risks to local residents, especially children. This study shows that risk assessment based on metal content in bulk soil could be overestimated when compared to fine-grained fraction. Our results provide important information that could be valuable in establishing risk assessment associated with residential soils within agricultural areas, where children can ingest and inhale dust.
• Yassine, H., Kimzey, M. J., Galligan, M. A., Gandolfi, A. J., Stump, C. S., & Lau, S. S. (2012). Adjusting for Urinary Creatinine Overestimates Arsenic Concentrations in Diabetics. Cardiorenal medicine, 2(1), 26-32.
  More info

  BACKGROUND/AIMS: Arsenic (As) is linked to insulin resistance in animal studies, but the effect of low-level As exposure on the prevalence of diabetes in humans is uncertain. An optimal method to report inorganic As in humans has not been established. Measurements of As in spot urine are usually adjusted to creatinine (Cr). However, urinary Cr is an independent variable in diabetes. Our aims are to optimize reporting of urinary As in the setting of diabetes and insulin resistance. METHODS: Urinary inorganic As was measured in 24-hour or first-void spot urine from diabetic (n = 31) and non-diabetic (n = 12) subjects and normalized to Cr or specific gravity (SG). The relation of normalized urinary inorganic As to glycemia and surrogate measures of insulin resistance was investigated. Blood pressure, waist circumference, and glycated hemoglobin were also assessed. Homeostasis model assessment was used to determine insulin resistance. RESULTS: A strong correlation was found between spot urinary As adjusted to Cr (R(2) = 0.82) or SG (R(2) = 0.61) to 24-hour urinary As (p < 0.001), while non-adjusted urinary As did not correlate well (R(2) = 0.03, p = 0.46). Adjusting for Cr revealed significant differences in total 24-hour urinary As when comparing diabetic to normal subjects. In contrast, no differences were found when As was adjusted to SG using either 24-hour or spot urine. Moreover, adjusted urinary spot or 24-hour As measures did not correlate with measures of glycemia or insulin resistance. Conclusions: Urinary Cr is an independent variable in diabetes, therefore adjusting spot As for SG is preferred.
• Gilbert-Diamond, D., Cottingham, K. L., Gruber, J. F., Punshon, T., Sayarath, V., Gandolfi, A. J., Baker, E. R., Jackson, B. P., Folt, C. L., & Karagas, M. R. (2011). Rice consumption contributes to arsenic exposure in US women. Proceedings of the National Academy of Sciences of the United States of America, 108(51), 20656-60.
  More info

  Emerging data indicate that rice consumption may lead to potentially harmful arsenic exposure. However, few human data are available, and virtually none exist for vulnerable periods such as pregnancy. Here we document a positive association between rice consumption and urinary arsenic excretion, a biomarker of recent arsenic exposure, in 229 pregnant women. At a 6-mo prenatal visit, we collected a urine sample and 3-d dietary record for water, fish/seafood, and rice. We also tested women's home tap water for arsenic, which we combined with tap water consumption to estimate arsenic exposure through water. Women who reported rice intake (n = 73) consumed a median of 28.3 g/d, which is ∼0.5 cup of cooked rice each day. In general linear models adjusted for age and urinary dilution, both rice consumption (g, dry mass/d) and arsenic exposure through water (μg/d) were significantly associated with natural log-transformed total urinary arsenic (βrice = 0.009, βwater = 0.028, both P < 0.0001), as well as inorganic arsenic, monomethylarsonic acid, and dimethylarsinic acid (each P < 0.005). Based on total arsenic, consumption of 0.56 cup/d of cooked rice was comparable to drinking 1 L/d of 10 μg As/L water, the current US maximum contaminant limit. US rice consumption varies, averaging ∼0.5 cup/d, with Asian Americans consuming an average of >2 cups/d. Rice arsenic content and speciation also vary, with some strains predominated by dimethylarsinic acid, particularly those grown in the United States. Our findings along with others indicate that rice consumption should be considered when designing arsenic reduction strategies in the United States.
• Wnek, S. M., Kuhlman, C. L., Camarillo, J. M., Medeiros, M. K., Liu, K. J., Lau, S. S., & Gandolfi, A. J. (2011). Interdependent genotoxic mechanisms of monomethylarsonous acid: role of ROS-induced DNA damage and poly(ADP-ribose) polymerase-1 inhibition in the malignant transformation of urothelial cells. Toxicology and applied pharmacology, 257(1), 1-13.
  More info

  Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA(III)), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA(III) exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA(III), PARP-1 activity does not increase despite the increase in MMA(III)-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA(III) exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA(III) indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA(III). The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA(III) to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA(III) to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA(III) exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA(III). Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA(III)-induced reactive oxygen species, or alter Cu-Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMA(III) may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMA(III)-induced DNA damage through the production of reactive oxygen species, and the direct MMA(III)-induced inhibition of PARP-1.
• Carranza-Rosales, P., Santiago-Mauricio, M. G., Guzmán-Delgado, N. E., Vargas-Villarreal, J., Lozano-Garza, G., Ventura-Juárez, J., Balderas-Rentería, I., Morán-Martínez, J., & Gandolfi, A. J. (2010). Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess. Experimental parasitology, 126(2), 117-25.
  More info

  Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.
• Escudero-Lourdes, C., Medeiros, M. K., Cárdenas-González, M. C., Wnek, S. M., & Gandolfi, J. A. (2010). Low level exposure to monomethyl arsonous acid-induced the over-production of inflammation-related cytokines and the activation of cell signals associated with tumor progression in a urothelial cell model. Toxicology and applied pharmacology, 244(2), 162-73.
  More info

  Human bladder cancer has been associated with chronic exposure to arsenic. Chronic exposure of an immortalized non-tumorigenic urothelial cell line (UROtsa cells) to arsenicals has transformed these cells to a malignant phenotype, but the involved mechanisms are not fully understood. Chronic inflammation has been linked with cancer development mainly because many pro-inflammatory cytokines, growth factors as well as angiogenic chemokines have been found in tumors. In this study the chronology of inflammatory cytokines production was profiled in UROtsa cells chronically exposed to the toxic arsenic metabolite, monomethylarsonous acid [50 nM MMA(III)] to know the role of inflammation in cell transformation. Acute 50 nM MMA(III) exposure induced over-production of many pro-inflammatory cytokines as soon as 12 h after acute exposure. The same cytokines remain over-regulated after chronic exposure to 50 nM MMA(III), especially after 3 mo exposure. At 3 mo exposure the sustained production of cytokines like IL-1, IL-6, IL-8 and TNF is coincident with the appearance of characteristics associated with cell transformation seen in other arsenic-UROtsa studies. The sustained and increased activation of NFkappaB and c-Jun is also present along the transformation process and the phosphorylated proteins p38 MAPK and ERK 1/2 are increased also through the time line. Taken together these results support the notion that chronic inflammation is associated within MMA(III)-induced cell transformation and may act as a promoting factor in UROtsa cell transformation.
• Wnek, S. M., Jensen, T. J., Severson, P. L., Futscher, B. W., & Gandolfi, A. J. (2010). Monomethylarsonous acid produces irreversible events resulting in malignant transformation of a human bladder cell line following 12 weeks of low-level exposure. Toxicological sciences : an official journal of the Society of Toxicology, 116(1), 44-57.
  More info

  Arsenic is a known human bladder carcinogen; however, the mechanisms underlying arsenical-induced bladder carcinogenesis are not understood. Previous research has demonstrated that exposure of a nontumorigenic human urothelial cell line, UROtsa, to 50 nM monomethylarsonous acid (MMA(III)) for 52 weeks resulted in malignant transformation. To focus research on the early mechanistic events leading to MMA(III)-induced malignancy, the goal of this research was to resolve the critical period in which continuous MMA(III) exposure (50 nM) induces the irreversible malignant transformation of UROtsa cells. An increased growth rate of UROtsa cells results after 12 weeks of MMA(III) exposure. Anchorage-independent growth occurred after 12 weeks with a continued increase in colony formation when 12-week exposed cells were cultured for an additional 12 or 24 weeks without MMA(III) exposure. UROtsa cells as early as 12 weeks MMA(III) exposure were tumorigenic in severe combined immunodeficiency mice with tumorigenicity increasing when 12-week exposed cells were cultured for an additional 12 or 24 weeks in the absence of MMA(III) exposure. To assess potential underlying mechanisms associated with the early changes that occur during MMA(III)-induced malignancy, DNA methylation was assessed in known target gene promoter regions. Although DNA methylation remains relatively unchanged after 12 weeks of exposure, aberrant DNA methylation begins to emerge after an additional 12 weeks in culture and continues to increase through 24 weeks in culture without MMA(III) exposure, coincident with the progression of a tumorigenic phenotype. Overall, these data demonstrate that 50 nM MMA(III) is capable of causing irreversible malignant transformation in UROtsa cells after 12 weeks of exposure. Having resolved an earlier timeline in which MMA(III)-induced malignant transformation occurs in UROtsa cells will allow for mechanistic studies focused on the critical biological changes taking place within these cells prior to 12 weeks of exposure, providing further evidence about potential mechanisms of MMA(III)-induced carcinogenesis.
• Alegría-Torres, J. A., Díaz-Barriga, F., Gandolfi, A. J., & Pérez-Maldonado, I. N. (2009). Mechanisms of p,p'-DDE-induced apoptosis in human peripheral blood mononuclear cells. Toxicology in vitro : an international journal published in association with BIBRA, 23(6), 1000-6.
  More info

  p,p'-Dichlorodiphenyldichloroethylene (DDE), the most stable metabolite of organochlorine insecticide p,p'-dichlorodiphenyltrichloroethane (DDT), has been detected in human populations living in malaria-endemic areas of México where this insecticide was used. DDE induces apoptosis in peripheral blood mononuclear cells (PMBC); however, the molecular mechanism of cell death induced by this compound is poorly understood. In the present study, PBMC isolated from healthy individuals (not exposed to DDE) were incubated in the presence of increasing concentrations of p,p'-DDE (0-80 microg/ml) over time. When PBMC were treated with low p,p'-DDE concentration (10 microg/ml) an antioxidant response and biomarkers of inflammation were induced, indicating a pro-inflammatory state. Moreover, when PBMC were treated with high p,p'-DDE concentration (80 microg/ml) several apoptotic biochemical events were triggered, such as activation of caspase-8, Bid, caspase-9 and caspase-3, as well as degradation of PARP and ubiquitination. The results described in this study show a possible inflammatory condition and the involvement of both extrinsic and intrinsic pathways in the induction of apoptosis in DDE-treated PBMC.
• Eblin, K. E., Jensen, T. J., Wnek, S. M., Buffington, S. E., Futscher, B. W., & Gandolfi, A. J. (2009). Reactive oxygen species regulate properties of transformation in UROtsa cells exposed to monomethylarsonous acid by modulating MAPK signaling. Toxicology, 255(1-2), 107-14.
  More info

  UROtsa cells exposed to 50 nM monomethylarsonous acid [MMA(III)] for 52 wk (MSC52) achieved hyperproliferation, anchorage independent growth, and enhanced tumorgenicity. MMA(III) has been shown to induce reactive oxygen species (ROS), which can lead to activation of signaling cascades causing stress-related proliferation of cells and even cellular transformation. Previous research established the acute activation of MAPK signaling cascade by ROS produced by MMA(III) as well as chronic up regulation of COX-2 and EGFR in MSC52 cells. To determine if ROS played a role in the chronic pathway perturbations by acting as secondary messengers, activation of Ras was determined in UROtsa cells [exposed to MMA(III) for 0-52 wk] and found to be increased through 52 wk most dramatically after 20 wk of exposure. Ras has been shown to cause an increase in O2(-) and be activated by increases in O2(-), making ROS important to study in the transformation process. COX-2 upregulation in MSC52 cells was confirmed by real time RT-PCR. By utilizing both antioxidants or specific COX inhibitors, it was shown that COX-2 upregulation was dependent on ROS, specifically, O2(-). In addition, because previous research established the importance of MAPK activation in phenotypic changes associated with transformation in MSC52 cells, it was hypothesized that ROS play a role in maintaining phenotypic characteristics of the malignant transformation of MSC52 cells. Several studies have demonstrated that cancer cells have lowered superoxide dismutase (MnSOD) activity and protein levels. Increasing levels of MnSOD have been shown to suppress the malignant phenotype of cells. SOD was added to MSC52 cells resulting in slower proliferation rates (doubling time=42h vs. 31h). ROS scavengers of OH also slowed proliferation rates of MSC52 cells. To further substantiate the importance of ROS in these properties of transformation in MSC52 cells, anchorage independent growth was assessed after the addition of antioxidants, both enzymatic and non-enzymatic. Scavengers of OH, and O2(-) blocked the colony formation of MSC52 cells. These data support the role for the involvement of ROS in properties of transformation of UROtsa cells exposed to MMA(III).
• Jensen, T. J., Novak, P., Wnek, S. M., Gandolfi, A. J., & Futscher, B. W. (2009). Arsenicals produce stable progressive changes in DNA methylation patterns that are linked to malignant transformation of immortalized urothelial cells. Toxicology and applied pharmacology, 241(2), 221-9.
  More info

  Aberrant DNA methylation participates in carcinogenesis and is a molecular hallmark of a tumor cell. Tumor cells generally exhibit a redistribution of DNA methylation resulting in global hypomethylation with regional hypermethylation; however, the speed in which these changes emerge has not been fully elucidated and may depend on the temporal location of the cell in the path from normal, finite lifespan to malignant transformation. We used a model of arsenical-induced malignant transformation of immortalized human urothelial cells and DNA methylation microarrays to examine the extent and temporal nature of changes in DNA methylation that occur during the transition from immortal to malignantly transformed. Our data presented herein suggest that during arsenical-induced malignant transformation, aberrant DNA methylation occurs non-randomly, progresses gradually at hundreds of gene promoters, and alters expression of the associated gene, and these changes are coincident with the acquisition of malignant properties, such as anchorage independent growth and tumor formation in immunocompromised mice. The DNA methylation changes appear stable, since malignantly transformed cells removed from the transforming arsenical exhibited no reversion in DNA methylation levels, associated gene expression, or malignant phenotype. These data suggest that arsenicals act as epimutagens and directly link their ability to induce malignant transformation to their actions on the epigenome.
• Jensen, T. J., Wozniak, R. J., Eblin, K. E., Wnek, S. M., Gandolfi, A. J., & Futscher, B. W. (2009). Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation. Toxicology and applied pharmacology, 235(1), 39-46.
  More info

  Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.
• Wnek, S. M., Medeiros, M. K., Eblin, K. E., & Gandolfi, A. J. (2009). Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid. Toxicology and applied pharmacology, 241(2), 202-9.
  More info

  Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA(III)); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA(III) [URO-MSC(+)] and after subsequent removal of MMA(III) [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA(III), URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA(III). URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA(III) exposure, suggesting the presence of MMA(III) is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA(III) results in: the induction of DNA damage that remains elevated upon removal of MMA(III); increased levels of ROS that play a role in MMA(III) induced-DNA damage; and decreased PARP activity in the presence of MMA(III).
• Eblin, K. E., Bredfeldt, T. G., & Gandolfi, A. J. (2008). Immortalized human urothelial cells as a model of arsenic-induced bladder cancer. Toxicology, 248(2-3), 67-76.
  More info

  Arsenical-induced carcinogenesis in human bladder has been established through epidemiological evidence, and UROtsa cells, a normal, immortalized cell culture model of human urothelium, have proven to be a good model for the bladder epithelium. This cell line does not form tumors when injected into immuno-compromised mice nor does it have anchorage-independent growth. UROtsa can be easily manipulated for acute studies related to arsenical exposure. They have been shown to be sensitive to all arsenicals, in particular, the trivalent species, arsenite and monomethylarsonous acid. UROtsa cells have also opened the area of cellular signaling alterations following subcytotoxic exposure to arsenicals in both the acute and long-term time points. In addition, UROtsa cells were shown to be malignantly transformed following low-level exposure to both As(III) and MMA(III) providing additional models for studying arsenical-induced carcinogenesis of the bladder. These transformed cell lines allow researchers the ability to investigate the process of urothelial tumorigenesis at multiple time points of arsenical exposure. Overall, UROtsa cells are an effective model for cellular insult following arsenical exposure.
• Eblin, K. E., Hau, A. M., Jensen, T. J., Futscher, B. W., & Gandolfi, A. J. (2008). The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies. Toxicology, 250(1), 47-54.
  More info

  Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.
• Jensen, T. J., Novak, P., Eblin, K. E., Gandolfi, A. J., & Futscher, B. W. (2008). Epigenetic remodeling during arsenical-induced malignant transformation. Carcinogenesis, 29(8), 1500-8.
  More info

  Humans are exposed to arsenicals through many routes with the most common being in drinking water. Exposure to arsenic has been associated with an increase in the incidence of cancer of the skin, lung and bladder. Although the relationship between exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation state of 13 000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation occur during arsenical-induced malignant transformation that are linked to the expression state of the associated gene. DNA hypermethylation was detected in hypoacetylated promoters in the select cases analyzed. These epigenetic changes occurred frequently in the same promoters whether the selection was performed with arsenite [As(III)] or with monomethylarsonous acid, suggesting that these promoters were targeted in a non-random fashion, and probably occur in regions important in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by altering the epigenetic terrain of select genes.
• Carranza-Rosales, P., Guzmán-Delgado, N. E., Cruz-Vega, D. E., Balderas-Rentería, I., & Gandolfi, A. J. (2007). DMPS reverts morphologic and mitochondrial damage in OK cells exposed to toxic concentrations of HgCl2. Cell biology and toxicology, 23(3), 163-76.
  More info

  Mercuric chloride (HgCl(2)) is a highly toxic compound, which can cause nephrotoxic damage. In the present study effects of HgCl(2) on mitochondria integrity and energy metabolism, as well as antidotal effects of 2,3-dimercaptopropane-1-sulfonate (DMPS) were investigated in the opossum kidney derived cell line (OK). OK cell monolayers were incubated during 0, 1, 3, 6, and 9 h in serum-free culture medium containing 15 microM HgCl(2), either in the absence or in the presence of 60 microM DMPS in a 1:4 ratio. Intracellular ATP content, MTT reduction, and HSP70/HSP90 induction were studied; confocal, transmission electron microscopy, and light microscopy studies were also performed. For confocal analysis, a mitochondrial selective probe (MitoTracker Red CMXH2Ros) was used. Antioxidant activity of DMPS was also studied by the scavenging of the free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique. A decrease of ATP content, an impaired ability to reduce tetrazolium, and dramatic changes on cellular and mitochondrial morphology, and energetic levels were found after either 6 or 9 h of HgCl(2) exposure. Increased expression of HSP90 and HSP70 were also seen. When OK cells were co-incubated with HgCl(2) and DMPS, cellular morphology, viability, intracellular ATP, and mitochondrial membrane potential were partially restored; a protective effect on mitochondrial morphology was also seen. DMPS also showed potent antioxidant activity in vitro. Mitochondrial protection could be the cellular mechanism mediated by DMPS in OK cells exposed to a toxic concentration of HgCl(2).
• Catania, J. M., Pershing, A. M., & Gandolfi, A. J. (2007). Precision-cut tissue chips as an in vitro toxicology system. Toxicology in vitro : an international journal published in association with BIBRA, 21(5), 956-61.
  More info

  Precision-cut tissue slices mimic specific organ toxicity because normal cellular heterogeneity and organ architecture are retained. To optimize the use of the smaller tissues of the mouse and to establish easy assays for tissue viability, a tissue chip based system was used to generate large numbers of samples from a single organ. Iodoacetamide (IAM) was used as a model toxicant and assays for intracellular potassium (normalized to DNA content) were used to establish viability and toxicant susceptibility. Thereafter, assays that were more rapid and specific were pursued. Lysates from tissues incubated in 6-carboxyfluorescein fluoresced proportionately to concentrations of IAM, indicating disruption of cellular membranes. Similarly, FURA-2, a probe applied to lysates to measure calcium levels, fluoresced proportionately to IAM dosage. Monobromobimane, a fluorescent sulfhydryl probe, displayed a decrease in fluorescent intensity at higher IAM challenge-a finding confirmed with an absorbance assay with Ellman's reagent. Importantly, the number of samples per organ/mouse was increased at least threefold and a significant time reduction per analysis was realized.
• Eblin, K. E., Bredfeldt, T. G., Buffington, S., & Gandolfi, A. J. (2007). Mitogenic signal transduction caused by monomethylarsonous acid in human bladder cells: role in arsenic-induced carcinogenesis. Toxicological sciences : an official journal of the Society of Toxicology, 95(2), 321-30.
  More info

  Previous studies have shown that human bladder cells (UROtsa), a target of arsenic-induced cancer, can biotransform arsenite to monomethylarsonous acid (MMA(III)), which is more cytotoxic and capable of transforming the UROtsa cells following long-term, low-level exposure. Cyclooxygenase-2 (COX-2) causes hyperplasia in bladder cells and is considered a key biomarker in bladder cancer. To investigate the role of mitogenic pathway stimulation in MMA(III)-induced transformation, UROtsa cells were treated with 50nM MMA(III) for 12, 24, or 52 weeks and analyzed by Western blot for COX-2 expression. Elevations in COX-2 expression were noted following chronic MMA(III) exposure, and this induction increased with duration of exposure, suggesting that COX-2 or the signal transduction pathways responsible for COX-2 protein expression may play a role in MMA(III)-induced transformation. Acute exposure studies found MMA(III) treatment (10, 50, and 100nM, 4 h) induced COX-2 in UROtsa cells with the lowest doses (10 and 50nM) causing the strongest induction. Using pharmacological inhibitors of various pathways, it was shown that epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK-1/-2), phosphoinositide 3-kinase (PI3K), and src were important in the induction of COX-2 by MMA(III). ERK-2 phosphorylation was verified by Western blot analysis with a peak at 15 min, and c-jun was translocated to the nucleus following 50nM MMA(III) treatment. To determine MMA(III) targets, receptors of the erythroblastosis oncogene family (ErbB) family were further investigated. Chronic MMA(III) exposure led to upregulation of the EGFR or ErbB1. Short-term MMA(III) treatment caused the phosphorylation of ErbB2 in its autophosphorylation site. To verify the importance of these signaling pathways to the growth of the MMA(III)-transformed UROtsa cells in soft agar, various inhibitors were used to block pathways and monitor cells growth. Pathways of importance in anchorage-independent growth of UROtsa cells chronically exposed to MMA(III) are src, PI3K, and COX-1 and -2. As COX-2 is an important mediator that contributes to carcinogenesis via promotion of cell proliferation, inhibition of cell death, induction of angiogenesis, and facilitation of invasion, and it is highly upregulated both acutely and chronically in the MMA(III)-transformed cells, it is likely that activation of the mitogen-activated protein kinase pathway and increased COX-2 expression is a plausible mechanism for MMA(III) bladder carcinogenesis.
• Meza, M., Gandolfi, A. J., & Klimecki, W. T. (2007). Developmental and genetic modulation of arsenic biotransformation: a gene by environment interaction?. Toxicology and applied pharmacology, 222(3), 381-7.
  More info

  The complexity of arsenic toxicology has confounded the identification of specific pathways of disease causation. One focal point of arsenic research is aimed at fully characterizing arsenic biotransformation in humans, a process that appears to be quite variable, producing a mixture of several arsenic species with greatly differing toxic potencies. In an effort to characterize genetic determinants of variability in arsenic biotransformation, a genetic association study of 135 subjects in western Sonora, Mexico was performed by testing 23 polymorphic sites in three arsenic biotransformation candidate genes. One gene, arsenic 3 methyltransferase (AS3MT), was strongly associated with the ratio of urinary dimethylarsinic acid to monomethylarsonic acid (D/M) in children (7-11 years) but not in adults (18-79 years). Subsequent analyses revealed that the high D/M values associated with variant AS3MT alleles were primarily due to lower levels of monomethylarsonic acid as percent of total urinary arsenic (%MMA5). In light of several reports of arsenic-induced disease being associated with relatively high %MMA5 levels, these findings raise the possibility that variant AS3MT individuals may suffer less risk from arsenic exposure than non-variant individuals. These analyses also provide evidence that, in this population, regardless of AS3MT variant status, children tend to have lower %MMA5 values than adults, suggesting that the global developmental regulation of arsenic biotransformation may interact with genetic variants in metabolic genes to result in novel genetic effects such as those in this report.
• Wang, X. J., Sun, Z., Chen, W., Eblin, K. E., Gandolfi, J. A., & Zhang, D. D. (2007). Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity. Toxicology and applied pharmacology, 225(2), 206-13.
  More info

  Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using a UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2 pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III) and MMA(III). Furthermore, the wild-type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF, whereas neither tBHQ nor SF conferred protection in the Nrf2(-/-)MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.
• Bredfeldt, T. G., Jagadish, B., Eblin, K. E., Mash, E. A., & Gandolfi, A. J. (2006). Monomethylarsonous acid induces transformation of human bladder cells. Toxicology and applied pharmacology, 216(1), 69-79.
  More info

  Arsenic is a human bladder carcinogen. Arsenic is methylated to both monomethyl and dimethyl metabolites which have been detected in human urine. The trivalent methylated arsenicals are more toxic than inorganic arsenic. It is unknown if these trivalent methylated metabolites can directly cause malignant transformation in human cells. The goal of this study is determine if monomethylarsonous acid (MMA(III)) can induce malignant transformation in a human bladder urothelial cell line. To address this goal, a non-tumorigenic human urothelial cell line (UROtsa) was continuously exposed to 0.05 muM MMA(III) for 52 weeks. Hyperproliferation was the first phenotypic change observed in exposed UROtsa (URO-MSC). After 12 weeks of exposure, doubling time had decreased from 42 h in unexposed control cells to 27 h in URO-MSC. Hyperproliferation continued to be a quality possessed by the URO-MSC cells after both 24 and 52 weeks of exposure to MMA(III), which had a 40-50% reduction in doubling time. Throughout the 52-week exposure, URO-MSC cells retained an epithelial morphology with subtle morphological differences from control cells. 24 weeks of MMA(III) exposure was required to induce anchorage-independent growth as detected by colony formation in soft agar, a characteristic not found in UROtsa cells. To further substantiate that malignant transformation had occurred, URO-MSC cells were tested after 24 and 52 weeks of exposure to MMA(III) for the ability to form tumors in SCID mice. Enhanced tumorigenicity in SCID mouse xenografts was observed after 52 weeks of treatment with MMA(III). These observations are the first demonstration of MMA(III)-induced malignant transformation in a human bladder urothelial cell line and provide important evidence that MMA(III) may be carcinogenic in human tissues.
• Conley, S. M., McKay, B. S., Gandolfi, A. J., & Stamer, W. D. (2006). Alterations in human trabecular meshwork cell homeostasis by selenium. Experimental eye research, 82(4), 637-47.
  More info

  Epidemiological evidence indicates that selenium supplementation may increase risk for glaucoma and ocular hypertension. The purpose of this study was to determine the effects of selenium on trabecular meshwork cells, a likely site of pathology for glaucoma. Human trabecular meshwork (HTM) cells and human umbilical vein endothelial cells (HUVECs) were treated with selenium (MSeA) at or near physiologically relevant concentrations. Selenium uptake by cells was monitored using mass spectrometry. Alterations in protein secretion, intracellular signaling, and cell morphology were monitored; and the role of integrin signaling in MSeA-induced morphological alterations was investigated using divalent cation treatments. Radiolabeling was used to assess protein synthesis and secretion, while luciferase and MTT assays monitored total cellular ATP and cell viability, respectively. Whereas detectible changes in intracellular selenium were observed after exposure to 1-10 microM MSeA for 24hr, the majority remained in the conditioned medium. Selenium-induced morphological changes (< or =3 hr) occurred before alterations in protein secretion and intracellular signaling (3-6 hr). Zinc treatment prevented selenium-mediated alterations in protein secretion and changes in cell-matrix adhesion. MSeA treatment (5 microM) led to a 60% decrease in protein synthesis after 3 hr and a 30% reduction in secretion, although significant alterations in cell viability and total ATP were not observed after MSeA treatment. Selenium altered several indicators of HTM cell homeostasis, but did not affect viability at physiologically relevant doses. Similar results with HUVECs have implications for understanding selenium's mechanisms of action as an anti-angiogenic agent.
• Cortinas, I., Field, J. A., Kopplin, M., Garbarino, J. R., Gandolfi, A. J., & Sierra-Alvarez, R. (2006). Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids. Environmental science & technology, 40(9), 2951-7.
  More info

  Large quantities of arsenic are introduced into the environment through land application of poultry litter containing the organoarsenical feed additive roxarsone (3-nitro-4-hydroxyphenylarsonic acid). The objective of this study was to evaluate the bioconversion of roxarsone and related N-substituted phenylarsonic acid derivatives under anaerobic conditions. The results demonstrate that roxarsone is rapidly transformed in the absence of oxygen to the corresponding aromatic amine, 4-hydroxy-3-aminophenylarsonic acid (HAPA). The formation of HAPA is attributable to the facile reduction of the nitro group. Electron-donating substrates, such as hydrogen gas, glucose, and lactate, stimulated the rate of nitro group reduction, indicating a microbial role. During long-term incubations, HAPA and the closely related 4-aminophenylarsonic acid (4-APA) were slowly biologically eliminated by up to 99% under methanogenic and sulfate-reducing conditions, whereas little or no removal occurred in heat-killed inoculum controls. Arsenite and, to a lesser extent, arsenate were observed as products of the degradation. Freely soluble forms of the inorganic arsenical species accounted for 19-28% of the amino-substituted phenylarsonic acids removed. This constitutes the first report of a biologically catalyzed rupture of the phenylarsonic group under anaerobic conditions.
• Eblin, K. E., Bowen, M. E., Cromey, D. W., Bredfeldt, T. G., Mash, E. A., Lau, S. S., & Gandolfi, A. J. (2006). Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture. Toxicology and applied pharmacology, 217(1), 7-14.
  More info

  Arsenicals have commonly been seen to induce reactive oxygen species (ROS) which can lead to DNA damage and oxidative stress. At low levels, arsenicals still induce the formation of ROS, leading to DNA damage and protein alterations. UROtsa cells, an immortalized human urothelial cell line, were used to study the effects of arsenicals on the human bladder, a site of arsenical bioconcentration and carcinogenesis. Biotransformation of As(III) by UROtsa cells has been shown to produce methylated species, namely monomethylarsonous acid [MMA(III)], which has been shown to be 20 times more cytotoxic. Confocal fluorescence images of UROtsa cells treated with arsenicals and the ROS sensing probe, DCFDA, showed an increase of intracellular ROS within five min after 1 microM and 10 microM As(III) treatments. In contrast, 50 and 500 nM MMA(III) required pretreatment for 30 min before inducing ROS. The increase in ROS was ameliorated by preincubation with either SOD or catalase. An interesting aspect of these ROS detection studies is the noticeable difference between concentrations of As(III) and MMA(III) used, further supporting the increased cytotoxicity of MMA(III), as well as the increased amount of time required for MMA(III) to cause oxidative stress. These arsenical-induced ROS produced oxidative DNA damage as evidenced by an increase in 8-hydroxyl-2'-deoxyguanosine (8-oxo-dG) with either 50 nM or 5 microM MMA(III) exposure. These findings provide support that MMA(III) cause a genotoxic response upon generation of ROS. Both As(III) and MMA(III) were also able to induce Hsp70 and MT protein levels above control, showing that the cells recognize the ROS and respond. As(III) rapidly induces the formation of ROS, possibly through it oxidation to As(V) and further metabolism to MMA(III)/(V). These studies provide evidence for a different mechanism of MMA(III) toxicity, one that MMA(III) first interacts with cellular components before an ROS response is generated, taking longer to produce the effect, but with more substantial harm to the cell.
• Field, J. A., Gandolfi, A. J., Garbarino, J. R., Kopplin, M., Sierra-Alvarez, R., & Yenal, U. (2006). Anaerobic Biotransformation of Organoarsenical Pesticides Monomethylarsonic Acid and Dimethylarsinic Acid. Journal of Agricultural and Food Chemistry, 54(11), 3959-3966. doi:10.1021/jf053223n
  More info

  Monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) are extensively utilized as pesticides, introducing large quantities of arsenic into the environment. Once released into the environment, these organo-arsenicals are subject to microbial reactions. Aerobic biodegradation of MMAV and DMAV has been evaluated, but little is known about their fate in anaerobic environments. The objective of this study was to evaluate the biotransformation of MMAV and DMAV in anaerobic sludge. Biologically mediated conversion occurred under methanogenic or sulfate-reducing conditions but not in the presence of nitrate. Monomethylarsonous acid (MMAIII) was consistently observed as an important metabolite of MMAV degradation, and it was recovered in molar yields ranging from 5 to 47%. The main biotransformation product identified from DMAV metabolism was MMAV, which was recovered in molar yields ranging from 8 to 65%. The metabolites indicate that reduction and demethylation are important steps in the anaerobic bioconversion of MMAV and DMAV, respectively.
• Sierra-Alvarez, R., Yenal, U., Field, J. A., Kopplin, M., Gandolfi, A. J., & Garbarino, J. R. (2006). Anaerobic biotransformation of organo-arsenical pesticides monomethylarsonic acid and dimethylarsinic acid. Journal of agricultural and food chemistry, 54(11), 3959-66.
  More info

  Monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) are extensively utilized as pesticides, introducing large quantities of arsenic into the environment. Once released into the environment, these organo-arsenicals are subject to microbial reactions. Aerobic biodegradation of MMAV and DMAV has been evaluated, but little is known about their fate in anaerobic environments. The objective of this study was to evaluate the biotransformation of MMAV and DMAV in anaerobic sludge. Biologically mediated conversion occurred under methanogenic or sulfate-reducing conditions but not in the presence of nitrate. Monomethylarsonous acid (MMAIII) was consistently observed as an important metabolite of MMAV degradation, and it was recovered in molar yields ranging from 5 to 47%. The main biotransformation product identified from DMAV metabolism was MMAV, which was recovered in molar yields ranging from 8 to 65%. The metabolites indicate that reduction and demethylation are important steps in the anaerobic bioconversion of MMAV and DMAV, respectively.
• Carranza-Rosales, P., Said-Fernández, S., Sepúlveda-Saavedra, J., Cruz-Vega, D. E., & Gandolfi, A. J. (2005). Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage. Toxicology, 210(2-3), 111-21.
  More info

  Mercury produces acute renal failure in experimental animal models, but the mechanism of tubular injury has not completely been clarified. There is an increased interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. However, detailed studies of morpho-functional alterations induced by mercuric chloride in kidney cell lines are scarce. This work characterizes these alterations in OK cell cultures. Morphological alterations were profiled using light microscopy, transmission electron microscopy, and confocal microscopy, as well as mitochondrial functional assays in the cells exposed to low concentrations of HgCl2. At concentrations of 1 and 10 microM of HgCl2 there were no morphological or ultrastructural alterations, but the mitochondrial function (MTT assay) and intracellular ATP content was increased, especially at longer incubation times (6 and 9 h). At 15 microM HgCl2, both the mitochondrial activity and the endogenous ATP decreased significantly. At this concentration the OK cells rounded up, had increased number of cytoplasmic vacuoles, and detached from the cell monolayer. At 15 microM HgCl2 ultrastructural changes were characterized by dispersion of the ribosomes, dilatation of the cisterns of the rough endoplasmic reticulum, increase of number of cytoplasmic vacuoles, chromatin condensation, invaginations of the nuclear envelope, presence of cytoplasmic inclusion bodies, and alterations in the size and morphology of mitochondria. At 15 microM HgCl2 apoptotic signs included membrane blebbing, chromatin condensation, mitochondrial alterations, apoptotic bodies, and nuclear envelope rupture. Using confocal microscopy and the mitochondrial specific dye MitoTracker Red, it was possible to establish qualitative changes induced by mercury on the mitochondrial membrane potential after incubation of the cells for 6 and 9h with 15 microM HgCl2. This effect was not observed at short times (1 and 3h) with this same concentration, neither with 1 and 10 microM HgCl2 in all the studied times. Taken together, these findings indicate that low concentrations of HgCl2 induce apoptosis by inhibiting mitochondrial function, and the OK cell line may be considered a useful tool for the study of programmed cell death involving mercurial species and other heavy metals.
• Kirkpatrick, D. S., Weldon, S. F., Tsaprailis, G., Liebler, D. C., & Gandolfi, A. J. (2005). Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin. Proteomics, 5(8), 2104-11.
  More info

  A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.
• Meza, M. M., Yu, L., Rodriguez, Y. Y., Guild, M., Thompson, D., Gandolfi, A. J., & Klimecki, W. T. (2005). Developmentally restricted genetic determinants of human arsenic metabolism: association between urinary methylated arsenic and CYT19 polymorphisms in children. Environmental health perspectives, 113(6), 775-81.
  More info

  We report the results of a screen for genetic association with urinary arsenic metabolite levels in three arsenic metabolism candidate genes, PNP, GSTO, and CYT19, in 135 arsenic-exposed subjects from the Yaqui Valley in Sonora, Mexico, who were exposed to drinking water concentrations ranging from 5.5 to 43.3 ppb. We chose 23 polymorphic sites to test in the arsenic-exposed population. Initial phenotypes evaluated included the ratio of urinary inorganic arsenic(III) to inorganic arsenic(V) and the ratio of urinary dimethylarsenic(V) to monomethylarsenic(V) (D:M). In the initial association screening, three polymorphic sites in the CYT19 gene were significantly associated with D:M ratios in the total population. Subsequent analysis of this association revealed that the association signal for the entire population was actually caused by an extremely strong association in only the children (7-11 years of age) between CYT19 genotype and D:M levels. With children removed from the analysis, no significant genetic association was observed in adults (18-79 years). The existence of a strong, developmentally regulated genetic association between CYT19 and arsenic metabolism carries import for both arsenic pharmacogenetics and arsenic toxicology, as well as for public health and governmental regulatory officials.
• Schmelz, M., Moll, R., Hesse, U., Prasad, A. R., Gandolfi, J. A., Hasan, S. R., Bartholdi, M., & Cress, A. E. (2005). Identification of a stem cell candidate in the normal human prostate gland. European journal of cell biology, 84(2-3), 341-54.
  More info

  Stem cells of the human prostate gland have not yet been identified utilizing a structural biomarker. We have discovered a new prostatic epithelial cell phenotype-expressing cytokeratin 6a (Ck6a+ cells). The Ck6a+ cells are present within a specialized niche in the basal cell compartment in fetal, juvenile and adult prostate tissue, and within the stem cell-enriched urogenital sinus. In adult normal prostate tissue, the average abundance of Ck6a+ cells was 4.9%. With proliferative stimuli in the prostate organ culture model, in which the epithelial-stromal interaction was maintained, a remarkable increase of Ck6a expression was noticed to up to 64.9%. The difference in cytokeratin 6a expression between the normal adult prostate and the prostate organ culture model was statistically significant (p
• Bredfeldt, T. G., Kopplin, M. J., & Gandolfi, A. J. (2004). Effects of arsenite on UROtsa cells: low-level arsenite causes accumulation of ubiquitinated proteins that is enhanced by reduction in cellular glutathione levels. Toxicology and applied pharmacology, 198(3), 412-8.
  More info

  Chronic arsenic exposure increases risk for the development of diabetes, vascular disease, and cancers of the skin, lung, kidney, and bladder. This study investigates the effects of arsenite [As(III)] on human urothelial cells (UROtsa). As(III) toxicity was determined by exposing confluent UROtsa cells to As(III) (0.5-200 microM). Depleting cellular glutathione levels with buthionine sulfoximine (BSO) potentiated the toxicity of As(III). Cell viability was assessed with the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. UROtsa cell ability to biotransform As(III) was determined by dosing cells with environmentally relevant concentrations of As(III) followed by HPLC/ICP-MS analysis of cell media and lysate. Both pentavalent and trivalent monomethylated products were detected. Although cytotoxicity was observed at high doses of As(III) (approximately 100 microM) in UROtsa cells, perturbations of a variety of molecular processes occurred at much lower doses. Exposure to low-level As(III) (0.5-25 microM) causes an accumulation of ubiquitin (Ub)-conjugated proteins. This effect is enhanced when cellular glutathione levels have been reduced with BSO treatment. Because As(III) has many effects on UROtsa cells, a greater understanding of how As(III) is affecting cellular proteins in a target tissue will lead to a better understanding of the mechanism of toxicity and pathogenesis for low-level As(III).
• Field, J. A., Sierra-Alvarez, R., Cortinas, I., Feijoo, G., Moreira, M. T., Kopplin, M., & Gandolfi, A. J. (2004). Facile reduction of arsenate in methanogenic sludge. Biodegradation, 15(3), 185-96.
  More info

  Due to the recent enactment of a stricter drinking water standard for arsenate, large quantities of arsenate-laden drinking water residuals will be disposed in municipal landfills. The objective of this study was to determine the role of methanogenic consortia on the conversion of arsenate. Methanogenic conditions commonly occur in mature municipal solid waste landfills. The results indicate the rapid and facile reduction of arsenate to arsenite in methanogenic sludge. Endogenous substrates in the sludge were sufficient to support the reductive biotransformation. However the rates of arsenate reduction were stimulated by the addition of exogenous electron donating substrates, such as H2, lactate or a mixture of volatile fatty acids. A selective methanogenic inhibitor stimulated arsenate reduction in microcosms supplied with H2, suggesting that methanogens competed with arsenate reducers for the electron donor. Rates of arsenate reduction increased with arsenate concentration up to 2 mM, higher concentrations were inhibitory. The electron shuttle, anthraquinone-2,6-disulfonate, used as a model of humic quinone moieties, was shown to significantly increase rates of arsenate reduction at substoichiometric concentrations. The presence of sulfur compounds, sulfate and sulfide, did not affect the rate of arsenate transformation but lowered the yield of soluble arsenite, due to the precipitation of arsenite with sulfides. The results taken as a whole suggest that arsenate disposed into anaerobic environments may readily be converted to arsenite increasing the mobility of arsenic. The extent of the increased mobility will depend on the concentration of sulfides generated from sulfate reduction.
• Meza, M. M., Kopplin, M. J., Burgess, J. L., & Gandolfi, A. J. (2004). Arsenic drinking water exposure and urinary excretion among adults in the Yaqui Valley, Sonora, Mexico. Environmental research, 96(2), 119-26.
  More info

  The objective of this study was to determine arsenic exposure via drinking water and to characterize urinary arsenic excretion among adults in the Yaqui Valley, Sonora, Mexico. A cross-sectional study was conducted from July 2001 to May 2002. Study subjects were from the Yaqui Valley, Sonora, Mexico, residents of four towns with different arsenic concentrations in their drinking water. Arsenic exposure was estimated through water intake over 24 h. Arsenic excretion was assessed in the first morning void urine. Total arsenic concentrations and their species arsenate (As V), arsenite (As III), monomethyl arsenic (MMA), and dimethyl arsenic (DMA) were determined by HPLC/ICP-MS. The town of Esperanza with the highest arsenic concentration in water had the highest daily mean intake of arsenic through drinking water, the mean value was 65.5 microg/day. Positive correlation between total arsenic intake by drinking water/day and the total arsenic concentration in urine (r = 0.50, P < 0.001) was found. Arsenic excreted in urine ranged from 18.9 to 93.8 microg/L. The people from Esperanza had the highest geometric mean value of arsenic in urine, 65.1 microg/L, and it was statistically significantly different from those of the other towns (P < 0.005). DMA was the major arsenic species in urine (47.7-67.1%), followed by inorganic arsenic (16.4-25.4%), and MMA (7.5-15%). In comparison with other reports the DMA and MMA distribution was low, 47.7-55.6% and 7.5-9.7%, respectively, in the urine from the Yaqui Valley population (except the town of Cocorit). The difference in the proportion of urinary arsenic metabolites in those towns may be due to genetic polymorphisms in the As methylating enzymes of these populations.
• Begay, C. K., & Gandolfi, A. J. (2003). Late administration of COX-2 inhibitors minimize hepatic necrosis in chloroform induced liver injury. Toxicology, 185(1-2), 79-87.
  More info

  Our previous studies have described the protective effects of hepatoprotective agents against liver injury elicited by chloroform even when given 24 h after the toxicant, at a time when the liver injury is taking place and rapidly developing. However, the mechanisms involved in this protection remain unknown. The cytoprotective mechanism of these hepatoprotectants such as DMSO, may be due to a dramatic shift in the production of prostaglandins that are responsible for controlling the degree of inflammatory response that can affect blood flow in the liver. In this study, NS-398, a specific COX-2 inhibitor, and indomethacin, a COX-1 and COX-2 inhibitor, were administered 24 h after chloroform dosing to determine their effect on liver injury in Sprague-Dawley rats. The extent of necrosis was evaluated by H&E staining, while injury to hepatocytes was evaluated by measuring plasma levels of alanine transaminase (ALT). Both COX inhibitors, indomethacin and NS-398, prevented an increase in (ALT) at 48 h after initial toxicant insult and attenuated further liver necrosis. No changes in cellular proliferative activity occurred in all the treatment groups, which indicates that protection from the Cyclooxygenase (COX) inhibitors did not have an effect on regeneration of cells at 32 and 48 h. These results indicate COX inhibitors provide a significant protective effect on liver cells against CHCl(3) injury and may provide further insight into therapeutic interventions against hepatotoxicants.
• Carter, D. E., Aposhian, H. V., & Gandolfi, A. J. (2003). The metabolism of inorganic arsenic oxides, gallium arsenide, and arsine: a toxicochemical review. Toxicology and applied pharmacology, 193(3), 309-34.
  More info

  The aim of this review is to compare the metabolism, chemistry, and biological effects to determine if either of the industrial arsenicals (arsine and gallium arsenide) act like the environmental arsenic oxides (arsenite and arsenate). The metabolism of the arsenic oxides has been extensively investigated in the past 4 years and the differences between the arsenic metabolites in the oxidation states +III versus +V and with one or two methyl groups added have shown increased importance. The arsenic oxide metabolism has been compared with arsine (oxidation state -III) and arsenide (oxidation state between 0 to -III). The different metabolites appear to have different strengths of reaction for binding arsenic (III) to thiol groups, their oxidation-reduction reactions and their forming an arsenic-carbon bond. It is unclear if the differences in parameters such as the presence or absence of methyl metabolites, the rates of AsV reduction compared to the rates of AsIII oxidation, or the competition of phosphate and arsenate for cellular uptake are large enough to change biological effects. The arsine rate of decomposition, products of metabolism, target organ of toxic action, and protein binding appeared to support an oxidized arsenic metabolite. This arsine metabolite was very different from anything made by the arsenic oxides. The gallium arsenide had a lower solubility than any other arsenic compound and it had a disproportionate intensity of lung damage to suggest that the GaAs had a site of contact interaction and that oxidation reactions were important in its toxicity. The urinary metabolites after GaAs exposure were the same as excreted by arsenic oxides but the chemical compounds responsible for the toxic effects of GaAs are different from the arsenic oxides. The review concludes that there is insufficient evidence to equate the different arsenic compounds. There are several differences in the toxicity of the arsenic compounds that will require substantial research.
• Catania, J. M., Parrish, A. R., Kirkpatrick, D. S., Chitkara, M., Bowden, G. T., Henderson, C. J., Wolf, C. R., Clark, A. J., Brendel, K., Fisher, R. L., & Gandolfi, A. J. (2003). Precision-cut tissue slices from transgenic mice as an in vitro toxicology system. Toxicology in vitro : an international journal published in association with BIBRA, 17(2), 201-5.
  More info

  In these experiments precision-cut tissue slices from two existing transgenic mouse strains, with transgenes that couple promoting or binding elements to a reporter protein, were used for determination of reporter induction. This approach combines the power of transgenic animals with the practicality of in vitro systems to investigate the biological impact of xenobiotics. Additionally, the normal cellular architecture and heterogeneity is retained in precision-cut tissue slices. Two transgenic mouse strains, one of which couples the promoting region of CYP 1A1 to beta-galactosidase, and another which couples two forward and two backward 12-O-tetradecanoyl phorbol-13-acetate (TPA) repeat elements (TRE) to luciferase (termed AP-1/luciferase), were used to determine the feasibility of this approach. Precision-cut kidney and liver slices from both transgenic strains remain viable as determined by slice K(+) ion content and LDH enzyme release. Liver slices harvested from the CYP 1A1/beta-galactosidase transgenic mice exhibit a 14-fold increase in beta-galactosidase activity when incubated with beta-napthoflavone for 24 h. Kidney and liver slices obtained from the AP-1/luciferase transgenic mice demonstrate induction of luciferase (up to 2.5-fold) when incubated with phorbol myristate acetate (PMA or TPA) up to 4 h. These data indicate that precision-cut tissue slices from transgenic mice offer a novel in vitro method for toxicity evaluation while maintaining normal cell heterogeneity.
• Kirkpatrick, D. S., Dale, K. V., Catania, J. M., & Gandolfi, A. J. (2003). Low-level arsenite causes accumulation of ubiquitinated proteins in rabbit renal cortical slices and HEK293 cells. Toxicology and applied pharmacology, 186(2), 101-9.
  More info

  Arsenic is a known human carcinogen that affects a variety of processes within the cell. In this study, the effects of environmentally relevant As(III) exposures on the ubiquitin (Ub)-proteasome pathway have been investigated. Low-level As(III) exposure (0.5 - 10 microM) causes an accumulation of high-molecular-weight ubiquitin protein conjugates in both precision-cut rabbit renal-cortical slices and human embryonic kidney (HEK) 293 cells. The As(III) doses that induced these molecular changes were subcytotoxic in both model systems. Doses of 10 microM As(III) decreased cellular activity of the 20S proteasome by 40 and 15% in slices and HEK293 cells, respectively. As(III) did not cause any notable difference in Ub-conjugating activity of rabbit renal slices or HEK293 cells. Since ubiquitination plays such a vital role in maintaining cellular homeostasis, this noticeable perturbation of cellular ubiquitination is likely to have a multitude of signaling effects within the cells and may contribute to the pathogenesis of low-level arsenic.
• Peraza, M. A., Carter, D. E., & Gandolfi, A. J. (2003). Toxicity and metabolism of subcytotoxic inorganic arsenic in human renal proximal tubule epithelial cells (HK-2). Cell biology and toxicology, 19(4), 253-64.
  More info

  Arsenic is an environmental toxicant and a human carcinogen. The kidney, a known target organ of arsenic toxicity, is critical for both in vivo arsenic biotransformation and elimination. This study investigates the potential of an immortalized human proximal tubular epithelial cell line, HK-2, to serve as a representative model for low level exposures of the human kidney to arsenic. Subcytotoxic concentrations of arsenite (< or = 10 micromol/L) and arsenate (< 100 micromol/L) were determined by leakage of LDH from cells exposed for 24 h. Threshold concentrations of arsenite (between 1 and 10 micromol/L) and arsenate (between 10 and 25 micromol/L) were found to affect MTT processing by mitochondria. Biotransformation of subcytotoxic arsenite or arsenate was determined using HPLC-ICP-MS to detect metabolites in cell culture media and cell lysates. Following 24 h, analysis of media revealed that arsenite was minimally oxidized to arsenate and arsenate was reduced to arsenite. Only arsenite was detected in cell lysates. Pentavalent methylated arsenicals were not detected in media or lysates following exposure to either inorganic arsenical. The activities of key arsenic biotransformation enzymes--MMAV reductase and AsIII methyltransferase--were evaluated to determine whether HK-2 cells could reduce and methylate arsenicals. When compared to the activities of these enzymes in other animal tissues, the specific activities of HK-2 cells were indicative of a robust capacity to metabolize arsenic. It appears this human renal cell line is capable of biotransforming inorganic arsenic compounds, primarily reducing arsenate to arsenite. In addition, even at low concentrations, the mitochondria are a primary target for toxicity.
• Quanrud, D. M., Arnold, R. G., Lansey, K. E., Begay, C., Ela, W., & Gandolfi, A. J. (2003). Fate of effluent organic matter during soil aquifer treatment: biodegradability, chlorine reactivity and genotoxicity. Journal of water and health, 1(1), 33-44.
  More info

  Hydrophobic acid (HPO-A) and transphilic acid (TPI-A) fractions of dissolved organic matter (DOM) were isolated from a domestic secondary wastewater effluent that was polished via soil aquifer treatment (SAT). Fractions were isolated using XAD resin adsorption chromatography from samples obtained along the vadose zone flowpath at a full-scale basin recharge facility in Tucson, Arizona. Changes in isolate character during SAT were established via biodegradability (batch test), specific ultraviolet light absorbance (SUVA), trihalomethane formation potential (THMFP), and Ames mutagenicity assays. The dissolved organic carbon (DOC) concentration decreased by >90% during SAT. A significant fraction (up to 20%) of isolated post-SAT HPO-A was biodegradable. The (apparent) refractory nature of DOM that survives SAT may be a consequence of low DOC concentration in groundwater as well as the nature of the compounds themselves. Specific THMFP (microg THM per mg DOC) of HPO-A and TPI-A varied little as a consequence of SAT, averaging 52 and 49 microg THM per mg DOC, respectively. The nonbiodegradable fractions of HPO-A and TPI-A exhibited higher reactivities: 89 and 95 microg THM per mg DOC, respectively. Genotoxicity of HPO-A (on a per mass basis) increased after SAT, suggesting that responsible compounds are removed less efficiently than bulk organics during vadose zone transport.
• Zheng, X. H., Watts, G. S., Vaught, S., & Gandolfi, A. J. (2003). Low-level arsenite induced gene expression in HEK293 cells. Toxicology, 187(1), 39-48.
  More info

  Chronic, low-level exposure to arsenic frequently results in skin, lung, bladder, and kidney cancer. Since arsenic is primarily excreted via the kidney, this study focused on this target tissue. Gene array was used as a sensitive low-level monitor of the impact of arsenic on this target tissue. Arsenite [As(III)] was chosen as the chemical species of arsenic since As(III) species are touted as the cellular toxic form of arsenic. Human embryonic kidney cell line HEK293 cells were incubated with 1, 10, and 25 microM arsenite [As(III)] for 6 or 24 h. Total RNA from treated and control cells was isolated, reverse transcribed, and labeled with Cy3 or Cy5, and hybridized to a human cDNA microarray. Hybridizations were performed four times using independent total RNA preparations to ensure reproducibility. Raw data from 10 and 25 microM treated cells exposed for 6 h was normalized within, and between, hybridizations followed by identification of genes affected by arsenite exposure based on practical significance (2-fold change up or down) and reproducibility (affected in four of six measurements). In these studies, 20 genes (HMOX1, MT1E, or FOSL1, etc.) were up-regulated, and 19 genes (MYC, JAK1, or CENPE, etc.) were down-regulated. Genes identified at 10 and 25 microM arsenic exposure were then examined after 1 microM treatment for 6 or 24 h. Expression of affected genes showed a dose-dependent (1-25 microM) trend that was apparently not time-dependent (6 vs. 24 h). The affected genes indicate that even this realistic, low-level arsenite exposure was recognized by the HEK293 cells (e.g. metallothionein genes) and produced an oxidative stress (e.g. heme oxygenase gene). These affected genes were characterized as stress response genes, proto-oncogene, signaling molecules, transcription factors, chemokine receptors, proteolytic enzymes, ESTs, and unknown genes. These findings imply that arsenite induces complex cellular injury and the cellular adaptation to As(III) is associated with alterations in the expression of many genes.
• Fisher, R. L., Hasal, S. J., Lipscomb, J. C., Gandolfi, A. J., & Brendel, K. (2002). Cold and cryopreservation of monkey liver slices. Toxicology mechanisms and methods, 12(2), 119-33.
  More info

  Both cynomolgus and rhesus monkeys are utilized in chemical toxicity screening and drug development studies by industry and government laboratories. Along with better utilization of their tissue for in vivo studies, it would be advantageous if the tissue could be cold-preserved or cryopreserved for future in vitro experimentation. Therefore, livers were excised from control monkeys and precision-cut tissue slices were prepared. The objective of this study was twofold: to compare cold-preservation solutions (V-7 and Viaspan) and to compare controlled-rate and vitrification cryopreservation protocols. Monkey liver slices were cold-stored in V-7 or Viaspan preservation solutions for 7 days. V-7 maintained slice viability for 5 days whereas Viaspan maintained slice viability for 1 day. In the controlled-rate freezing procedure, slices were exposed to 10% dimethyl sulfoxide and 90% fetal calf serum (FCS); cooled at the rate of 0.5 degrees C, 1 degrees C, or 12 degrees C per min to -70 degrees C; then placed into liquid nitrogen. Vitrification was accomplished by exposing slices stepwise to increasing concentrations of 1,2-propanediol (1.2, 2.4, and 4 M) in FCS with direct submersion into liquid nitrogen. In both protocols, slices were rewarmed quickly to 37 degrees C and then incubated in FCS for 4 h. Three viability parameters were used to measure slice viability - retention of potassium, leakage of lactate dehydrogenase, and synthesis of protein. Liver slices cryopreserved at a rate of 0.5 degrees C per min and vitrified successfully retained 80 to 90% of their viability. These results confirm the feasibility of functional cold preservation and cryopreservation protocols for monkey liver slices that would allow for a more efficient use of monkey tissue.
• Parrish, A. R., Sallam, K., Nyman, D. W., Orozco, J., Cress, A. E., Dalkin, B. L., Nagle, R. B., & Gandolfi, A. J. (2002). Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology. Cell biology and toxicology, 18(3), 205-19.
  More info

  Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCI2, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl2 demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology.
• Catania, J. M., Parrish, A. R., & Gandolfi, A. J. (2001). Toxicity of a sevoflurane degradation product incubated with rat liver and renal cortical slices. Drug and chemical toxicology, 24(4), 347-57.
  More info

  Compound A (2-fluoromethoxy-1,1,3,3,3-pentafluoro-1-propene) is a degradation product of the anesthetic sevoflurane which is created in closed-circuit anesthetic machines. Past in vivo and in vitro studies have implied that Compound A is nephrotoxic via bioactivation through the cysteine conjugate beta-lyase pathway. Although glutathione (GSH) conjugates of Compound A have been reported, it is not clear if they are formed enzymatically or via direct reaction with GSH. To determine if these metabolites are produced and toxic, a tissue slice system that first exposes male Fischer 344 rat liver slices to volatilized Compound A followed by exposure of rat kidney slices to the liver incubate was employed. Liver slices exposed to volatilized Compound A (6-12 microM medium conc.; approximately 23 ppm) exhibited a loss of K+ by 6 h, which was not seen in kidney slices exposed to Compound A. Aminobenzotriazole, a cytochrome P 450 suicide inhibitor, initially inhibits the cytotoxicity of Compound A to liver slices (at these times and concentrations). The sequential liver/kidney slice experiments using Compound A have not demonstrated nephrotoxic results. GSH conjugates were synthesized and was found to be nephrotoxic at concentrations above 91 microM (18 h), with higher concentrations showing toxicity at earlier times. Additionally, non-enzymatic reactions of Compound A with GSH or sulfhydryl-containing medium produces nephrotoxic products. These studies show that Compound A is directly toxic to the liver, possibly via P 450 activation, and Compound A can react with sulfhydryls directly to produce a nephrotoxic.
• Fisher, R. L., Gandolfi, A. J., & Brendel, K. (2001). Human liver quality is a dominant factor in the outcome of in vitro studies. Cell biology and toxicology, 17(3), 179-89.
  More info

  Donated human liver in the form of precision-cut tissue slices or isolated hepatocytes, is increasingly being used to predict metabolism and toxicity of xenobiotics in man. These tissue slices or hepatocytes can also be cold-preserved and cryopreserved to prolong their use for biological experiments. The viability of human liver could substantially affect the outcome of such experimentation. The goal of this investigation was to assess the viability of donated human livers, in the form of tissue slices, as they were received and to determine how varying degrees of liver quality affect experimental outcomes. Over one hundred human livers were categorized according to initial viability, as assessed by ATP content, K+ retention, protein synthesis, and LDH leakage. Each liver was placed in a low-, a medium-, or a high-quality group. The results showed that 76% of transplant-grade tissue (procured for transplantation) fell into the high-viability classification while the majority of research-grade tissue (not procured for transplantation) fell into the lowest viability classification. It was also found that only tissue slices prepared from highly viable human liver could be cold-preserved and cryopreserved. Dichlorobenzene metabolism was also greater in slices from highly viable human livers as compared to less viable livers. This study showed that human liver tissue acquired for medical research substantially varies in its viability and that these differences will affect the experimental data obtained.
• Wijeweera, J. B., Gandolfi, A. J., Parrish, A., & Lantz, R. C. (2001). Sodium arsenite enhances AP-1 and NFkappaB DNA binding and induces stress protein expression in precision-cut rat lung slices. Toxicological sciences : an official journal of the Society of Toxicology, 61(2), 283-94.
  More info

  Arsenic is a known human carcinogen. These studies were designed to examine the impact of low arsenite concentrations on immediate early gene expression in precision-cut rat lung slices. Precision-cut lung slices are a versatile in-vitro system for toxicity studies, as they preserve the architecture and cellular heterogeneity of the lung. Since 0.1-100 microM arsenite did not compromise slice viability at 4 hours, effects of arsenite on the expression of c-jun/AP-1, NFkappaB, HSP 32, HSP 72, HSP 60, and HSP 90 were studied, using these concentrations of arsenite at 4 h. Nuclear c-jun was increased by 10 and 100 microM arsenite, while NFkappaB was not affected. Gel-shift assays indicated that 10 microM arsenite resulted in an enhanced DNA-binding activity of both AP-1 and NFkappaB. Confocal microscopic analysis of AP-1 indicated nuclear localization of this transcription factor, mainly in type-II epithelial cells and alveolar macrophages. Nuclear localization of NFkappaB was lower than that observed for AP-1, while most of the NFkappaB was localized to cytoplasm of type-II epithelial cells and alveolar macrophages. HSP 32 was increased by 1.0 and 10 microM arsenite, while HSP 72 was increased by only 100 microM arsenite. HSP 60 and HSP 90 were not changed by arsenite. These studies indicate that noncytotoxic concentrations of arsenite are capable of affecting signal transduction pathways and gene expression in the lung.
• Zheng, X. H., Begay, C., Lind, R. C., & Gandolfi, A. J. (2001). Humoral immune response to a sevoflurane degradation product in the guinea pig following inhalation exposure. Drug and chemical toxicology, 24(4), 339-46.
  More info

  Compound A (2-fluoromethoxy-1,1,3,3,3-pentafluoro-1-propene) is produced by reaction of the inhalation anesthetic, sevoflurane, with CO2 absorbents. Compound A has been reported to directly react with protein. Since adduction of proteins can transform them into antigenic material, Compound A was assessed for its ability to produce a humoral immune response. Male outbred Hartley guinea pigs (500-600 g, N = 7) were exposed via inhalation for 4 h to a subtoxic level (100 ppm) of Compound A, 3 times, at 42 day intervals. Blood samples obtained at 2, 14, 28 and 40 days after each exposure were measured for ALT, creatinine, and urea nitrogen and for the presence of antibodies to trifluoroacetylated guinea pig albumin (TFA-GSA). All indicators of liver and kidney injury remained within normal range throughout the course of the study. A humoral immune response to TFA-GSA was observed following each exposure to Compound A with a titer appearing by day 14 after exposure, peaking near day 28, and resolving to normal levels by day 40. The titer levels were approximately equivalent after each exposure and about one-third that previously seen in guinea pigs after multiple exposures to halothane. Compound A would appear to have the ability to form antigenic adducts during inhalation exposure. These findings are similar to those observed for halogenated inhalation anesthetics that have been linked to cases of immune-medicated idiosyncratic hepatitis and indicate that Compound A exposure may pose the same hazard.
• Ito, ., Lind, . C., Begay, . K., Gandolfi, . J., McCuskey, . K., & McCuskey, . S. (2000). Late administration of dimethyl sulfoxide minimizes the hepatic microvascular inflammatory response to chloroform in rats. Hepatology research : the official journal of the Japan Society of Hepatology, 18(3), 203-217.
  More info

  Dimethyl sulfoxide (DMSO) protects against liver injury elicited by chloroform (CHCl(3)) in rats even when given 24 h after the toxicant. Aminobenzotriazole (ABT) is a cytochrome P-450 inhibitor as is DMSO. The present study was conducted to examine the effect of DMSO or ABT on the hepatic microvascular inflammatory response to CHCl(3) using in vivo microscopy. Rats received 0.75 ml/kg CHCl(3) by oral administration followed 24 h later by either 2.0 ml/kg DMSO, 30 mg/kg ABT, or saline injected intraperitoneally. Untreated animals served as controls. At 28 h after CHCl(3) administration, the number of leukocytes adhering to the sinusoidal wall was significantly increased associated with a reduction in the numbers of perfused sinusoids. DMSO, but not ABT, caused a restoration of perfused sinusoids to 85% of normal. At 32 h after, leukocyte adhesion and Kupffer cell phagocytic activity were significantly increased. The numbers of perfused sinusoids were decreased by 32% in periportal regions and 50% in centrilobular regions. Sinusoidal perfusion and Kupffer cell activity were modified significantly by either DMSO or ABT, while leukocyte adhesion was reduced by DMSO alone. The results suggest that attenuation of the hepatic microvascular response to CHCl(3) is a potential mechanism of post-treatment with DMSO.
• Lind, R. C., Begay, C. K., & Gandolfi, A. J. (2000). Hepatoprotection by dimethyl sulfoxide. III. Role of inhibition of the bioactivation and covalent bonding of chloroform. Toxicology and applied pharmacology, 166(2), 145-50.
  More info

  Dimethyl sulfoxide (DMSO) has previously been shown to have the ability to attenuate chloroform (CHCl(3))-induced liver injury in the naive rat even when administered 24 h after the toxicant. These studies were undertaken to determine if the protective action by late administration of DMSO is due to an inhibition of the bioactivation of CHCl(3). This was done by comparing the cytochrome P450 inhibitors, diallyl sulfide (DAS), and aminobenzotriazole (ABT) to DMSO for their protective efficacy when administered 24 h after CHCl(3) exposure. In addition, (14)CHCl(3) was utilized to measure the effect of DMSO and ABT on the covalent binding of CHCl(3) in the liver following their late administration. Male Sprague-Dawley rats (300-350 g) received 0.75 ml/kg CHCl(3) po. Twenty-four hours later, they received ip injection of 2 ml/kg DMSO, 100 mg/kg DAS, or 30 mg/kg ABT. Plasma ALT activities and quantitation of liver injury by light microscopy at 48 h after CHCl(3) dosing indicated that all three treatments were equally effective at protecting the liver. A detailed study of the time course of injury development indicated that the protective action of DMSO was occurring within 10 h of its administration. Therefore, in the radiolabel studies, rats were killed 24-34 h after receiving 0.75 ml/kg CHCl(3) (30 microCi/kg (14)CHCl(3)) po. Treatment with ABT at 24 h after (14)CHCl(3) dosing decreased the covalent binding of (14)C to hepatic protein by 35% and reduced the amount of (14)C in the blood by 50% by 10 h after its administration. DMSO treatment did not significantly affect any of these parameters. The lack of effect by late administration of DMSO on the covalent binding of CHCl(3) would indicate that DMSO may offer protection by mechanisms other than inhibition of the bioactivation of CHCl(3). These studies also indicate that specific cytochrome P450 inhibitors may be of benefit in clinical situations to help treat the delayed onset hepatitis that can result following poisoning with an organohalogen, even if the antidotes are administered a number of hours after the initial exposure.
• Lind, R. C., & Gandolfi, A. J. (1999). Hepatoprotection by dimethyl sulfoxide. II. Characterization of optimal dose and the latest time of administration for effective protection against chloroform and bromobenzene induced injury. Experimental and toxicologic pathology : official journal of the Gesellschaft fur Toxikologische Pathologie, 51(6), 537-43.
  More info

  Dimethyl sulfoxide (DMSO) has previously been shown to attenuate chloroform (CHCl3) and bromobenzene (BB) induced hepatotoxicity in the rat when a dose of 2.0 ml/kg is given 24 hr after the toxicants. However, the optimal dose of DMSO and the latest time at which DMSO can be administered and still provide effective protection have not been determined. In order to determine the latest time at which DMSO can interrupt the development of necrosis, male Sprague Dawley rats received either 0.75 ml/kg CHCl3 or 0.5 ml/kg BB, 20% in corn oil, p.o., followed by single dose of 2 ml/kg DMSO, 50% in saline, i.p., at 24, 26, 28 or 30 hr later. Positive control groups received either CHCl3 or BB and then 4.0 ml/kg saline, i.p., 24 hr later. All of the animals were then killed 48 hr after toxicant dosing. The extent of liver injury present when DMSO was administered was examined by killing animals at 24, 26, 28 or 30 hr after toxicant dosing. The optimal dose of DMSO for providing protection was estimated by administering either 0, 1.0, 2.0, 3.0 or 4.0 ml/kg DMSO at 24 hr after toxicant dosing and then killing the animals at 48 hr. Delaying DMSO administration to times later than 24 hr after toxicant dosing led to a loss of protection as indicated by both plasma ALT activity and the light microscopic appearance of liver tissue. The distinctive liver lesions present at 24 hr after CHCl3 or BB dosing rapidly expanded from being limited around central veins to bridging between centrilobular areas in only a few hours. This was accompanied by large increases in plasma ALT. With both toxicants, doses of DMSO greater than 2 ml/kg did not enhance its protective action while the lower dose of 1 ml/kg DMSO was not as effective. The loss of DMSO's antidotal action when given at times later than 24 hr after the toxicants indicates irreversible changes were underway as the centrilobular lesions progressed from being limited to more bridging in nature. Hopefully, further elucidation of the mechanism(s) by which DMSO interrupts the rapid progression of injury will both help to understand the steps involved in lesion development and provide insights into therapeutic interventions for drug and chemical induced hepatitis.
• Parrish, A. R., Catania, J. M., Orozco, J., & Gandolfi, A. J. (1999). Chemically induced oxidative stress disrupts the E-cadherin/catenin cell adhesion complex. Toxicological sciences : an official journal of the Society of Toxicology, 51(1), 80-6.
  More info

  The impact of xenobiotics on intercellular adhesion, a fundamental biological process regulating most, if not all, cellular pathways, has been sparsely investigated. Cell-cell adhesion is regulated in the epithelium primarily by the E-cadherin/catenin complex. To characterize the impact of oxidative stress on the E-cadherin/catenin complex, precision-cut mouse liver slices were challenged with two model compounds for the generation of oxidative stress, diamide (DA; 25-250 microM) or t-butylhydroperoxide (tBHP; 5-50 microM), for 6 h. At the concentrations used, neither compound elicited cytotoxicity, as assessed by intracellular K+ content and leakage of lactate dehydrogenase into the culture media. However, a 25% reduction in non-protein sulfhydryl levels, an indication of oxidative perturbation, was seen in liver slices treated with DA or tBHP. Total protein expression of E-cadherin, beta-, or alpha-catenin was not affected by challenge with DA or tBHP. A decrease of beta-catenin in the SDS-soluble fraction of slices, an indicator of the formation of the adhesion complex, was observed. Additionally, a decrease in beta-catenin interactions with E-cadherin and alpha-catenin, as assessed by immunoprecipitation and Western blot analysis, was seen. Disruption of the E-cadherin/catenin complex by tBHP, but not DA, correlated with enhanced tyrosine phosphorylation of beta-catenin. These results suggest that noncytotoxic oxidative stress disrupts the E-cadherin/catenin cell adhesion complex in precision-cut mouse liver slices.
• Parrish, A. R., Zheng, X. H., Turney, K. D., Younis, H. S., & Gandolfi, A. J. (1999). Enhanced transcription factor DNA binding and gene expression induced by arsenite or arsenate in renal slices. Toxicological sciences : an official journal of the Society of Toxicology, 50(1), 98-105.
  More info

  Although the kidney represents a target for the accumulation and toxicity of arsenic, little is known about the molecular targets of arsenic in this organ. Therefore, these studies were designed to examine the molecular impact of arsenite [As(III)] and arsenate [As(V)] at low (nanomolar) concentrations. Precision-cut rabbit renal cortical slices were challenged with As(III) or As(V) for up to 8 h. Neither form of the metal induced overt cytotoxicity as assessed by intracellular K+ levels over this time period at concentrations from 0.01-10 microM. In addition, no alterations in the expression of Hsp 60, 70, or 90 were observed. However, induction of heme oxygenase-1 (Hsp 32) was seen following a 4-h challenge with As(III), but not with As(V). As(III) and As(V) induced DNA binding of AP-1 at 2- and 4-h exposure; following a 6-h exposure there was no difference. Although no alteration in the DNA binding activity of ATF-2 was induced by As(III) or As(V), both forms enhanced the DNA binding activity of Elk-1. Enhanced DNA binding activity of AP-1 and Elk-1 correlated with increased gene expression of c-fos, but not c-jun, at 2 h. c-myc gene expression was also induced by As(III) and As(V), albeit at a later time point (6 h). These results suggest that acute arsenic challenge, by either As(III) or As(V), is associated with discrete alterations in the activity of signaling pathways and gene expression in renal tissue.
• Turney, K. D., Parrish, A. R., Orozco, J., & Gandolfi, A. J. (1999). Selective activation in the MAPK pathway by Hg(II) in precision-cut rabbit renal cortical slices. Toxicology and applied pharmacology, 160(3), 262-70.
  More info

  The kidneys are the primary organ for the accumulation and toxicity of inorganic mercury. In these studies the molecular response of precision-cut rabbit renal cortical slices to low levels of inorganic mercury was examined. Cortical slices (275 microm) were obtained from 1.0 kg NZW rabbits and exposed to mercuric chloride [Hg(II)] at concentrations of 0.01-10 microM for 2-8 h. Overt cytotoxicity, as assessed by intracellular K(+) levels, was not observed following exposure to these concentrations of Hg(II). However, an induction of heme-oxygenase-1 (Hsp32) was seen following a 2-h challenge to Hg(II). A dose-dependent induction of the DNA binding activity of the AP-1 transcription factor after 4 h of Hg(II) exposure correlated with a dose-dependent enhancement of c-jun gene expression following 2 h of Hg(II) exposure. Additionally, an increase in phosphorylated c-Jun NH(2)-terminal protein kinase (JNK) was observed following 2 h of Hg(II) exposure. These results suggest activation of the mitogen-activated protein (MAP) signal transduction pathway, specifically the c-Jun NH(2)-terminal protein kinase (JNK) pathway. No changes were observed, however, in the DNA binding activity of ATF2 and Elk-1, transcription factors involved in both the JNK and p38 pathways of MAP signal transduction, nor in the gene expression of c-myc. This selectivity of alterations in molecular signaling suggests an acute response in signal transduction, specifically activation of the JNK pathway in renal tissue following exposure to nanomolar concentrations of Hg(II).
• Wei, H., Qiu, L., Divine, K. K., Ashbaugh, M. D., McIntyre, L. C., Fernando, Q., & Gandolfi, A. J. (1999). Toxicity and transport of three synthesized mercury-thiol-complexes in isolated rabbit renal proximal tubule suspensions. Drug and chemical toxicology, 22(2), 323-41.
  More info

  Previous work has suggested that endogenous sulfhydryls, such as glutathione (GSH) and cysteine, are involved in the uptake and toxicity of HgCl2. To study this possibility, uptake and toxicity of synthesized Hg(SG)2, Hg(cysteinylglycine)2 [Hg(CYS-GLY)2] and Hg(CYS)2 were investigated in rabbit renal proximal tubule suspensions (RPT). The intracellular K+ was used as a toxicity indicator, and the mercury content in the tubules was measured by proton induced x-ray emission analysis. The toxicity rank order of the three synthesized mercury-thiol-complexes from the highest to the lowest was: Hg(CYS)2 > Hg(CYS-GLY)2 > Hg(SG)2. However, no significant difference among the mercury contents in the tubules exposed to these synthesized mercury-thiol-complexes was detected. Acivicin (0.25 mM), an inhibitor of gamma-glutamyltranspeptidase (GGT), decreased the toxicity of Hg(SG)2 in a manner that did not decrease the uptake of mercury in the tubules. This suggests that the toxicity of Hg(SG)2 requires processing to Hg(CYS-GLY)2 or Hg(CYS)2, while Hg(SG)2 may be taken up by the tubules via Na(+)-dependent GSH transporter since 10 mM acivicin, an inhibitor of this transporter dramatically decreased the uptake of Hg(SG)2. Organic anion transporter plays a minor role, if any, in the toxicity and uptake of Hg(SG)2 and Hg(CYS)2 since p-aminohippuric acid (PAH), an inhibitor of organic anion transporter, did not have significant effect on their uptake and toxicity. L-phenylalanine, an inhibitor of the neutral amino acid decreased the uptake of mercury, but to a lesser extent. This suggested that neutral amino acid transporter seemed to play a role, in part, in the toxicity and uptake of synthesized Hg(CYS)2. In summary, the data suggested that basolateral transport is important for the toxicity of the three synthesized mercury-thiol-complexes, and a variety of mechanisms are involved in the toxicity and uptake of these complexes in isolated rabbit RPT.
• Parrish, A. R., Fisher, R., Bral, C. M., Burghardt, R. C., Gandolfi, A. J., Brendel, K., & Ramos, K. S. (1998). Benzo(a)pyrene-induced alterations in growth-related gene expression and signaling in precision-cut adult rat liver and kidney slices. Toxicology and applied pharmacology, 152(2), 302-8.
  More info

  Benzo(a)pyrene (BaP) and related aromatic hydrocarbons are suspected carcinogens; however, the molecular basis underlying tumorigenesis remains unclear. To identify acute molecular targets of BaP within the liver and kidney, precision-cut slices harvested from naive, adult female Sprague-Dawley rats were challenged with BaP (0.3-30 microM) for 0.5 to 24 h. BaP did not elicit cytotoxicity, as assessed by intracellular K+ and ATP content and histological evaluation over the 24-h period. To determine if molecular signaling pathways were maintained in precision-cut slices, induction of the aryl hydrocarbon receptor (AhR) pathway was assessed following BaP challenge. Induction of cytochrome P450IA1 (P450IA1) mRNA and protein expression was observed in both liver and kidney slices. c-fos and c-Ha-ras gene expression was enhanced in liver, but not kidney, slices by BaP. c-jun mRNA levels were decreased in liver and kidney slices, although the effect was earlier (0.5 h) in liver slices compared to kidney slices. BaP increased the DNA binding of nuclear proteins to the AP-1 consensus recognition element in liver, but decreased DNA binding in kidney slices. In contrast, DNA binding of NF-kappa B was not affected by BaP in either liver or kidney slices. These results suggest that acute BaP challenge is associated with altered expression of several growth-related genes and AP-1 signaling and establish precision-cut slices as a useful in vitro system to investigate the molecular basis of BaP-induced tumorigenesis, including organ-specific differences.
• Wijeweera, J., Gandolfi, J., & Zheng, X. H. (1998). Interactive toxicity and stress protein expression by vinylidene chloride and monochloroacetate in precision-cut rat liver slices. Environmental health perspectives, 106 Suppl 6, 1319-23.
  More info

  Vinylidene chloride (VDC) is a groundwater and drinking water contaminant. Monochloroacetic acid (MCA) is a chlorination by-product of drinking water. Because environmental or occupational exposure to chemicals takes place at low concentrations, a sensitive in vitro system of liver slices was used to examine the interactive toxicity of MCA and VDC. Liver slices from Sprague-Dawley rats were exposed to 100 microM MCA for 1 hr before exposure to 20 or 48 microM VDC and incubated for 1 to 8 hr. MCA + 48 microM VDC resulted in a significant leakage of K+ by 4 hr, while MCA+ 20 microM VDC did not. At 4 hr, MCA + 48 microM VDC resulted in centrilobular necrosis. MCA caused a significant depletion of slice glutathione (GSH) at 1 hr, which was maintained up to 3 hr. As reactive VDC metabolites are detoxified by conjugation with GSH, the increase in VDC toxicity by MCA is possibly due to GSH-depleting effects of MCA. Heat shock protein (HSP) 72 was increased 2.5-fold by MCA + 20 microM VDC as early as 2 hr, although K+ leakage was not increased. MCA + 48 microM VDC resulted in a 3-fold increase in HSP 72 by 2 hr, while there were modest increases in HSPs 60 and 32. Therefore, HSP 72 is an early sensitive indicator of interactive toxicity of nontoxic concentrations of MCA and VDC. This is the first time that micromolar concentrations of these drinking water contaminants were observed to affect cellular homeostasis in the liver.
• Furst, S. M., & Gandolfi, A. J. (1997). Interaction of lymphocytes with Kupffer cells from halothane-exposed guinea pigs. International archives of allergy and immunology, 114(1), 46-53.
  More info

  The purpose of this study was to investigate lymphocyte adhesion to Kupffer cells as a component of an immune-mediated mechanism for halothane hepatitis.
• Furst, S. M., Luedke, D., & Gandolfi, A. J. (1997). Kupffer cells from halothane-exposed guinea pigs carry trifluoroacetylated protein adducts. Toxicology, 120(2), 119-32.
  More info

  The anesthetic, halothane, is bioactivated by the liver cytochrome P450 system to trifluoroacetyl-chloride, which can readily acylate liver protein. Covalent binding of the trifluoroacetyl moiety may result in hapten formation leading to the induction of an immune response and ultimately halothane hepatitis. In this study the presence of trifluoroacetylated-protein adducts in Kupffer cells was investigated to learn how the immune system might come in contact with the proteins. Guinea pigs were exposed to 1.0% halothane, 40% oxygen for 4 h. Kupffer cells were isolated on days 1 through 9 post-exposure, by liver perfusion and purification by elutriation. Using gel electrophoresis and Western blotting techniques, it has been demonstrated that Kupffer cells obtained from halothane-treated guinea pigs, do carry trifluoroacetyl-protein adducts as recognized by an anti-trifluoroacetyl-rabbit serum albumin antibody. Apparent molecular weights of polypeptides bound by trifluoroacetyl were of a wide range, 25-152 kDa. Bands were most prominent in the larger Kupffer cells with more appearing at lower molecular weights. Trifluoroacetyl-protein adducts were not detected in lung, spleen, lymph node or peripheral blood macrophages. This work suggests a role for Kupffer cells in the presentation of altered proteins in the liver to cells of the immune system.
• Furst, S. M., Luedke, D., Gaw, H. H., Reich, R., & Gandolfi, A. J. (1997). Demonstration of a cellular immune response in halothane-exposed guinea pigs. Toxicology and applied pharmacology, 143(2), 245-55.
  More info

  Halothane hepatitis is considered to be a result of an idiosyncratic autoimmune reaction brought about by the formation of neoantigens that have been generated by covalent binding of halothane biotransformation intermediates. The guinea pig is being examined as an animal model to investigate an immune-mediated mechanism for halothane hepatotoxicity. Male Hartley guinea pigs were exposed to 1% halothane/40% oxygen for 4 hr, three times with 40-day intervals. Kupffer cells and splenocytes were isolated from animals on various days after each halothane exposure. Splenocytes were cocultured in a lymphocyte transformation test with various concentrations of TFA(trifluoroacetylated)-antigens for 7 days and proliferation was measured by 3H-thymidine incorporation. In a second experiment, Kupffer cells were cocultured with autologous as well as allogeneic splenocytes with or without concanavalin A to determine whole cell sensitization and accessory function by Kupffer cells from treated animals. A 4-fold increase in splenocyte proliferation occurred in response to TFA-guinea pig albumin. No significant increase in proliferation could be detected with TFA-lysine or guinea pig albumin. A 14-fold increase in splenocyte proliferation also occurred in response to Kupffer cells from halothane-exposed animals. Autologous splenocytes demonstrated more of a response from treated versus control animals, indicating possible involvement of major histocompatibility complex II antigens. These results indicate recognition of TFA-antigens and Kupffer cells as antigen-presenting cells in halothane-exposed guinea pigs. This study provides good evidence that a cellular immune response is involved in the guinea pig after halothane exposure.
• Keith, R. L., Setiarahardjo, I., Fernando, Q., Aposhian, H. V., & Gandolfi, A. J. (1997). Utilization of renal slices to evaluate the efficacy of chelating agents for removing mercury from the kidney. Toxicology, 116(1-3), 67-75.
  More info

  Mercury is an environmental contaminant that preferentially accumulates in the kidney. It has been previously shown using proton-induced X-ray emission analysis that mercury (HgCl2) accumulated in precision-cut rabbit renal cortical slices. In this study, the efficacy of seven chelating agents for the removal of Hg from renal slices has been examined. Rabbits were injected with HgCl2 (10 mg/kg) and 3 h later kidneys were sliced, or renal slices were exposed in vitro to a mildly toxic concentration of HgCl2 (5 x 10(-5)M, 4 h). The slices were then treated in vitro with 10 mM concentrations of EDTA, lipoic acid (LA), penicillamine (PA), glutathione (GSH), 1,4-dithiothreitol (DTT), DMSA, or DMPS. DMPS proved to be the most effective in mobilizing Hg from in vivo or in vitro HgCl2-exposed renal tissue ( > 85% of control after 3 h incubation). Relative efficacies for the seven agents were DMPS > DMSA, PA > DTT, GSH > LA, EDTA. The use of renal slices appears to be a useful in vitro tool for assessing the efficacy of chelating agents on mobilizing accumulated Hg from renal tissue.
• Lind, R. C., & Gandolfi, A. J. (1997). Late dimethyl sulfoxide administration provides a protective action against chemically induced injury in both the liver and the kidney. Toxicology and applied pharmacology, 142(1), 201-7.
  More info

  Dimethyl sulfoxide (DMSO) can protect the liver from injury produced by a variety of hepatotoxicants when administered prior to or concomitant with the toxicants. This protective action has previously been attributed to DMSO-induced inhibition of bioactivation of the compounds to toxic intermediates. In these studies, the ability of DMSO to provide protection when administered 10 hr after a toxicant was evaluated in several animal models of xenobiotic-induced liver and kidney injury. In the guinea pig model of halothane-associated hepatotoxicity, male outbred Hartley guinea pigs received 2 ml/kg DMSO 10 hr after an inhalation exposure to 1.0% halothane, 40% O2 for 4 hr. DMSO decreased the extent of liver necrosis as indicated by a threefold decrease in plasma alanine aminotransferase activity 48 hr after exposure and a reduction in the incidence and extent of zone 3 necrosis. These results do not appear to be due to alterations in halothane biotransformation since DMSO administered at 10 hr after halothane had no affect on plasma concentrations of the halothane metabolite tritluoroacetic acid or covalent binding by reactive halothane biotransformation intermediates to hepatic protein. In addition, administration of the structurally analogous biotransformation inhibitor diallyl sulfide at 10 hr after halothane also had no affect on biotransformation or covalent binding but provided no protection from liver injury. Hepatic glutathione concentrations in the guinea pigs 24 hr after halothane exposure were also unaffected by late treatment with DMSO. Further studies in male Sprague-Dawley rats demonstrated the ability of DMSO to decrease the hepatic injury resulting from oral administration of 1.0 ml/kg chloroform or 0.5 ml/kg bromobenzene when administered 10 hr after either toxicant. The chloroform-treated rats also developed renal tubular necrosis with large increases in plasma creatinine and urea nitrogen, which were completely ameliorated by DMSO. Elucidating the mechanism(s) of this protective action of late DMSO administration should provide insight into the cascade of events that lead to liver and kidney injury from toxicants and, hopefully, therapeutic modalities for individuals suffering from acute, progressing, xenobiotic-induced hepatitis.
• Fisher, R. L., Hasal, S. J., Sanuik, J. T., Hasal, K. S., Gandolfi, A. J., & Brendel, K. (1996). Cold- and cryopreservation of dog liver and kidney slices. Cryobiology, 33(1), 163-71.
  More info

  The use of tissue slices in culture could decrease the number of animals used in health-related research and decrease experimental variation. This reduction may come about particularly if the methods of cold- and cryopreserving tissue slices are perfected, and one can conduct sequential in vitro experiments into xenobiotic metabolism, organ-specific toxicity, or organ-specific biochemical processes with tissue slices. With this goal in mind, dog liver and kidney slices were placed in cold storage at 0 degrees C using Viaspan (UW), Euro-Collins (EC), Sacks + prostacyclin (SP), and V-7 (V7) cold-preservation solutions for 10 days. Viability was assessed each day by measuring K+ content and protein synthesis after 4 h of incubation in Waymouth + 10% fetal calf serum (FCS). Dog liver slices can be cold-preserved in V7 for up to 7 days using K+ retention as the viability criterion but only up to 4 days using protein synthesis. Dog kidney slices can be cold-preserved in UW, EC, and V7 for up to 10 days using K+ retention, but only V7 could maintain protein synthesis for 10 days. Cryopreserved dog liver and kidney slices retained 63-68% of control viability after 4 h of incubation in FCS. The cryopreservation regimen included using 10% dimethyl sulfoxide in FCS as the cryoprotectant, a freezing rate of 0.5 degrees C/min for liver slices and 12 degrees C/min for kidney slices, and thawing in 37 degrees C FCS. Continued development of cold- and cryopreserving tissue slices could reduce the numbers of animals used and provide accurate and reproducible data.
• Hoivik, D. J., Fisher, R. L., Brendel, K., Gandolfi, A. J., Khairallah, E. A., & Cohen, S. D. (1996). Protein arylation precedes acetaminophen toxicity in a dynamic organ slice culture of mouse kidney. Fundamental and applied toxicology : official journal of the Society of Toxicology, 34(1), 99-104.
  More info

  Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals. In vivo studies suggest that in situ activation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution of in situ activation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0-25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K+ retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier, in vivo results and demonstrates the importance of in situ activation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity.
• Wueweera, J. B., Gandolfi, A. J., Badger, D. A., Sipes, I. G., & Brendel, K. (1996). Vitamin A potentiation of vinylidene chloride hepatotoxicity in rats and precision-cut rat liver slices. Fundamental and applied toxicology : official journal of the Society of Toxicology, 34(1), 73-83.
  More info

  Pretreatment of large doses of vitamin A (VA) is known to potentiate the hepatotoxicity of carbon tetrachloride. Therefore the effects of 1-day VA pretreatment on VDC hepatotoxicity was examined both in vivo and in an in vitro system of precision-cut rat liver slices. Male Sprague-Dawley rats were pretreated with 250,000 IU/kg VA by oral gavage. After 24 hr rats were administered 50, 100, or 200 mg/kg VDC ip. Precision-cut liver slices were prepared from VA pretreated rats 24 hr later and the liver slices were exposed for 2-8 hr to 0.025-1.0 microliter VDC evaporated into the gas phase of the incubation vials. VA pretreatment resulted in an enhancement of VDC toxicity, both in vivo and in vitro. There was a dose-dependent increase in plasma ALT 24 hr after VDC treatment of rats and an increase in K+ leakage from liver slices after VDC exposure. Histological analysis of the liver or the liver slices revealed that VA + VDC treatment resulted in centrilobular necrosis of the liver. When GdCl3 (10 mg/kg iv) was administered just before VA pretreatment of rats, VDC toxicity was partially reversed as observed by a decrease in ALT in vivo and a decrease in the loss of K+ in vitro. These results indicated that Kupffer cells, the resident macrophages of the liver, were partially responsible for the VA-potentiated VDC hepatotoxicity. One-day pretreatment of VA induced cytochrome P450IIE1 protein content as well as its enzymatic activity as measured by pnitrophenol hydroxylation. Because VDC is bioactivated by cytochrome P450IIE1, the increase in VDC hepatotoxicity after VA may be due to an increased bioactivation of VDC in the liver and in precision-cut liver slices. Thus, more than one mechanism may be involved in the VA enhancement of VDC hepatotoxicity.
• Burton, C. A., Hatlelid, K., Divine, K., Carter, D. E., Fernando, Q., Brendel, K., & Gandolfi, A. J. (1995). Glutathione effects on toxicity and uptake of mercuric chloride and sodium arsenite in rabbit renal cortical slices. Environmental health perspectives, 103 Suppl 1, 81-4.
  More info

  The mechanism of renal uptake of nephrotoxic heavy metals such as HgCl2 and NaAsO2 is not clear. The metals are known to react with endogenous sulfhydryls such as glutathione (GSH), so metal-GSH conjugates may be delivered to the kidney. To study this possibility, renal cortical slices from male New Zealand white rabbits were incubated with 10(-4) M HgCl2 or 10(-3) M NaAsO2 +/- stoichiometric amounts (1-3x) of GSH; or synthetic metal-GSH conjugates [10(-4) M Hg(SG)2 or 10(-3) M As(SG)3]. Incubations were performed at 37 degrees C in DME-F12 buffer (95/5 O2/CO2) for 8 hr. Hg(SG)2 reduced slice K+/DNA content, as an indicator of viability, significantly less than HgCl2. As(SG)3 exhibited a 2-hr delay in K+/DNA content reduction compared to NaAsO2. This delay in toxicity was not correlated to changes in uptake. Arsenic and mercury accumulation, determined by proton-induced X-ray emission, were also identical between the metal salts and the metal-GSH conjugates. Exogenous GSH decreased HgCl2 cytotoxicity and was correlated to a decrease in Hg accumulation in the slice. Exogenous GSH had limited if any protective effects against cytotoxicity by NaAsO2 and a decrease in As accumulation was not observed. Complex metal-GSH interactions appear to exist and impact on the uptake and toxicity of these metals.
• Clarke, J. B., Thomas, C., Chen, M., Hastings, K. L., & Gandolfi, A. J. (1995). Halogenated anesthetics form liver adducts and antigens that cross-react with halothane-induced antibodies. International archives of allergy and immunology, 108(1), 24-32.
  More info

  Two halogenated anesthetics, enflurane and isoflurane, have been associated with an allergic-type hepatic injury both alone and following previous exposure to halothane. Halothane hepatitis appears to involve an aberrant immune response. An antibody response to a protein-bound biotransformation product (trifluoroacetyl adduct) has been detected on halothane hepatitis patients. This study was performed to determine cross-reactivity between enflurane and isoflurane with the hypersensitivity induced by halothane. The subcellular and lobular production of hepatic neoantigens recognized by halothane-induced antibodies following enflurane and isoflurane, and the biochemical nature of these neoantigens was investigated in two animal models. Enflurane administration resulted in neoantigens detected in both the microsomal and cytosolic fraction of liver homogenates and in the centrilobular region of the liver. In the same liver, biochemical analysis detected fluorinated liver adducts that were up to 20-fold greater in guinea pigs than in rats. This supports and extends previous evidence for a mechanism by which enflurane and/or isoflurane could produce a hypersensitivity condition similar to that of halothane hepatitis either alone or subsequent to halothane administration. The guinea pig would appear to be a useful model for further investigations of the immunological response to these antigens.
• Fisher, R. L., Hasal, S. J., Sipes, I. G., Gandolfi, A. J., & Brendel, K. (1995). Comparative metabolism and toxicity of dichlorobenzenes in Sprague-Dawley, Fischer-344 and human liver slices. Human & experimental toxicology, 14(5), 414-21.
  More info

  1. Precision-cut liver slices, prepared from Sprague-Dawley and Fischer-344 rats and donated human liver tissue, were used to identify differences in 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB) and 1,4-dichlorobenzene (1,4-DCB) metabolism and how it may relate to toxicity. 2. Rat and human liver slices were incubated with 1 mM of either dichlorobenzene to determine metabolism and toxicity, at 2 and 6 h of organ culture. 3. The human liver slices metabolised the dichlorobenzenes to a greater extent than those from either of the rat strains. Liver slices from the Fischer-344 strain had a higher metabolic rate than the slices from the Sprague-Dawley rat strain. 4. The metabolic rate of dichlorobenzene isomers did not consistently correlate with its toxicity. For example, human slices did not exhibit any hepatoxicity, even though they metabolised these compounds to a greater extent than either rat strain. 5. Cross species covalent binding did not correlate with toxicity endpoints measured in this study. 6. The phase two metabolite profiles for each of the isomers in human and rat slices were similar in that the glutathione-cysteine conjugate was the major metabolite. 7. The use of an in vitro system which utilises human liver slices might provide an important bridge between animal derived data and the human situation.
• Gandolfi, A. J., Brendel, K., Fisher, R. L., & Michaud, J. P. (1995). Use of tissue slices in chemical mixture toxicology and interspecies investigations. Toxicology, 105(2-3), 285-90.
  More info

  Precision-cut tissue slices have proven to be a useful in vitro system for biotransformation and toxicity studies. Since tissue slices can be readily prepared from a variety of tissues and species, they can easily be used for interspecies investigations and comparisons. Furthermore, slices can be readily prepared from human tissue, thus comparisons (extrapolation) can be made between laboratory animals and humans. Slices can also be used to examine the toxic interactions of chemicals in vitro. It is important to use the correct experimental design to demonstrate toxic interactions and to assure that the tissue slices are properly exposed to the chemicals. Overall, tissue slices offer a valid in vitro system for performing species comparisons and chemical-chemical interaction studies.
• Groves, C. E., Morales, M. N., Gandolfi, A. J., Dantzler, W. H., & Wright, S. H. (1995). Peritubular paraquat transport in isolated renal proximal tubules. The Journal of pharmacology and experimental therapeutics, 275(2), 926-32.
  More info

  To better understand the characteristics of peritubular transport of organic cations (OCs), the uptake of the polyvalent OC dimethylbipyridinium (paraquat) and the structurally similar monovalent OC 1-methyl-4-phenylpyridinium (MPP+) was measured in suspensions of rabbit renal proximal tubules. Compared to the uptake of MPP+, the uptake of paraquat across the peritubular membrane was a low affinity, low capacity carrier-mediated process with a Jmax of 0.52 +/- 0.19 nmol.mg of protein.-1 min-1 and a Km of 162 +/- 25 microM. The uptake of MPP+ was a carrier-mediated process with a measured Jmax and Km of 1.8 +/- 0.09 nmol.mg of protein.-1min-1 and 28 +/- 8 microM, respectively. To determine whether paraquat is a substrate for the monovalent OC pathway, the effect of unlabeled MPP+ and tetraethylammonium (TEA) on paraquat uptake was examined. A 1 mM concentration of the monovalent OC MPP+ and TEA reduced the uptake of [14C]paraquat and [3H]MPP+ by approximately 30 and 90%, respectively, whereas 1 mM paraquat had no effect on [3H]MPP+ or [14C]TEA uptake. Thus, MPP+, but not paraquat, appears to interact with the monovalent OC transporter. On the other hand, the polyvalent OC substrates, including the polyamines putrescine and spermine, the herbicide diquat and the divalent hexamethonium bromidehydrate had no effect on either paraquat or MPP+ uptake. However, the synthetic polyamine methylglyoxal bis(guanyl-hydrazone)dihydrochloride (MGBG; 1 mM) reduced both paraquat and MPP+ uptake (by 60 and 90%, respectively). The ability of MGBG, unlike the other polyvalent substrates, to interact with paraquat transport may be related to structural similarities in the relative location of the two charged nitronium moieties in paraquat and MGBG.(ABSTRACT TRUNCATED AT 250 WORDS)
• Gunaratnam, N. T., Benson, J., Gandolfi, A. J., & Chen, M. (1995). Suspected isoflurane hepatitis in an obese patient with a history of halothane hepatitis. Anesthesiology, 83(6), 1361-4.
• Hastings, K. L., Thomas, C., Brown, A. P., & Gandolfi, A. J. (1995). Trifluoroacetylation potentiates the humoral immune response to halothane in the guinea pig. Immunopharmacology and immunotoxicology, 17(1), 201-13.
  More info

  Halothane hepatitis appears to result from an inappropriate immune response to the products of halothane metabolism. Attempts to produce an animal model for halothane hepatitis have been largely unsuccessful. Although guinea pigs produce neoantigens following treatment with halothane, the subsequent antibody response is weak, possibly accounting for the failure to produce halothane hepatitis in these animals. In order to increase the antibody response to halothane neoantigens, three methods for trifluoroacetylating proteins were used. Guinea pigs were either treated with S-ethylthiotrifluoroacetate, autologous lymphocytes trifluoroacetylated ex vivo, or immunized with trifluoroacetylated mycobacterial protein, followed by exposure to halothane, and examined for anti-halothane metabolite antibodies (anti-TFA antibodies). Animals treated with S-ethylthiotrifluoroacetate developed anti-TFA antibodies, and following exposure to halothane exhibited an enhanced antibody response. Treatment with trifluoroacetylated lymphocytes also resulted in an enhanced anti-TFA antibody response following halothane exposure. Immunization with trifluoroacetylated mycobacterial proteins resulted in very high anti-TFA antibody titers. However, subsequent exposure to halothane had no observable effect on specific antibody titers. Exposure to halothane, regardless of treatment, resulted in the production of anti-microsomal protein antibodies. Signs of halothane hepatitis were not observed, indicating that enhancement of the humoral immune response does not appear to be sufficient for production of halothane hepatitis.
• Keith, R. L., McGuinness, S. J., Gandolfi, A. J., Lowe, T. P., Chen, Q., & Fernando, Q. (1995). Interaction of metals during their uptake and accumulation in rabbit renal cortical slices. Environmental health perspectives, 103 Suppl 1, 77-80.
  More info

  The uptake and accumulation of metals occurs in the kidney, which is a key site for interaction between metal nephrotoxicants. The uptake/accumulation and interaction of CdCl2, HgCl2, K2Cr2O7, and NaAsO2 was examined in precision-cut rabbit renal cortical slices. Slices were incubated with 10(-6) to 10(-3) M of a single metal toxicant or combinations of metal toxicants for 12 hr in DME-F12 media. Slices were blotted and sandwiched between two mylar films stretched across XRF sample cups. Quantitation of the metal in the slices was performed by proton-induced X-ray emission analysis (PIXE). The uptake of the metals was rapid, often reaching a maximum between 3 to 6 hr; the accumulation of Hg was highest, followed in order by Cd, Cr, and As. When two metals were present together, substantial alterations were observed in the uptake of the metals in the slices. HgCl2 hindered the uptake of K2Cr2O7, NaAsO2, CdCl2 (in this order), whereas these metals facilitated the uptake of HgCl2. However, a decreased uptake of both metals was often noted after exposure to other combinations of metals. PIXE analysis of metal content in slices is attractive since all elements (atomic number > 20) can be determined simultaneously. This information will be particularly useful in studying potential toxic interactions.
• Lind, R. C., Gandolfi, A. J., & Hall, P. D. (1995). Biotransformation and hepatotoxicity of HCFC-123 in the guinea pig: potentiation of hepatic injury by prior glutathione depletion. Toxicology and applied pharmacology, 134(1), 175-81.
  More info

  The chlorofluorocarbon substitute 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) is a structural analog of halothane. Both are oxidatively metabolized by CYP2EI, producing a reactive trifluoroacyl acid chloride intermediate and have been shown to cause acute liver necrosis in the guinea pig. With halothane, liver injury has been associated with the degree of reactive intermediate binding to hepatic protein. This injury can be potentiated by prior glutathione (GSH) depletion. Thus, the combination of GSH depletion and HCFC-123 exposure was evaluated for its hepatotoxic potential in this species. Male outbred Hartley guinea pigs were injected with either 0.8 g/kg l-buthionine-(S,R)-sulfoximine (BSO) to deplete hepatic glutathione or vehicle control solution 24 hr before a 4-hr inhalation exposure to 1.0% (v/v) HCFC-123 with 40% O2. HCFC-123 caused minimal liver injury with only 1 of 8 exposed animals displaying confluent zone 3 necrosis. GSH depletion potentiated injury producing submassive to massive liver necrosis in some animals. This potentiation was associated with a 36% increase in covalent binding of reactive HCFC-123 intermediates to hepatic protein. These results were not due to alterations in the biotransformation of HCFC-123 as indicated by plasma concentrations of the metabolites trifluoroacetic acid and fluoride ion which were not affected by BSO pretreatment. HCFC-123 was also found to cause a decrease in liver GSH concentrations following exposure. These findings demonstrate a role for hepatic GSH in helping to prevent covalent binding by the trifluoroacyl acid chloride intermediate. Inhalation of HCFC-123 can cause acute hepatic injury in the guinea pig that is worsened by low hepatic GSH concentrations.
• Parrish, A. R., Gandolfi, A. J., & Brendel, K. (1995). Precision-cut tissue slices: applications in pharmacology and toxicology. Life sciences, 57(21), 1887-901.
  More info

  Almost a decade has passed since the first paper describing the isolation and maintenance of precision-cut liver slices produced using a mechanical tissue slicer was published (1). Although tissue slices of various organs have been employed as an in vitro system for several decades, the lack of reproducibility within the slices and the relatively limited viability of the tissue preparations has prevented a widespread acceptance of the technique. The production of an automated slicer, capable of reproducibly producing relatively thin slices of tissue, as well as the development of a dynamic organ culture system, overcame several of these obstacles. Since that time, significant advances in the methods to produce and culture tissue slices have been made, as well as the application of the technique to several other organs, including kidney, lung and heart. This review will i) summarize the historical use of tissue slices prior to the development of the precision-cut tissue slice system; ii) briefly analyze current methods to produce precision-cut liver, kidney, lung and heart slices; and iii) discuss the applications of this powerful in vitro system to the disciplines of pharmacology and toxicology.
• Payne, A. K., Morgan, S. E., Gandolfi, A. J., & Brendel, K. (1995). Biotransformation of sevoflurane by rat neonate liver slices. Drug metabolism and disposition: the biological fate of chemicals, 23(4), 497-500.
  More info

  Sevoflurane [CF3-CH(OCH2F)-CF3] is biotransformed to inorganic fluoride (F-) and hexafluoroisopropanol, which forms a glucuronide conjugate. Although sevoflurane may be used in newborns without fully developed biotransformation activity, studies were performed using liver slices from rat neonates to determine sevoflurane disposition. Sevoflurane was vaporized in sealed roller culture vials to produce a continuous saturating dose (0.5 mM). After incubation, slices and incubation media were sonicated and centrifuged to remove debris. The supernatant fraction was analyzed for F-, hexafluoroisopropanol, and hexafluoroisopropanol-glucuronide conjugate. The metabolism of sevoflurane by liver slices increased proportionately with time with a stoichiometric production (1:1) of hexafluoroisopropanol and F- in all age groups. Only glucuronide conjugates of hexafluoroisopropanol were found. The rate of sevoflurane biotransformation measured as fluoride production was similar among slices prepared from all neonate age groups. Although no hexafluoroisopropanol-glucuronide was generated by slices from 4-, 6-, and 8-day-old neonates, by day 21, 17% of the total hexafluoroisopropanol is glucuronidated. This contrasts with the lower levels of free hexafluoroisopropanol typically seen in adults liver slices, wherein 51% of the hexafluoroisopropanol was glucuronidated. These studies indicate that sevoflurane is equally metabolized to hexafluoroisopropanol and F-, but a deficiency in glucuronosyltransferase occurs in neonates.
• Wijeweera, J. B., Thomas, C. M., Gandolfi, A. J., & Brendel, K. (1995). Sodium arsenite and heat shock induce stress proteins in precision-cut rat liver slices. Toxicology, 104(1-3), 35-45.
  More info

  The present study was undertaken to investigate the usefulness of stress proteins as early, sensitive indicators of hepatotoxicity. Induction of stress protein synthesis in precision-cut rat liver slices was examined following in vitro exposure to sodium arsenite or heat shock. Precision-cut rat liver slices were incubated with 10(-5) or 10(-6) M sodium arsenite for 2, 4 or 8 h in the presence of 35S-methionine or exposed to hyperthermia (42.5 +/- 0.5 degrees C) for 45 min and then incubated with 35S-methionine for 2, 4 or 8 h. Fluorographic analysis indicated an increase in the synthesis of HSP 70 and HSP 90 family of proteins by both treatments. Immunoblot analysis demonstrated that there was a specific induction of HSP 72 and HSP 90. Induction of HSP 70 was greater than that of HSP 90 by both treatments. Stress protein induction occurred at earlier times by concentrations of arsenite which did not alter other viability parameters such as leakage of intracellular K+ or total protein synthesis. The results indicated that induction of stress proteins has the potential usefulness as an early biomarker of arsenite toxicity.
• Azri-Meehan, S., Mata, H. P., Gandolfi, A. J., & Brendel, K. (1994). The interactive toxicity of CHCl3 and BrCCl3 in precision-cut rat liver slices. Fundamental and applied toxicology : official journal of the Society of Toxicology, 22(2), 172-7.
  More info

  The interactive toxicity of two nontoxic concentrations of chloroform (CHCl3) and bromotrichloromethane (BrCCl3) was examined in precision-cut rat liver slices. Liver slices were prepared from male Sprague-Dawley rats (220-250 g) pretreated with phenobarbital for 4 days. Toxicants were administered 1 hr apart. Intracellular K+ levels were similar to untreated controls in slices treated with 0.2 mM CHCl3 or 0.125 microliters (0.25 mg, 1.26 mumol) BrCCl3 alone, indicating that these concentrations were nontoxic. However, addition of both toxicants, irrespective of order, resulted in a time-dependent loss of intracellular K+ which was significant at 9 hr following administration. This was interpreted as evidence of synergistic toxicity. Cytochrome P450 loss was significant as early as 3 hr following exposure to BrCCl3, alone or when added with CHCl3. This loss may be attributed to BrCCl3-induced suicide inactivation of cytochrome P450. Centrilobular hepatocytes may be more susceptible to the interactive toxicity of CHCl3 and BrCCl3. Activity of enzymes found predominantly in this area was significantly decreased in slices exposed to both toxicants relative to controls. Conversely, activity of enzymes found predominantly in the periportal region was similar to that of untreated and treated controls. Interactive toxicity of BrCCl3 and CHCl3 was not a consequence of increased lipid peroxidation or depletion of slice glutathione content. Further studies need to be conducted to elucidate the mechanisms mediating the interactive toxicity of BrCCl3 and CHCl3.
• Brown, A. P., & Gandolfi, A. J. (1994). Glutathione-S-transferase is a target for covalent modification by a halothane reactive intermediate in the guinea pig liver. Toxicology, 89(1), 35-47.
  More info

  The anesthetic halothane is bioactivated by the liver cytochrome P450 system to the reactive intermediate, trifluoroacetyl chloride, which can acylate liver protein. Cytosolic glutathione-S-transferase (GST) was identified as a major target for protein adduct formation in guinea pig liver slices exposed to halothane. To determine if GST is also a target in vivo, male Hartley guinea pigs were exposed to 1% halothane in 40% O2 for 4 h. At 10 h post exposure, livers were removed and microsomal and cytosolic fractions prepared. Past studies have shown these conditions resulted in maximal covalent binding of halothane intermediates to hepatic protein. Protein was isolated by ethanol precipitation and washed with trichloroacetic acid to remove unbound metabolites. Cytosolic GST was isolated by gel filtration and S-hexyl-glutathione affinity chromatography to electrophoretic purity. Protein adducts were quantified using a covalently bound fluorine assay. Covalent binding of a halothane intermediate to cytosolic and microsomal protein was determined as 2.0 +/- 0.4 and 13.2 +/- 2.3 nmol F/mg protein, respectively. Liver glutathione depletion by buthionine sulfoximine pretreatment produced an increase in covalent binding only to cytosolic proteins (3.3 +/- 0.4 nmol F/mg protein). Adduct formation to cytosolic GST was determined to be 4.7 +/- 1.6 nmol F/mg protein. Glutathione-S-transferase is a target for covalent modification in the liver following an inhalation exposure to halothane.
• Christen, U., Quinn, J., Yeaman, S. J., Kenna, J. G., Clarke, J. B., Gandolfi, A. J., & Gut, J. (1994). Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis. Molecular mimicry of trifluoroacetyl-lysine by lipoic acid. European journal of biochemistry, 223(3), 1035-47.
  More info

  Trifluoroacetylated (CF3CO-) proteins, elicited upon exposure of animals or humans to halothane, were recognized by anti-CF3CO antibody, monospecific for the hapten derivative N6-trifluoroacetyl-L-lysine. Anti-CF3CO antibodies cross-reacted with the dihydrolipoamide acetyltransferase (E2 subunit) of pyruvate dehydrogenase, indicating that epitopes on the E2 subunit of pyruvate dehydrogenase molecularly mimic those on CF3CO-proteins. Lipoic acid, the prosthetic group of the E2 subunit of pyruvate dehydrogenase was essential in this process, in that only the lipoylated form of the recombinantly expressed inner lipoyl domain of the human E2 subunit of pyruvate dehydrogenase, but not the unlipolyated form, was recognized by anti-CF3CO antibody. Furthermore, based on a high degree of structural relatedness, both CF3CO-Lys and (6RS)-lipoic acid, as well as the lipoylated peptide ETDK(lipoyl)ATIG specifically inhibited the recognition by anti-CF3CO antibody of the E2 subunit of pyruvate dehydrogenase, of trifluoroacetylated rabbit serum albumin and of human liver CF3CO-proteins. In sera of patients with halothane hepatitis, autoantibodies with properties identical to those of anti-CF3CO antibody were identified which could not discriminate between CF3CO-proteins and the E2 subunit of pyruvate dehydrogenase. These data suggest that the E2 subunit pyruvate of dehydrogenase is an autoantigen in halothane hepatitis and that molecular mimicry of CF3CO-proteins by the E2 subunit of pyruvate dehydrogenase is due to the similar structures of CF3CO-Lys and lipoic acid.
• Fisher, R. L., Sanuik, J. T., Gandolfi, A. J., & Brendel, K. (1994). Toxicity of cisplatin and mercuric chloride in human kidney cortical slices. Human & experimental toxicology, 13(8), 517-23.
  More info

  1. Organ specific toxicity such as nephrotoxicity is often investigated with the use of in vivo or in vitro animal models. 2. It would be beneficial if these findings could be verified in a human in vitro system which utilizes non-transplantable human kidneys. 3. Non-transplantable human kidneys were decapsulated, cut in half along the long axis, cores made perpendicular to the hemisphere, and precision-cut renal cortical slices produced. 4. These human kidney slices were incubated for 3, 6, 12, 18 and 24 h, viability assessed using intracellular K+ content, protein synthesis and organic ion transport and the potential nephrotoxicity of cisplatin (0.25, 0.5 and 1.0 mM) and mercuric chloride (10, 50 and 100 microM) on these slices were examined. 5. Control human kidney slices were viable for up to 24 h using all viability parameters while a dose- and time-dependent toxic response was seen using both cisplatin and mercuric chloride. 6. Cisplatin was more nephrotoxic in this human in vitro system than in previously investigated in vitro animal systems whereas mercuric chloride was similar in both systems. 7. These results indicate that human renal cortical slices are useful in predicting and verifying potentially nephrotoxic compounds in man.
• Fisher, R. L., Sanuik, J. T., Nau, H., Gandolfi, A. J., & Brendel, K. (1994). Comparative toxicity of valproic acid and its metabolites in liver slices from adult rats, weanling rats and humans. Toxicology in vitro : an international journal published in association with BIBRA, 8(3), 371-9.
  More info

  The incidence of fatal hepatic failure associated with valproic acid (VPA) treatment is low but when it occurs, 90% of the affected patients are below the age of 20 yr. At present the mechanism of VPA hepatotoxicity is unclear; however, it may be a combination of the formation and action of toxic metabolites in developing tissues. In the study reported here we investigated the action of VPA and its metabolites in liver slices prepared from adult and weanling Sprague-Dawley rats and from human livers (non-transplantable livers from organ donors, or biopsy material from patients undergoing surgical liver resection). VPA, 2-propyl-2-pentenoic acid (2-en-VPA or Delta(2)-VPA), 2-propyl-4-pentenoic acid (4-en-VPA or Delta(4)-VPA) were incubated for various times at concentrations of 100 or 300 mug/ml. Protein synthesis and K(|) content were used to assess functional integrity or general viability. The question addressed was whether there were differences in the in vitro toxicity of VPA and its metabolites that were related to the age of the livers from which the slices were taken. Liver slices from weanling rats were significantly more sensitive to VPA and its metabolites than the slices from livers of adult rats. The rank order of toxicities (4-en-VPA > VPA > 2-en-VPA) was the same in both sets of rat slices. The human liver slices were significantly affected by VPA and its metabolites but these compounds were equal in their ability to produce this toxicity. There was also an indication of differences in sensitivity to the toxicity of VPA in slices from livers of donors of different ages.
• Fisher, R. L., Smith, M. S., Hasal, S. J., Hasal, K. S., Gandolfi, A. J., & Brendel, K. (1994). The use of human lung slices in toxicology. Human & experimental toxicology, 13(7), 466-71.
  More info

  1. Successful use of agar-filled precision-cut rat lung slices in dynamic organ culture prompted the use of this technology with human lung. 2. The larger tissue mass of a human lung required that the trachea be cannulated with a balloon catheter and subsequently inflated with 4 liters of warm agar/medium mixture and then cooled before being precision-cut into 500 microns thick slices. 3. To characterize the human lung slices, viability and the effects of acrolein and nitrofurantoin were assessed over a period of 24 h using protein synthesis and nonprotein sulfhydryl content. 4. Control human lung slices synthesized protein at a linear rate and maintained a stable nonprotein sulfhydryl content for 24 h. 5. Slices incubated with acrolein exhibited no significant decrease in protein synthesis or nonprotein sulfhydryl levels until 24 h. 6. Incubation with nitrofurantoin exhibited a definite time- and dose-dependent inhibition of protein synthesis, and depletion of the cellular thiol pool. 7. These results indicate that this human lung tissue slice system may be used as an in vitro model to identify and screen pneumotoxicants.
• Lind, R. C., Gandolfi, A. J., & Hall, P. M. (1994). A model for fatal halothane hepatitis in the guinea pig. Anesthesiology, 81(2), 478-87.
  More info

  In the guinea pig, depleting hepatic glutathione before inhaling subanesthetic 0.1% halothane increases covalent binding of halothane biotransformation intermediates to hepatic protein and potentiates resultant liver injury. Because inhalation of a higher concentration of halothane is known to produce greater levels of covalent binding than with subanesthetic halothane, this study was undertaken with 0.25-1.0% halothane concentrations to further examine glutathione depletion as an etiology for halothane hepatitis.
• Mehendale, H. M., Roth, R. A., Gandolfi, A. J., Klaunig, J. E., Lemasters, J. J., & Curtis, L. R. (1994). Novel mechanisms in chemically induced hepatotoxicity. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 8(15), 1285-95.
  More info

  This review focuses on cellular events that modulate hepatotoxicity subsequent to initial liver insult. Cellular events that determine the nature and extent of hepatotoxic injury and the ultimate outcome of that injury are also discussed. The roles of cell types other than hepatocytes, hepatocyte organelle-specific processes, and regeneration in progression or recovery from liver injury are emphasized. Leukocyte activities are key events in two distinct hepatotoxicities. Neutrophil-mediated, periportal inflammation appears to play a primary role in progression of alpha-naphthylisothiocyanate-induced cholangiolitic hepatitis. However, a humorally mediated autoimmune response to protein adducts that occurs after anesthesia is critical in onset of halothane-induced hepatitis. New insights into specific events at the hepatocyte level are also emerging. Although reducing gap junctional communication between hepatocytes can protect against progression of liver injury, down-regulation of the subunit proteins (connexins) can isolate neoplastic cells from growth regulation. Acidic intracellular pH characteristic of hypoxia is protective against both hypoxic and toxicant-induced cell injury. In oxidative injury, a pH-mediated mitochondrial permeability transition causes mitochondrial uncoupling and ATP loss and leads to cell death. The ultimate outcome of hepatotoxic injury depends on the extent of tissue repair. Stimulation of tissue repair after a sublethal dose of CCl4 appears to be the central mechanism in protection against death from a subsequent large dose. Taken together, these examples illustrate the importance of events subsequent to initial liver injury as determinants of extent of liver damage.
• Michaud, J. P., Gandolfi, A. J., & Brendel, K. (1994). Toxic responses to defined chemical mixtures: mathematical models and experimental designs. Life sciences, 55(9), 635-51.
  More info

  The problem and relevance of assessing biological responses to chemical mixtures is presented with reference to the literature on this problem and its possible solutions. This review is intended for a general audience as an introduction to, and comment on, assessing the interactions of defined mixtures of xenobiotics. The focus is on experimental toxicology, however, the methods are also applicable to pharmacology. Much of the literature on this topic is quite specialized in statistics, theory, or specific applications. This may deter a significant portion of the growing number of investigators in this field from using this literature, and may partially account for the persistent use of methods which have been shown to permit precarious conclusions. References are given for some of the most comprehensive and recent work and reviews on the subject. The reader is given some familiarity with this topic's basic problems and ideas, and the controversy on terminology. One example is presented of a popular experimental design and data analysis method which while applicable in some situations, has been shown to lead to precarious and even erroneous conclusions. Eight other methods of data analysis are briefly presented and some of their advantages, disadvantages, assumptions, and limitations are discussed. These methods were selected to illustrate similarities and differences in the various approaches taken in addressing this problem. Three basic types of experimental design appropriate to these kinds of studies are outlined. General considerations, suggested guidelines, and possible pitfalls in experimental design, and data analysis of biological responses to chemical mixtures are discussed.
• Morgan, S. E., Frink, E. J., & Gandolfi, A. J. (1994). A simplified gas chromatographic method for quantifying the sevoflurane metabolite hexafluoroisopropanol. Anesthesiology, 80(1), 201-5.
  More info

  The results of sevoflurane biotransformation (fluoromethyl-1,1,1,3,3,3,-hexafluoro-2-propyl ether) to inorganic fluoride have been examined. However, these investigations have lacked a simplified assay for determining the primary organic metabolite, hexafluoroisopropanol. Previous attempts have involved extensive extraction steps, complicated derivatization techniques, or sophisticated detectors.
• Parrish, A. R., Dorr, R. T., Gandolfi, A. J., & Brendel, K. (1994). Adult rat myocardial slices: A tool for studies of comparative cardiotoxicity. Toxicology in vitro : an international journal published in association with BIBRA, 8(6), 1233-7.
  More info

  The applicability of myocardial slices in comparative cardiotoxicity studies was investigated using the known cardiotoxicants allylamine (AAM) and doxorubicin (DOX). Precision-cut adult rat myocardial slices are a recently developed in vitro system. Previously, it has been demonstrated that myocardial slices are viable for up to 24 hr in organ culture. Myocardial slices exhibited a concentration- and time-dependent loss of viability in response to exposure to AAM or DOX (10(-7), 10(-6) or 10(-5)m) during 24 hr in culture, as assessed by biochemical parameters including protein synthesis, ATP content, lipid peroxidation and the loss of the cytosolic enzyme creatine kinase. Protein synthesis and ATP content were sensitive indicators of slice viability, while lipid peroxidation was affected only by 10(-5)m DOX. Myocardial slices appear to be a useful in vitro system for the study of the cardiotoxic potential of chemicals. The maintenance of normal cellular architecture makes myocardial slices uniquely attractive for these studies.
• Reid, L. L., Hastings, K. L., Gandolfi, A. J., & Van Ert, M. (1994). Use of staphylococcal enterotoxin A-induced lymphoproliferation and interleukin 2 production as indicators of immunotoxicity. Drug and chemical toxicology, 17(1), 1-14.
  More info

  Suppression of mitogen-induced splenocyte lymphoproliferation and interleukin 2 (IL-2) production can be used as indicators of immunotoxicity. Staphylococcal enterotoxin A (SEA) is both a potent mitogen and the most potent in vitro inducer of IL-2 production that has been described. An in vitro system was used to measure impairment of SEA-induced lymphoproliferation and IL-2 production using splenocytes from female C57BL/6 mice dosed with either cyclosporin A (30 mg/kg/day, 14 days), benzene (220, 440, or 880 mg/kg/day, 14 days), or vehicle. Splenocytes were stimulated with either concanavalin A (con A) or SEA. Benzene- and cyclosporin A-treated mice demonstrated significant decreases in splenocyte proliferation. IL-2 production was determined by incubating splenocyte culture supernatants with IL-2 dependent cytotoxic T-cells (CTLL-2), pulsing with 3H-thymidine, and determining amount of incorporated label. Cell proliferation and IL-2 production were inhibited by both benzene and cyclosporin A, effects more clearly demonstrated using SEA than con A. SEA was a superior mitogen compared to con A in the assays evaluated here.
• Sheevers, H. V., Brendel, K., Wright, S. H., Nagle, R. B., & Gandolfi, A. J. (1994). Nephrotoxicity and uptake of 1-benzyl quinolinium in precision-cut rabbit renal cortical slices. Toxicology in vitro : an international journal published in association with BIBRA, 8(6), 1243-51.
  More info

  Nephrotoxicity studies have not always meshed toxicity with transport and uptake, although the two are interdependent. To begin to address this issue, a series of model organic cations (quaternary amines) was examined which revealed differences in toxicity that were not explained by slight structural variations. Thus, a single model organic cation, 1-benzyl quinolinium (BQ), was used to assess organic cations in rabbit renal cortical slices. Histopathological evaluation revealed that BQ produced site-specific injury in the S(3) region of the proximal tubule. Biochemical assays (K(+), qO(2) and ATP) revealed dose- and time-dependent decreases of BQ-exposed slices over 2-12 hr. Cation transport (uptake of tetraethylammonium) was decreased by BQ at sub-toxic doses within 2 hr, although anion transport (uptake of p-aminohippurate) was not affected. Understanding the toxicity and transport of model cations such as BQ will help to identify the mechanisms associated with organic cation nephrotoxicity as well as to facilitate the use of transport parameters to prevent toxicity or prolong drug action of cations.
• Wysynski, A. M., Martínez-Zaguilán, R., Alden, C., Gandolfi, A. J., & Gillies, R. J. (1994). Phenobarbital induces cytosolic acidification in an established liver epithelial cell line. Toxicology letters, 74(2), 157-66.
  More info

  Phenobarbital (PB), a long-acting barbiturate, is also a known tumor promoter. The mechanism responsible for the promoting effect of PB has not yet been elucidated. Changes in intracellular pH (pHin) have been associated with both early and late events of multistage carcinogenesis. We conducted this study to evaluate whether PB alters pH homeostasis. Adult rat liver (ARL) epithelial cells were cultured for 48 h on glass coverslips, loaded with the intracellular pH indicator SNARF-1, and perfused with various concentrations of PB to evaluate its effect on pHin. Our results show that PB treatment of cultured cells induces a concentration-dependent cytosolic acidification. These results indicate that its ability to decrease pHin may be involved in the mechanism of tumor promotion by PB.
• Dale, O., Frink, E. J., Thommesen, L., & Gandolfi, A. J. (1993). Hepatic elimination of diazepam: interactions with albumin, desflurane and sevoflurane. British journal of anaesthesia, 70(4), 454-5.
  More info

  We have studied the effect of sevoflurane and desflurane on the hepatic elimination of diazepam, by incubating slices of rat livers in a closed system. Protein free and protein containing (albumin 10 g litre-1) buffers were used to examine the effect of the anaesthetics on enzyme activity and diazepam binding to albumin. Both anaesthetics (in concentrations of 0.5, 1.5 and 3.0 mmol litre-1) reduced the elimination of diazepam slightly in the absence of albumin, while the presence of the protein increased elimination to a maximum of 30% at the greatest concentration of the anaesthetics. These data support previous observations that volatile anaesthetics may interact pharmacokinetically with both liver enzyme activity and drug binding to albumin.
• Fisher, R. L., Gandolfi, A. J., Sipes, I. G., & Brendel, K. (1993). Culture medium composition affects the relative toxicities of chlorobenzenes in rat liver slices and the isolated perfused liver. Drug and chemical toxicology, 16(4), 321-39.
  More info

  The effects of different media composition on the hepatotoxicity produced by monochlorobenzene (MCB), 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB) and 1,4-dichlorobenzene (1,4-DCB) were examined in two different in vitro systems. The toxicity of these chlorobenzenes was investigated in the perfused rat liver and liver slices using Krebs-Henseleit buffer. Significant differences between the chlorobenzenes were apparent in the perfused liver but not in the tissue slices. However, a dose and time related response of rat liver slices to the chlorobenzenes was observed. Partial amelioration of the chlorobenzene toxicity was observed when the Krebs-Henseleit buffer was supplemented with vitamins, amino acids, and/or bovine serum albumin. 1,2-DCB and 1,3-DCB toxicity was affected by amino acids and vitamins. The toxicity produced by 1,4-DCB was suppressed by amino acids, vitamins and 1% BSA. MCB hepatoxicity could only be suppressed by 1% BSA. This data suggests that tissue culture media composition plays a major role in the hepatotoxicity of the chlorobenzenes.
• Fisher, R. L., Hasal, S. J., Sanuik, J. T., Scott, K. S., Gandolfi, A. J., & Brendel, K. (1993). Cold- and cryopreservation of human liver and kidney slices. Cryobiology, 30(3), 250-61.
  More info

  Tissue slices may provide a rapid and economical way of determining cold ischemic effects on human liver and kidney cell viability and metabolism. In contrast to isolated hepatocyte cultures, tissue slices offer an in vitro system which more closely resembles the in vivo situation because of the differentiation and functional heterogeneity of the slice. In this study, human liver and kidney slices were cold stored for 10 days in Belzers University of Wisconsin (UW), Euro-Collins, and Modified Sacks solutions. Another set of slices was cryopreserved at 1 degree C/min for liver and 12 degrees C/min for kidney using a 10% dimethyl sulfoxide/fetal calf serum (FCS) cryoprotectant solution. The cold- and cryopreserved slices were incubated in roller culture for 4 h using FCS as the media. Liver slice viability was assessed by K+ content, protein synthesis, gluconeogenesis, and urea synthesis. Kidney slice viability was assessed using K+ content, protein synthesis, and organic ion transport (PAH and TEA). Human kidney slices were cold preserved in UW for 4-6 days, while the human liver slices were preserved for 12-24 h depending on the viability parameter. Following cryopreservation, human liver slice viability was retained at between 65 and 90% of control values, while kidney slice viability was maintained between 70 and 90% of control values depending on the viability parameter. These results indicate that this human in vitro tissue slice system can be used to optimize preservation solutions and methods. The ability to cold- and cryopreserve human slices could facilitate the more efficient utilization of human tissue.
• Hubbard, A. K., Lohr, C. L., Hastings, K., Clarke, J. B., & Gandolfi, A. J. (1993). Immunogenicity studies of a synthetic antigen of alpha methyl dopa. Immunopharmacology and immunotoxicology, 15(5), 621-37.
  More info

  Since the idiosyncratic liver toxicity of methyl dopa (L-alpha-methyl-3,4- dihydroxy-phenylalanine) may be due to immune mediated mechanisms, immunologic tools are needed to detect methyl dopa induced antibody and antigen. Hapten (methyl dopa)--carrier (albumin) conjugates were synthesized to generate antibodies reactive with this drug. Studies were also conducted to test the immunogenicity of this hapten-carrier conjugate and its cross reactivity with methyl catechol and levodopa. Methyl dopa (MD), levodopa (LD) or methyl catechol (MC) were conjugated to rabbit serum albumin (RSA) under high pH (base) conditions or by a tyrosinase (tyr) catalyzed reaction. Under the base conjugation conditions, MD-RSA, LD-RSA and MC-RSA conjugates were produced at higher hapten: carrier ratios than conjugates produced by the enzyme catalyzed reaction. Rabbits were subsequently immunized with either MD-RSA(base) or MD-RSA(tyr). Antibodies elicited by MD-RSA(base) had marked reactivity to the carrier protein, albumin, whereas antibodies elicited by MD-RSA(tyr) did not. In addition, reactivity of anti-MD antibody was equal to or greater with MC-RSA than reactivity with either MD-RSA or LD-RSA. This work suggests that the conjugation method using the tyr catalyzed reaction produces the optimal immunogen with minimal modification of the carrier protein. In addition, the catechol moiety of MD, MC and LD appears to be the immunogenic epitope on these haptens.
• Lowe, T., Chen, Q., Fernando, Q., Keith, R., & Gandolfi, A. J. (1993). Elemental analysis of renal slices by proton-induced X-ray emission. Environmental health perspectives, 101(4), 302-8.
  More info

  We optimized proton-induced X-ray emission (PIXE) for tissue analysis in a toxicity-disposition study. We used cultured rabbit renal slices as the biological system to demonstrate the use of PIXE analysis. The renal slices were exposed to HgCl2, CdCl2, K2Cr2O7, or NaAsO2 alone or in a mixture. The PIXE analysis provides information on concentrations of elements above atomic number 11, and it is the only analytical technique that can determine 20-30 elements nondestructively in a single, small sample (approximately 5 mg) with detection limits of 1-5 ppm (dry weight). The renal slices are thin targets that yield X-ray emission spectra with low backgrounds and high elemental sensitivities. The nondestructive nature of PIXE and the ability to simultaneously measure uptake of multiple metals and endogenous elements are unique to this methodology.
• Stijntjes, G. J., Commandeur, J. N., te Koppele, J. M., McGuinness, S., Gandolfi, A. J., & Vermeulen, N. P. (1993). Examination of the structure-toxicity relationships of L-cysteine-S-conjugates of halogenated alkenes and their corresponding mercapturic acids in rat renal tissue slices. Toxicology, 79(1), 67-79.
  More info

  Rat kidney slices were produced using a modified version of a mechanical tissue slicer. The slices were incubated with various concentrations of L-cysteine conjugates and mercapturic acids of halogenated alkenes in a submersion incubation system. The slices showed a time- and concentration-dependent toxicity to the nephrotoxic conjugates. The five L-cysteine conjugates tested: S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), S-(1-chloro-1,2,2-trifluoroethyl)-L-cysteine (CTFEC), S-(1,1-dichloro-2,2-difluoroethyl)-L-cysteine (DCDFEC) and S-(1,1-dibromo-2,2-difluoroethyl)-L-cysteine (DBDFEC) were more toxic compared to the corresponding mercapturic acids. Comparing the in vitro toxicity data with the in vivo data for the same compounds results in similar ranking for the relative nephrotoxicity of the conjugates.
• Brown, A. P., Hastings, K. L., Gandolfi, A. J., Liebler, D. C., & Brendel, K. (1992). Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothane. Toxicology, 73(3), 281-95.
  More info

  The anesthetic halothane can be bioactivated to the reactive intermediate, trifluoroacetyl chloride, which can covalently bind to liver protein. The product of this reaction is trifluoroacetyl-N-epsilon-lysine which can act as a foreign epitope in altering both protein immunogenicity and antigenicity. An in vitro liver slice system was used to study the formation of protein adducts following exposure to halothane. Liver slices (30-35 mg wet weight, 250-300 microns thick) from adult male Hartley guinea pigs (600-800 g) were exposed to [14C]halothane (0.6-0.9 microCi, 1.0-1.7 mM) in 95% O2/5% CO2 for 1, 6 and 12 h. The slices were homogenized and subcellular fractions prepared. Proteins were resolved by electrophoresis and bound radioactivity was detected by scintillation counting and autoradiography. Greater than 80% of detectable radioactivity to whole liver cell protein was localized in the 20-30-kDa range and increased in a linear fashion over the 12-h incubation period. Covalent binding was localized to two proteins of 27 kDa and 26 kDa present in the cytosolic compartment. Purification followed by N-terminal amino acid sequence analysis of the 27-kDa protein has identified it to be homologous with glutathione-S-transferase b. This cytosolic protein appears to be the major target for trifluoroacetylation in liver slices exposed to halothane.
• Frink, E. J., Ghantous, H., Malan, T. P., Morgan, S., Fernando, J., Gandolfi, A. J., & Brown, B. R. (1992). Plasma inorganic fluoride with sevoflurane anesthesia: correlation with indices of hepatic and renal function. Anesthesia and analgesia, 74(2), 231-5.
  More info

  The biotransformation and plasma inorganic fluoride ion production of sevoflurane (the new volatile anesthetic) during and after surgical anesthesia was studied in 50 ASA I or II surgical patients. Twenty-five additional patients served as controls by receiving isoflurane. Sevoflurane or isoflurane was administered with a semiclosed (total gas flow, 2 L/min O2) circle absorption system for durations of 1.0 to greater than 7.0 minimal alveolar concentration (MAC) hours for surgical anesthesia (sevoflurane MAC, 2.05%; isoflurane MAC, 1.15%). Preoperative and postoperative blood urea nitrogen and creatinine concentrations were determined. Blood samples were obtained during and after anesthesia in both groups for determining anesthetic blood concentration analysis and plasma fluoride level. Plasma fluoride concentrations did not significantly increase during isoflurane anesthesia. Sevoflurane biotransformation produced a mean peak plasma inorganic fluoride concentration of 29.3 +/- 1.8 mumol/L, 2 h after anesthesia, which decreased to 18 mumol/L concentration by 8 h after anesthesia. The peak plasma inorganic fluoride ion concentration correlated with duration of sevoflurane anesthetic exposure. Five patients given sevoflurane had peak levels transiently exceeding 50 mumol/L, and one of these had a history of ingesting drugs potentially producing hepatic enzyme induction. No increases in postoperative levels of creatinine, blood urea nitrogen, direct bilirubin, or hepatic transaminase and no changes in serum electrolyte level occurred in either anesthetic group. Indirect bilirubin concentration increased significantly after sevoflurane anesthesia, but the increase was not of clinical significance (from 0.30 +/- 0.03 to 0.38 +/- 0.06 mg/dL). Indirect bilirubin concentrations did not increase after isoflurane anesthesia; the concentrations reached 0.31 +/- 0.04 mg/dL and did not differ significantly from those found with sevoflurane.(ABSTRACT TRUNCATED AT 250 WORDS)
• Ghantous, H. N., Fernando, J. L., Keith, R. L., Gandolfi, A. J., & Brendel, K. (1992). Effects of halothane and other volatile anaesthetics on protein synthesis and secretion in guinea pig liver slices. British journal of anaesthesia, 68(2), 172-7.
  More info

  We have investigated the effect of volatile anaesthetics on protein synthesis and secretion in Hartley male guinea pig liver slices. The slices (250-300 microns thick) were incubated in sealed roller vials containing Krebs-Henseleit buffer at 37 degrees C under 95% oxygen. Volatile anaesthetics were vaporized in the vials to produce constant concentrations in the medium. Halothane 1-2.1 mmol litre-1 produced a concentration-related decrease in protein synthesis (3H-leucine incorporation) and secretion. Deuterated halothane (d-halothane), which is less biotransformed, was less inhibiting than halothane: uptake of the 3H-leucine was not affected but its incorporation into the nascent peptide was inhibited. Enflurane 2.2 mmol litre-1, isoflurane 2.2 mmol litre-1 and sevoflurane 2.1 mmol litre-1 also inhibited protein synthesis, but to a lesser extent than halothane and d-halothane. We conclude that alterations in protein synthesis and secretion are an early and sensitive indicator of cellular injury by volatile anaesthetics in liver slices.
• Ghantous, H. N., Fernando, J., Gandolfi, A. J., & Brendel, K. (1992). Sevoflurane is biotransformed by guinea pig liver slices but causes minimal cytotoxicity. Anesthesia and analgesia, 75(3), 436-40.
  More info

  Guinea pig liver slices were used to evaluate the biotransformation and hepatotoxic potential of sevoflurane. Precision-cut liver slices (250-300 microns thick) were incubated in sealed roller vials in buffer at 37 degrees C under 95% O2. Sevoflurane was added to produce 0.9 or 2.1 mM medium concentrations. After incubation (6-24 h), the intracellular K+ content and protein synthesis were determined, along with the defluorination of sevoflurane. Isoflurane was included for comparative purposes. Sevoflurane (2.1 mM) and isoflurane (2.3 mM) had no effect on slice K+ content, but both anesthetics depressed protein synthesis. The biotransformation of sevoflurane was maximal at 95% O2, with threefold more F- produced from sevoflurane than isoflurane. Sevoflurane appears to have a minimal effect on the guinea pig liver slices, which is consistent with in vivo studies in which minimal or no hepatotoxicity has been observed.
• Lind, R. C., Gandolfi, A. J., & Hall, P. M. (1992). Glutathione depletion enhances subanesthetic halothane hepatotoxicity in guinea pigs. Anesthesiology, 77(4), 721-7.
  More info

  Reduced glutathione has a potential role in protecting the liver against the reactive acyl acid chloride intermediate generated during the oxidative biotransformation of halothane. Glutathione is also important in maintaining the integrity of an injured cell. Thus, the effect of decreased hepatic glutathione concentrations on covalent binding of halothane metabolic intermediates to hepatic protein and lipid and the resultant hepatic injury were investigated in male, outbred Hartley guinea pigs. The animals were injected with either 1.6 g.kg-1 dl-buthionine-S,R-sulfoximine to deplete hepatic glutathione or vehicle-control solution 24 h before exposure to 0.1% (subanesthetic) halothane for 4 h (fractional inspired oxygen tension = 0.40). Buthionine sulfoximine pretreatment depleted liver glutathione concentrations by 85% at the time of halothane exposure, without affecting the degree of halothane biotransformation or causing hepatic injury. Glutathione depletion caused a significant increase in the level of organic fluorine covalently bound to hepatic protein but not lipid after halothane exposure. Glutathione-depleted animals also exhibited a significant enhancement of hepatotoxicity after halothane exposure; plasma isocitrate dehydrogenase activity was 25-fold greater than the increase observed 48 h after exposure in animals treated with vehicle plus halothane, and the incidence and severity of hepatic injury were significantly greater, as observed by light microscopic examination of tissue 96 h after exposure. These findings are in agreement with a previously proposed mechanism of halothane-associated hepatotoxicity in guinea pigs and indicate that hepatic glutathione status may play an important role in the susceptibility of patients to halothane-induced liver injury.
• Brown, A. P., Hastings, K. L., & Gandolfi, A. J. (1991). Generation and detection of neoantigens in guinea pig liver slices incubated with halothane. International journal of immunopharmacology, 13(4), 429-35.
  More info

  The volatile anesthetic halothane can be biotransformed by the hepatic cytochrome P-450 system to produce a reactive intermediate, trifluoroacetyl chloride, capable of covalently binding to liver proteins. The product of this reaction, the trifluoroacetyl lysinyl moiety, can act as an epitope to alter protein antigenicity. An in vitro system has been developed to produce halothane induced neoantigens and to study conditions for formation in the liver. Liver slices, capable of halothane biotransformation, provide a viable means for mechanistic studies. Liver slices (1 cm diameter, 300 microns thick) from male Hartley guinea pigs (600-800 g) were exposed to either 1.0 or 1.7 mM halothane (media concentration) in 95% O2/5% CO2 for 12 h. Covalent binding was determined using 14C-halothane. Neoantigens were detected by Western immunoblot analysis using rabbit anti-trifluoroacetylated albumin antiserum. Covalent binding was detected by 1 h of incubation and increased linearly through 12 h (20.7-48.5 nmole equiv/mg protein). Covalent binding preceded and correlated with the appearance of neoantigens. By 12 h of incubation, five neoantigens were seen with molecular weights ranging from 51 to 97 kD. These neoantigens have molecular weights similar to those seen in vivo. Liver slices exposed to deuterated halothane, which is oxidatively metabolized to a lower extent, did not develop neoantigens. This in vitro model system can be used to examine the mechanism for covalent binding and neoantigen production in the hepatocyte.
• Brown, A. P., Hastings, K. L., Gandolfi, A. J., & Brendel, K. (1991). Covalent binding of a halothane metabolite and neoantigen production in guinea pig liver slices. Advances in experimental medicine and biology, 283, 693-7.
• Ghantous, H. N., Fernando, J., Gandolfi, A. J., & Brendel, K. (1991). Minimal biotransformation and toxicity of desflurane in guinea pig liver slices. Anesthesia and analgesia, 72(6), 796-800.
  More info

  Biotransformation and hepatotoxicity of desflurane were evaluated in the guinea pig liver slice culture system. Liver slices (250-300 microns) were prepared from 600-650-g male Hartley guinea pigs. The slices were incubated in sealed vials in a Krebs-Henseleit buffer at 37 degrees C under 95% O2. Desflurane was vaporized to produce media concentrations of 0.7-2.3 mM. After incubation (3-24 h) viability of the slices was determined (K+ content; protein synthesis secretion) along with the biotransformation of desflurane (F-). Isoflurane (2.3 mM) was included in the studies for comparative purposes. Although desflurane caused a mild concentration-related reduction in slice K+ content (1.1-2.2 mM; 20%-40% of control), the effects were less than those produced by 2.3 mM isoflurane (50% of control). High concentrations of desflurane decreased protein synthesis at the first 9 h of incubation, and isoflurane decreased protein synthesis throughout the incubation period. Neither anesthetic affected protein secretion. The biotransformation of desflurane was minimal with threefold less F- produced from desflurane than isoflurane.
• Ghantous, H., Fernando, J., Gandolfi, A. J., & Brendel, K. (1991). Inhibition of protein synthesis and secretion by volatile anesthetics in guinea pig liver slices. Advances in experimental medicine and biology, 283, 725-30.
  More info

  The decrease in protein synthesis and secretion caused by volatile anesthetics was investigated using Hartley male guinea pig liver slices. Precision-cut liver slices (250-300 mM thick) were incubated in sealed roller vials (3 slices/vial) containing Krebs-Hensleit buffer at 37 degrees C under 95% O2 atmosphere. Volatile anesthetics were injected through a teflon septa cap on a filter paper wick and vaporized to produce constant concentration in the medium. A concentration (1-2.1 mM) and time related (0-24) decrease in protein synthesis (3H-leucine incorporation) and secretion by halothane and d-halothane was observed. d-Halothane was less inhibiting than halothane. Inhibition was not on the uptake of the 3H-leucine but with its incorporation in the nascent peptide. The effects of enflurane (2.2 mM), isoflurane (2.2 mM), and sevoflurane (1.3 mM) on protein synthesis and secretion were also studied. The rank order of decrease in protein synthesis caused by the volatile anesthetics studied was halothane greater than isoflurane greater than enflurane greater than sevofluane greater than d-halothane. Enflurane, isoflurane, and sevoflurane increased the protein secretion while halothane and d-halothane caused a pronounced decrease. Alterations in protein synthesis and secretion appears to be an early and sensitive indicator of cytotoxin injury.
• Hastings, K. L., Schuman, S., Brown, A. P., Thomas, C., & Gandolfi, A. J. (1991). S-ethylthiotrifluoroacetate enhancement of the immune response to halothane in the guinea pig. Advances in experimental medicine and biology, 283, 739-44.
• Hastings, K. L., Thomas, C., Hubbard, A. K., & Gandolfi, A. J. (1991). Screening for antibodies associated with halothane hepatitis. British journal of anaesthesia, 67(6), 722-8.
  More info

  The diagnosis of halothane hepatitis (HH) may be assisted by detection of antibodies reacting to trifluoroacetylated proteins (anti-TFA antibodies). An enzyme-linked immunosorbent assay (ELISA) utilizing trifluoroacetylated rabbit serum albumin (TFA-RSA) as antigen detected anti-TFA antibodies in 67% of sera from patients for whom a clinical diagnosis of HH was made. Anti-TFA antibodies were detected in 33% of sera when using an ELISA with liver microsomal protein from halothane-treated rabbits as antigen. Absorption of the sera with untreated rabbit liver microsomal protein before using the microsomal protein ELISA resulted in detection of anti-TFA antibodies in 42% of sera. Using the presumptive hapten N-epsilon-trifluoroacetyl-1-lysine to block antibody binding in an ELISA resulted in positive detection in 50% of sera: the results did not always agree with the other ELISA methods. The TFA-RSA ELISA was the most sensitive method and, combined with the TFA-lysine blocking ELISA, resulted in 92% of sera from HH patients testing positive for HH-associated antibodies.
• Gandolfi, A. J., & Brendel, K. (1990). In vitro systems for nephrotoxicity studies. Toxicology in vitro : an international journal published in association with BIBRA, 4(4-5), 337-45.
  More info

  A variety of in vitro systems, including the perfused kidney, microperfused nephron(s), cultured renal slices, renal tubule suspensions/cultures, isolated nephron components (glomeruli and tubule segments) and renal cell suspensions/cultures can be utilized to evaluate chemicals for their potential nephrotoxicity and mechanisms of action. These systems have varying degrees of relevance to the intact kidney or portions thereof; which model to use depends on the specific question asked. If maintenance of physiological flow conditions is important then only the perfused kidney (or tubule) can be used. If the toxicity can be studied in the absence of normal fluid flow, then the other systems may be utilized. Kidney subfractions also have the advantage of multiple biological units that can be examined simultaneously and separately. Closely defined renal slice systems allow for enrichment and evaluation of multiple target cells within the slice. Suspensions or cultures of nephron subfractions are now frequently used to assess the potency and/or mechanism of renal toxins. Disadvantages include the utilization of collagenase, which scars cell and basement membranes and affects membrane transport proteins, which leads to inadequate maintenance of transport mechanisms. Isolation of tubule segments (P(1), P(2), P(3)), allows site-specific toxicity studies. Lastly, there has been a dramatic increase in renal cell culture to study toxic events in the kidney. Because of the multiple cell types, minimal availability of specific cell markers, and de-differentiation phenomena, this technique should be used with caution. These in vitro systems have allowed us to examine the role of transport and biotransformation, and to compare potency and mechanism of nephrotoxic chemicals. Although in vitro systems cannot totally duplicate or replace in vivo toxicity studies, they are a very useful and valuable complement.
• Ghantous, H. N., Fernando, J., Gandolfi, A. J., & Brendel, K. (1990). Toxicity of halothane in guinea pig liver slices. Toxicology, 62(1), 59-69.
  More info

  Guinea pigs have proven to be a reliable model of halothane associated hepatotoxicity. An in vitro system with Hartley male guinea pig liver tissue was designed to assess the toxicity of halothane and other volatile anesthetics in the target organ. Precision-cut guinea pig liver slices (250-300 microns) were incubated in sealed roller vials containing Krebs-Henseleit buffer (plus vitamins, amino acids, glutamine, gentamycin) at 37 degrees C, under 95%, 21% and 5% O2/CO2 atmospheres. Halothane (10-15 microliters) was injected through a Teflon septa cap on a filter paper wick and vaporized. Viability of the slices was monitored by measuring intracellular K+ content which was maintained under 95% O2 up to 24 h. A dose- and time-related decrease in intracellular slice K+ by 1.9, 2.1, 2.7 mM halothane in the media was observed. At 2.7 mM halothane a direct physio-chemical effect may be occurring since incubating liver slices from allylisopropyl-acetamide-treated animals did not protect against the drop in intracellular K+. Concentration/time responses of halothane, d-halothane, enflurane, isoflurane and sevoflurane were compared. Sevoflurane had no effect on the liver slice K+ content up to 24 h while the other anesthetics caused the following rank-order decrease in intracellular K+ content: halothane greater than isoflurane and enflurane greater than d-halothane. Precision-cut cultured guinea pig liver slices offer a system where the target tissue for intoxication by anesthetics can be examined for its susceptibility and mechanism of intoxication.
• Hubbard, A. K., Levy, J. P., Roth, T. P., & Gandolfi, A. J. (1990). Use of structural alterations in the synthesis of halothane metabolite antigens to mimic halothane-induced immunogen. Drug and chemical toxicology, 13(2-3), 93-112.
  More info

  Four hapten-carrier conjugates were synthesized to evaluate any potential antigenic similarities between these synthetic compounds and the immunogens induced in vivo by the anesthetic, halothane and, thus, be used eventually as a more sensitive probe to detect the presence of these halothane-induced antibodies in halothane-exposed individuals. In this study, antibodies from five halothane hepatitis patients were used to evaluate these antigenic alterations since the specificity of these antibodies would most accurately reflect the antigenic structure of halothane-induced immunogens. Quantitation of antibody binding to these synthetic proteins was determined in an enzyme linked immunosorbent assay and immunoblot techniques. Trifluoroacetylated rabbit serum albumin was 5 times more reactive with these antibodies and thus more antigenic than the homologous acetylated moiety confirming the importance of the trifluoromethyl moiety as an epitope in the immunogen in vivo. Insertion of a spacer arm, aminocaproic acid, between the hapten and carrier moieties and an epitope density of 40% acetylation also increased antigenicity. Through these structural alterations produced in vitro, antigenic compounds have been produced which may resemble more closely the immunogen elicited in vivo and which may ultimately serve as more sensitive probes for halothane-induced antibodies from exposed individuals.
• Wolfgang, G. H., Gandolfi, A. J., Nagle, R. B., Brendel, K., & Stevens, J. L. (1990). Assessment of S-(1,2-dichlorovinyl)-L-cysteine induced toxic events in rabbit renal cortical slices. Biochemical and histological evaluation of uptake, covalent binding, and toxicity. Chemico-biological interactions, 75(2), 153-70.
  More info

  A renal cortical slice system was utilized to investigate the events leading to site-specific nephrotoxicity induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC uptake into renal cortical slices was shown to be rapid (5-15 min) as well as time- and concentration-dependent. Of the total amount taken up at 1 h, 40% was subsequently covalently bound. These observations were confirmed by autoradiography, illustrating uptake and binding in the proximal tubule cells. Following these events, toxicity was evidenced by alterations in ATP content and O2 consumption between 4 and 8 h as well as leakage of the brush border enzymes (gamma glutamyl transpeptidase and alkaline phosphatase) as early as 4 h. Light microscopy provided a sequence of histopathological changes from an initial S3 lesion between 4 and 8 h to a lesion encompassing all proximal tubule segments (by 12 h). Electron microscopy demonstrated not only the specificity of DCVC toxicity (at 6 h) but also illustrated mitochondrial damage and loss of brush borders. A comparison of continuous versus short-term exposure to DCVC indicated that an irreversible sequence of events was initiated as early as 30 min. By utilizing an in vitro model which allows correlation of biochemical and histological changes, a sequence of events leading to DCVC induced toxicity was established.
• Gandolfi, A. J., Lind, R. C., & de la M. Hall, P. (1989). Age and Gender Influence Halothane-Associated Hepatotoxicity in Strain 13 Guinea Pigs. Anesthesiology, 71(6), 878-884. doi:10.1097/00000542-198912000-00011
• Hubbard, A. K., Roth, T. P., Schuman, S., & Gandolfi, A. J. (1989). Localization of halothane-induced antigen in situ by specific anti-halothane metabolite antibodies. Clinical and experimental immunology, 76(3), 422-7.
  More info

  Multiple or single halothane exposure of rabbits or guinea pigs induces an antibody reactive with trifluoroacetylated (TFA) proteins. The antigen that initiates this immune response was investigated in halothane-exposed rabbits and guinea pigs for its anatomical location in the liver, the chronology of its expression in situ and exposure conditions which would modulate its expression. Using an immuno-staining technique, binding by an anti-TFA antibody to the antigen was detected in liver tissue from all halothane-exposed rabbits and guinea pigs. Antigen could be detected only in the centrilobular area around the central vein where staining intensity was concentrated in an area seven to nine cells deep. In halothane-exposed rabbits, the appearance of TFA antigen was most predominant on the first and second days following a single exposure. Multiple exposures induced TFA antigen in a larger area around the central vein than did a single exposure. Though maximal expression of TFA antigen occurred following two or three exposures, subsequent exposures did not potentiate antigen expression. In halothane-exposed guinea pigs, exposure to deuterated halothane, which reduces the extent and metabolites of oxidative halothane metabolism, elicited the appearance of TFA antigen around the central veins, although to a lesser extent than during halothane exposure. Halothane-induced antigen was evident in guinea pigs as early as 6 h post-exposure and was still apparent 90 h later. Thus, halothane exposure by inhalation elicits the appearance of TFA protein conjugates which may, in turn, evoke the anti-TFA immune response.
• Wolfgang, G. H., Gandolfi, A. J., & Brendel, K. (1989). Evaluation of organic nephrotoxins in rabbit renal cortical slices. Toxicology in vitro : an international journal published in association with BIBRA, 3(4), 341-50.
  More info

  An in vitro cortical-slice system was used to assess the toxicity of several organic nephrotoxins that require transport and/or bioactivation in order to induce toxicity. The toxins cephaloridine, hexachlorobutadiene, S-(1,2- dichlorovinyl)- l -cysteine and gentamicin all produce site-specific proximal tubular injury when administered in vivo. Damage was assessed in vitro by observing alterations in intracellular potassium, intracellular lactate dehydrogenase, and organic anion and cation accumulation. Histopathology was studied to assess the localization of injury. All four compounds produced dose- and time-dependent decreases in the biochemical parameters as well as site-specific lesions in the S(3) region. The results illustrate the usefulness of renal cortical slices in acute studies of organic nephrotoxins.
• Wolfgang, G. H., Gandolfi, A. J., Stevens, J. L., & Brendel, K. (1989). In vitro and in vivo nephrotoxicity of the L and D isomers of S-(1,2-dichlorovinyl)-cysteine. Toxicology, 58(1), 33-42.
  More info

  The toxicity of the optical isomers S-(1,2-dichlorovinyl)-L-cysteine (L-DCVC) and S-(1,2-dichlorovinyl)-D-cysteine (D-DCVC) was investigated in vivo and in vitro. In vitro studies, utilizing a rabbit renal cortical slice system, demonstrated toxicity due to both forms with the L-form being more toxic. Dose- and time-dependent decreases in intracellular K+ and LDH were observed. Both compounds produced an initial S3 lesion, L-DCVC at 10(-5) M (12 h), D-DCVC at 10(-4) M (8 h), followed by a lesion encompassing all proximal tubules. In vivo studies demonstrated elevated blood urea nitrogen values at 24 and 48 h with 25 mg/kg of either isomer. Histopathology indicated both D and L-DCVC produced a straight proximal tubular lesion by 48 h, the lesion produced by L-DCVC being more severe. The D and L isomers of DCVC were both shown to be toxic, the toxicity assessed in vitro corresponded well with the toxicity in vivo.
• Wolfgang, G. H., Gandolfi, A. J., Stevens, J. L., & Brendel, K. (1989). N-acetyl S-(1,2-dichlorovinyl)-L-cysteine produces a similar toxicity to S-(1,2-dichlorovinyl)-L-cysteine in rabbit renal slices: differential transport and metabolism. Toxicology and applied pharmacology, 101(2), 205-19.
  More info

  Renal cortical slices were used to determine the toxicity of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-acetyl-DCVC) as well as to investigate the transport and metabolism of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and the N-acetyl derivative. N-Acetyl-DCVC produced dose- and time-dependent decreases in intracellular K+ content and lactate dehydrogenase activity. Histopathology demonstrated an initial S3 lesion followed by a lesion inclusive of all proximal tubules. N-Acetyl-DCVC was shown to be transported via the organic anion system by its ability to inhibit PAH transport by the cells and the ability of probenecid to decrease uptake (80%) and toxicity of N-acetyl-DCVC. DCVC, in contrast, was not transported by the organic anion system, but may be transported by one or more amino acid systems. N-Acetyl-DCVC must be deacetylated before undergoing metabolism by beta-lyase. This process must occur since covalent binding of a 35S-labeled reactive product from N-acetyl [35S]DCVC is observed within 1 hr. Both the uptake inhibitor, probenecid, and aminooxyacetic acid (AOAA), a beta-lyase inhibitor, decreased the covalent binding from N-acetyl [35S]DCVC (80 and 50%, respectively), but only AOAA inhibited the covalent binding of DCVC. AOAA also partially inhibited the toxicity of DCVC and N-acetyl-DCVC as determined by intracellular K+ content, lactate dehydrogenase activity, and histopathology. Despite the fact that a separate transport system and an additional enzymatic step (deacetylation) are required, N-acetyl-DCVC produces a lesion with similar intratubular specificity to that seen with DCVC. Therefore, the S3 specificity seen in vivo could be produced by either compound.
• Hubbard, A. K., Gandolfi, A. J., & Brown, B. R. (1988). Immunological basis of anesthetic-induced hepatotoxicity. Anesthesiology, 69(6), 814-7.
• Hubbard, A. K., Roth, T. P., Gandolfi, A. J., Brown, B. R., Webster, N. R., & Nunn, J. F. (1988). Halothane hepatitis patients generate an antibody response toward a covalently bound metabolite of halothane. Anesthesiology, 68(5), 791-6.
• Becker, G. M., Gandolfi, A. J., & Nagle, R. B. (1987). Effects of cyclosporin A on a kidney epithelial cell line (LLC-PK1). Research communications in chemical pathology and pharmacology, 56(2), 277-80.
  More info

  Cyclosporin A (CSA), a potent immunosuppressant with the adverse side effect of nephrotoxicity, inhibited cell growth of pig kidney tubule cells (LLC-PK1) in culture. CSA (10(-5) M) also induced intense cytoplasmic vacuolation and the formation of dense granules. At the same concentration an analogue of CSA, cyclosporin G, had much less effect. This cell line may prove useful for revealing the mechanism of CSA-nephrotoxicity and testing the nephrotoxic potential of new analogues of cyclosporine.
• Brendel, K., McKee, R. L., Hruby, V. J., Johnson, D. G., Gandolfi, A. J., & Krumdieck, C. L. (1987). Precision cut tissue slices in culture: a new tool in pharmacology. Proceedings of the Western Pharmacology Society, 30, 291-3.
• Brown, B. R., & Gandolfi, A. J. (1987). Adverse effects of volatile anaesthetics. British journal of anaesthesia, 59(1), 14-23.
  More info

  The volatile inhalation anaesthetics have been implicated in a variety of adverse viscerotoxic reactions. In general, they have been proven to produce very few non-predicted toxicities. Hepatitis caused by halothane now seems to be the only major problem in this regard with these drugs in current practice. The evidence is convincing that this reaction is based initially on biotransformation. Thus decreases in the amount of biotransformation and lessened production of reactive metabolic products would theoretically produce a safer anaesthetic. While not perfect in all circumstances, enflurane and isoflurane come close to achieving the goal of decreased adverse viscerotoxic events.
• Callis, A. H., Brooks, S. D., Roth, T. P., Gandolfi, A. J., & Brown, B. R. (1987). Characterization of a halothane-induced humoral immune response in rabbits. Clinical and experimental immunology, 67(2), 343-51.
  More info

  An animal model of halothane-induced liver injury has been developed in the rabbit to study the production of humoral immunity towards a biotransformation intermediate of halothane. Rabbits exposed many times to halothane in a 75% O2/25% N2 atmosphere produce an antibody that cross-reacts with the trifluoroacetyl moiety of trifluoroacetylated rabbit serum albumin (TFA-RSA). The generation of this halothane-induced immunogen is dependent upon high oxygen tension as shown by the minimal anti-TFA antibody response seen in rabbits exposed to halothane in a 14% O2/86% N2 atmosphere. In addition, halothane exposure of rabbits specifically immunized with the metabolite-carrier complex, TFA-RSA, induces a secondary antibody response toward the immunogen. In rabbits, either immunized with TFA-RSA or not, multiple halothane exposures induce populations of antibodies with varying specificities. Evidence suggests that predominance of the metabolic intermediate, the ensuing immunogen, and the subsequent antibody response depends upon the oxygen tension during successive exposures to halothane. These successive exposures could potentially generate many different immunogens resulting in varied antibody specificities.
• Phelps, J. S., Gandolfi, A. J., Brendel, K., & Dorr, R. T. (1987). Cisplatin nephrotoxicity: in vitro studies with precision-cut rabbit renal cortical slices. Toxicology and applied pharmacology, 90(3), 501-12.
  More info

  Severe nephrotoxic side effects limit the use of cisplatin, a potent anticancer drug. In this study, precision-cut renal cortical slices from rabbits were evaluated as a cisplatin nephrotoxicity model. Cortical slices accumulated approximately 180 ppm (195 ppm Pt = 10(-3) M) of platinum(II) after 18 hr of incubation in medium containing 10(-3) M cisplatin. Dose- and time-dependent toxic responses for clinically relevant concentrations of cisplatin (10(-3)-10(-5) M) were apparent using leakage of intracellular K+, ATP, and lactate dehydrogenase (LDH) to determine cell damage. Histopathologic changes were also produced. Intracellular ATP levels dropped significantly after 6 hr of incubation in 10(-3) M cisplatin, and after 12 hr with 10(-4) M cisplatin. Similarly, intracellular K+ levels decreased significantly by 6 hr of incubation with 10(-3) M cisplatin but remained at control levels for 18 hr in the presence of 10(-4) M cisplatin. Decrements in intracellular LDH levels were not seen until after 12 hr of incubation in 10(-3) M cisplatin. The noncytotoxic isomer transplatin at 10(-3) M was not accumulated by slices; however, intracellular ATP levels were depressed. Of the viability parameters evaluated, intracellular K+ and ATP were found to be optimal indicators. Other active platinum analogs, carboplatin and iproplatin, also caused dose- and time-dependent leakage of intracellular K+ and ATP from renal cortical slices. The ranking of nephrotoxicity of the platinate compounds within this system at concentrations adjusted to approximate equivalent therapeutic activity was similar to that observed in vivo (cisplatin = iproplatin greater than carboplatin greater than transplatin). These results suggest that precision-cut renal cortical slices comprise a viable in vitro model for platinum-induced nephrotoxicity studies.
• Ruegg, C. E., Gandolfi, A. J., Nagle, R. B., & Brendel, K. (1987). Differential patterns of injury to the proximal tubule of renal cortical slices following in vitro exposure to mercuric chloride, potassium dichromate, or hypoxic conditions. Toxicology and applied pharmacology, 90(2), 261-73.
  More info

  The innate susceptibility of renal cell types to these agents was investigated using precision-cut rabbit renal cortical slices made perpendicular to the cortical-papillary axis. Slices were incubated in DME/F12 medium containing 10 microM, 100 microM, or 1 mM concentrations of either metal for 12 hr or in Krebs-Hepes buffer gassed with nitrogen (100%) for 0.75 to 5 hr of hypoxic exposure. To simulate postischemic reperfusion, some slices were transferred to vessels gassed with oxygen after an initial hypoxic period. Mercuric chloride (100 microM) exposure resulted in damage to the straight regions of proximal tubules by 12 hr leaving convoluted regions unaffected. Hypoxia (2.25 hr) and potassium dichromate (100 microM for 12 hr) both caused injury to the convoluted proximal tubules without affecting straight proximal tubular regions. Mercury concentrations of 10 microM and 1 mM had no effect or injured all cell types within the slice, respectively. Similar results were observed for hypoxic periods less than 1.5 hr or greater than 3 hr of exposure. Potassium dichromate had no measurable affect at 10 microM, but at 1 mM focal lesions were observed after 4 hr of exposure, and by 12 hr all cell types within the slice were affected. Intracellular potassium content normalized to DNA correlated well, but always preceded the pathological lesions observed. These results demonstrate that injury to specific regions of the proximal tubule by these agents relates to an innate susceptibility of the intoxicated cell type independent of physiologic feedback or blood delivery patterns proposed as mechanisms of selective injury from in vivo studies.
• Ruegg, C. E., Gandolfi, A. J., Nagle, R. B., Krumdieck, C. L., & Brendel, K. (1987). Preparation of positional renal slices for study of cell-specific toxicity. Journal of pharmacological methods, 17(2), 111-23.
  More info

  To reduce structural complexity, rabbit kidneys were sliced perpendicular to their cortical-papillary axis to isolate four distinct cell groupings. This positional orientation allows identification of each renal cell type based on its location within the slice. A mechanical slicer was used to make several precision-cut slices rapidly from an oriented cylindrical core of renal tissue, with minimal tissue trauma. Slices were then submerged under a gently circulating oxygenated media in a fritted glass support system that maintains viability (intracellular K+/DNA ratio) and structural integrity (histology) for at least 30 h. A high dose of mercuric chloride (10(-3) M) was used to demonstrate the structural and biochemical changes of intoxicated slices. This method provides a controlled subchronic in vitro system for the study of the individual cell types involved in cell-specific renal toxicities and may also be a useful tool for addressing other pharmacological and physiological research questions.
• Sipes, I. G., Fisher, R. L., Smith, P. F., Stine, E. R., Gandolfi, A. J., & Brendel, K. (1987). A dynamic liver culture system: a tool for studying chemical biotransformation and toxicity. Archives of toxicology. Supplement. = Archiv fur Toxikologie. Supplement, 11, 20-33.
• DiRenzo, A. B., Adamson, C. R., Gandolfi, A. J., Brendel, K., & Krack, G. (1986). Effect of volatile anesthetics on protein synthesis and secretion by rat hepatocyte suspensions. Drug and chemical toxicology, 9(3-4), 223-37.
  More info

  The effect of volatile anesthetics on protein synthesis and secretion by isolated rat hepatocytes in suspension was investigated. Halothane and enflurane inhibited protein synthesis in a dose-dependent manner. Diethyl ether had little effect on protein synthesis while isoflurane caused a mild inhibition. This effect was more pronounced in hepatocytes from phenobarbital treated male rats when compared to hepatocytes from control rats. Protein synthesis in hepatocytes from phenobarbital treated female rats was inhibited similar to that seen with control male rat hepatocytes. Isoflurane, enflurane, and halothane also caused a dose-dependent inhibition of protein secretion, while diethyl ether was only mildly inhibitory. From these studies it appears that inhibition of protein synthesis and secretion might be an early and sensitive indicator of cellular injury by volatile anesthetics.
• MacDonald, J. R., Gandolfi, A. J., & Sipes, I. G. (1986). Amelioration of carbon tetrachloride-induced hepatic necrosis by post-toxicant treatment with cystamine. Toxicology, 39(2), 135-48.
  More info

  A quantitative animal model was developed to study amelioration of carbon tetrachloride-induced hepatic injury by post-toxicant administration of cystamine. Amelioration of CCl4-induced injury by post-toxicant cystamine treatment was compared to prevention of injury by cystamine pretreatment and possible mechanisms of the post-toxicant cytoprotective effect were investigated. Pretreatment of rats with cystamine dihydrochloride (300 mg/kg, p.o.) 30 min prior to CCl4 (0.25 ml/kg, i.p.) prevented CCl4-induced hepatic necrosis, plasma enzyme elevations, and hepatic calcium accumulation. When administered up to 12 h after CCl4, a single oral dose of cystamine inhibited necrosis in a dose-dependent manner, but did not reduce CCl4-induced plasma enzyme elevation or hepatic calcium accumulation. Cystamine post-treatment, therefore, does not appear to inhibit toxicant-induced influx of extracellular calcium into toxicant-damaged cells. This also suggests that the influx of extracellular calcium does not necessarily constitute an irreversible event leading to cell death. The mild hypothermia induced by post-toxicant treatment with cystamine did not delay the appearance of the lesion. Evidence for a slightly earlier regeneration of hepatic tissue was noted when cystamine was administered 12 h after CCl4. However, this effect was observed too long after exposure to the toxicant to account for the protection from necrosis observed 24 h after CCl4.
• Silber, P. M., Gandolfi, A. J., & Brendel, K. (1986). Adaptation of a gamma-glutamyl transpeptidase assay to microtiter plates.. Analytical biochemistry, 158(1), 68-71. doi:10.1016/0003-2697(86)90590-7
  More info

  A kinetic assay for measuring gamma-glutamyl transpeptidase (GGT) activity has been adapted to microtiter plates and an automated microtiter plate reader. This method permits the simultaneous analysis of enzyme activity in a large number of samples incubated with the chromogenic GGT substrate gamma-glutamyl-p-nitroanilide. A major advantage of this assay over previously reported methods is the substantial reduction in the time needed for measuring sample enzyme activity. In addition, reduction of the total assay volume to 0.28 ml conserves both sample and reagents. This method has been calibrated at 23 degrees C using purified GGT, and used to analyze GGT activity in human sera. The assay is sensitive over a range of 3-200 U/liter.
• Cater, K. C., Gandolfi, A. J., & Sipes, I. G. (1985). Characterization of dimethylnitrosamine-induced focal and nodular lesions in the livers of newborn mice. Toxicologic pathology, 13(1), 3-9.
  More info

  Newborn Swiss-Webster mice were given an intraperitoneal injection of 25 micrograms of dimethylnitrosamine. At weaning they began receiving 0.05% phenobarbital in the drinking water to promote the lesions for the term of the study. Preneoplastic foci and hyperplastic nodules were identified histologically by two markers, resistance to exogenous iron accumulation and an increase in gamma-glutamyltranspeptidase activity. AT 8, 12, and 16 weeks of age, livers of affected male mice exhibited 12, 18, and 12 iron-resistant foci/cm2 and 13, 9, and 9 gamma-glutamyltranspeptidase-positive foci/cm2, respectively (average for median right and right anterior sublobes). Iron-resistant nodules were first observed at 8 weeks; however, gamma-glutamyltranspeptidase-positive nodules were not noted until 12 weeks. In animals that received dimethylnitrosamine but were not placed on phenobarbital, there was an average of 5 foci/cm2 (iron-resistant or gamma-glutamyltranspeptidase-positive) at 12 weeks while no nodules were noted. This model could provide a practical short-term in vivo tool for the detection of early sequential cellular alterations produced by initiators, inhibitors, and promoters of carcinogenesis.
• DiRenzo, A. B., Gandolfi, A. J., Brooks, S. D., & Brendel, K. (1985). Toxicity and biotransformation of volatile halogenated anesthetics in rat hepatocyte suspensions. Drug and chemical toxicology, 8(4), 207-18.
  More info

  This study examines the toxicity (as measured by reduced intracellular K+) and metabolism (defluorination) of halothane and enflurane in rat hepatocytes in suspension (RHS) with regards to O2 tension, time, and concentration. In 95% O2 halothane is more toxic than enflurane when RHS are exposed to 5-20 microliters of these anesthetics. At these levels halothane is not metabolized while enflurane is metabolized. At 21% O2 a similar pattern was seen with regards to toxicity. However, metabolism of halothane rapidly reached an elevated level while that of enflurane is reduced when compared to 95% O2. Thus toxicity of halothane and enflurane at these dose levels appears to be unrelated to metabolism and due solely to a solvent effect.
• Jackson, J. E., Bentley, J. B., Glass, S. J., Fukui, T., Gandolfi, A. J., & Plachetka, J. R. (1985). Effects of histamine-2 receptor blockade on lidocaine kinetics. Clinical pharmacology and therapeutics, 37(5), 544-8.
  More info

  The hypothesis that the H2-receptor blockers cimetidine and ranitidine have different effects on the disposition of lidocaine, a microsomally metabolized drug dependent on hepatic blood flow for elimination, was tested. Six normal men received lidocaine infusions (2 mg/kg over 10 minutes) and lidocaine levels were determined by HPLC. Lidocaine kinetics were studied in the untreated state (O) and in a double-blind, double-dummy design after 2 days of placebo (P), cimetidine (C, 300 mg every 6 hours by mouth), or ranitidine (R, 160 mg every 12 hours by mouth). Model-independent kinetics were estimated by the statistical moment theory. The steady-state volume of distribution was lower after cimetidine (means +/- SD: O, 156 +/- 39 L; P, 156 +/- 48 L; C, 123 +/- 20 L; and R, 174 +/- 38 L). A trend toward decreased lidocaine clearance after cimetidine was also noted (O, 1011 +/- 140 ml/min; P, 1087 +/- 227 ml/min; C, 886 +/- 214 ml/min; and R, 1143 +/- 225 ml/min). Elimination rate constants were of the same order in all four treatments. Only higher levels of alpha 1-acid glycoprotein appeared to limit the lidocaine steady-state volume of distribution. Cimetidine and ranitidine have distinctly different effects on lidocaine kinetics in normal subjects. The absence of ranitidine effects on the disposition of lidocaine, a high-extraction, high-clearance drug, suggests that H2-receptor blockade may not decrease hepatic blood flow, and that cimetidine impairs drug elimination only by inhibition of hepatic microsomal enzymes. Such interactions are not likely to occur with ranitidine.
• Jaffe, D. R., Hassall, C. D., Gandolfi, A. J., & Brendel, K. (1985). Production of DNA single strand breaks in rabbit renal tissue after exposure to 1,2-dichlorovinylcysteine. Toxicology, 35(1), 25-33.
  More info

  S-(trans-1,2-dichlorovinyl)-L-cysteine (DCVC) is a recognized nephrotoxin. To investigate the genotoxic effects of DCVC on the kidney, DNA strand breaks were measured as an indicator of DCVC induced damage. To ascertain if bioactivation of DCVC occurred in the kidney, 3 experimental systems were used: in vivo; isolated perfused kidneys; and isolated proximal tubules of albino male rabbits. A dose-dependent increase in strand breaks in the kidney tubular DNA occurred after in vivo dosing with 5-100 mg/kg DCVC and after in vitro exposure to 10(-5)-10(-2) M DCVC. These results demonstrate the genotoxic effect of this compound on renal tissue.
• MacDonald, J. R., Gandolfi, A. J., & Sipes, I. G. (1985). Hepatic cysteamine and non-protein sulfhydryl levels following cystamine or cysteamine treatment of galactosamine-poisoned rats. Drug and chemical toxicology, 8(6), 483-94.
  More info

  Hepatic cysteamine and non-protein sulfhydryl (NPSH) levels were determined in galactosamine (GAL)-poisoned rats following hepatoprotective cystamine or cysteamine treatments to determine whether alterations of hepatic NPSH status could contribute to their observed protective actions. D(+)-Galactosamine HC1 (400 mg/kg, ip) was administered to male Sprague-Dawley rats at 8 pm. Cystamine diHC1 (300 mg/kg, po) or cysteamine HC1 (170 mg/kg, ip) were administered 12 hr after GAL. Hepatic NPSH levels were determined using Ellman's reagent. Hepatic cysteamine levels were determined by separating NPSH Ellman's derivatives by reversed phase HPLC. Cystamine and cysteamine caused transient elevation of NPSH levels of 1-2 nanomoles/mg liver which correlated with the presence of 1-2 nanomoles of cysteamine/mg liver. However, neither cystamine nor cysteamine prevented NPSH levels from falling to 3 nanomoles/mg tissue 24 hr after GAL. Hepatoprotective treatments did not affect long term NPSH status in GAL-poisoned rats. However, transient NPSH increases, due to the intrahepatic presence of cysteamine, may contribute to the therapeutic effects of these hepatoprotective agents.
• MacDonald, J. R., Gandolfi, A. J., & Sipes, I. G. (1985). Structural requirements for cytoprotective agents in galactosamine-induced hepatic necrosis. Toxicology and applied pharmacology, 81(1), 17-24.
  More info

  A variety of compounds were tested for their ability to inhibit the development of galactosamine-induced hepatic necrosis when administered 12 hr after the toxicant. Hepatic necrosis in male Sprague-Dawley rats was quantified by histopathologic examination 24 hr after a hepatotoxic dose of D(+)-galactosamine HCl (400 mg/kg, ip). Compounds found to have antinecrotic activity were not able to eliminate the accumulation of calcium associated with galactosamine-induced hepatic necrosis. Potent calcium chelators (EDTA and EGTA), compounds with aminoethanethiol-chelating structures (cysteamine and penicillamine), compounds that may be metabolized to aminoethanethiol structures in vivo (N-acetylcysteine, 2-aminoethylisothiourea, and cystamine), and a compound known to alter subcellular calcium sequestration (taurine) all inhibited galactosamine-induced hepatic necrosis. Compounds without antinecrotic effects (S-methylcysteamine, thioproline, dimercaptopropanesulfonic acid, and dimercaptosuccinic acid) do not possess structural or functional characteristics of the antinecrotic agents. It is suggested that chelation of free intercellular calcium or enhanced subcellular sequestration of calcium could explain the reduction of cytotoxic consequences of hepatic calcium accumulation observed in this model.
• Wiggum, D. C., Cork, R. C., Weldon, S. T., Gandolfi, A. J., & Perry, D. S. (1985). Postoperative respiratory depression and elevated sufentanil levels in a patient with chronic renal failure. Anesthesiology, 63(6), 708-10.
• DiRenzo, A. B., Gandolfi, A. J., Sipes, I. G., Brendel, K., & Byard, J. L. (1984). Effect of O2 tension on the bioactivation and metabolism of aliphatic halides by primary rat-hepatocyte cultures. Xenobiotica; the fate of foreign compounds in biological systems, 14(7), 521-5.
  More info

  The covalent binding of CCl4 and CF3CHBrCl to cultured rat hepatocytes was enhanced by anoxic conditions, indicative of enhanced reductive biotransformation. The covalent binding of CHCl3 and CHCl2CH2Cl decreased as the O2 concentration was decreased, indicating a preference for oxidative metabolism. Lower O2 tensions decreased the formation of polar water-soluble metabolites with CHCl3 and CHCl2CH2Cl, while CCl4 and CF3CHBrCl were unaffected. The results indicate that primary cultured hepatocytes can reductively biotransform and bioactivate aliphatic halides under anoxic conditions.
• Durham, J. A., Gandolfi, A. J., & Bentley, J. B. (1984). Hepatotoxicological evaluation of dantrolene sodium. Drug and chemical toxicology, 7(1), 23-40.
  More info

  The clinical use of dantrolene has been associated with hepatotoxicity, thus the toxicity of dantrolene in Swiss-Webster mice was characterized. Animals were treated orally (po) with single or multiple doses of up to 400 mg/kg of D without any increases in SGPT or alterations in hepato-cellular architecture. To possibly enhance the hepatotoxicity of dantrolene, its biotransformation was altered by inhibiting acetylation, depleting glutathione, inducing biotransformation, and promoting reductive metabolism. None of the metabolic alterations elicited any toxicity of dantrolene. Hepatic microsomal incubations were used to detect the possible bioactivation and covalent binding of 14C-dantrolene. Analysis for covalent adducts found only 20-30 pmol bound/mg microsomal protein. Thus the suspected hepatotoxicity of dantrolene does not appear to be linked to its biotransformation or bioactivation. Additional studies will be necessary to clarify if other parameters are necessary for dantrolene to be a hepatotoxin.
• Hassall, C. D., Gandolfi, A. J., Duhamel, R. C., & Brendel, K. (1984). The formation and biotransformation of cysteine conjugates of halogenated ethylenes by rabbit renal tubules. Chemico-biological interactions, 49(3), 283-97.
  More info

  The nephrotoxicity of chlorotrifluoroethylene ( CTFE ) was examined using isolated rabbit renal tubules suspensions. Exposure of the tubules to CTFE resulted in consumption of CTFE , formation of a glutathione conjugate and inhibition of active organic acid transport. Synthetic cysteine, N-acetylcysteine or glutathione conjugates of CTFE inhibited transport indicating S-conjugation as a possible toxic pathway. 1,2-dichlorovinyl glutathione ( DCVG ), a model synthetic glutathione conjugate, was used to examine the degradation and toxicity of these conjugates. DCVG inhibited rabbit renal tubule transport in vivo and in vitro. The DCVG was found to be degraded with the evolution of glutamine and glycine to produce the ultimate nephro-toxicant, dichlorovinyl cysteine. Dichlorovinyl cysteine is then bioactivated with the release of ammonia. This sequential degradation explains the latency of DCVG -induced renal transport inhibition relative to dichlorovinyl cysteine. It is now evident that certain halogenated ethylenes are capable of being biotransformed to glutathione conjugates in the kidney with their subsequent hydrolysis to nephrotoxic cysteine conjugates.
• Jaffe, D. R., Gandolfi, A. J., & Nagle, R. B. (1984). Chronic toxicity of S-(trans-1,2-dichlorovinyl)-L-cysteine in mice. Journal of applied toxicology : JAT, 4(6), 315-9.
  More info

  S-(trans-1,2-Dichlorovinyl)-L-cysteine (DCVC) exposure causes acute renal tubular cytotoxicity. To further characterize the effects of DCVC, a chronic study was undertaken. Male Swiss-Webster mice received DCVC dissolved in their drinking water at 0.01, 0.05 and 0.1 mg ml-1. At 4, 8, 21 and 37 weeks, animals were terminated. Bladders, spleens, livers, kidneys and eyes were removed for histopathological examination. At 0.05 and 0.1 mg ml-1 DCVC, growth retardation was evident by 21 weeks. By 26 weeks, all animals in the 0.1 mg ml-1 group had developed cortical cataracts. Cytomegaly, nuclear hyperchromatism and multiple nucleoli were noted in the cells of the pars recta region of the kidney by 4 weeks and correlated to time and dose. At later time points, renal tubular atrophy and early interstitial fibrosis were evident. The epithelial cytological cellular abnormalities appear to be dose-related. Minor pathological changes were noted in the spleen, while there was no effect on the liver or bladder. Chronic ingestion of DCVC results in severe kidney injury.
• MacDonald, J. R., Gandolfi, A. J., & Sipes, I. G. (1984). Cystamine modulation of galactosamine-induced hepatotoxicity. Toxicology and applied pharmacology, 73(3), 551-8.
  More info

  The ability of cystamine treatment to protect against galactosamine-induced hepatic necrosis was investigated. Reduced hepatotoxicity was observed following galactosamine hydrochloride (400 mg/kg, ip) in male Sprague-Dawley rats that received cystamine dihydrochloride (300 mg/kg, po) 30 min prior to or 2, 4, 6, 8, or 12 hr after galactosamine. In contrast, uridine treatment only protected against galactosamine-induced liver damage if administered within 2 hr of galactosamine. Protection by cystamine was found to extend over 3 days during which time the lesion was resolving. The degree of protection was dose related when administered 12 hr after galactosamine. Cystamine did not prevent or alter the increase in hepatic Ca2+ seen following galactosamine administration. The results indicate that the protective effects of cystamine on galactosamine-induced hepatotoxicity are unrelated to prevention of the early biochemical events that initiate the injury. Furthermore, prevention of Ca2+ accumulation in damaged hepatocytes is not the mechanism of protection and hence is not necessarily an irreversible cytotoxic event.
• Gandolfi, A. J., Blitt, C. D., & Weldon, S. (1983). Discoloration and impurities in isoflurane vaporizer. Anesthesia and analgesia, 62(3), 366.
• Hassall, C. D., Brendel, K., & Gandolfi, A. J. (1983). Regulation of a S(trans-1,2-dichlorovinyl)-L-cysteine-induced renal tubular toxicity by glutathione. Journal of applied toxicology : JAT, 3(6), 321-5.
  More info

  The nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is cleaved in the renal tubules to produce a reactive electrophilic intermediate. If this intermediate is responsible for the toxicity, addition of the nucleophilic scavenger glutathione (GSH) should decrease toxicity, and depletion of tubular GSH should enhance toxicity. GSH was added to isolated rabbit renal tubules simultaneously with, 15 min before, and 15 min after the addition of DCVC. The active accumulation of the organic anion para-aminohippuric acid (PAH) and organic cation tetraethylammonium bromide (TEA) was used as an index of renal toxicity. Incubation of renal tubules with 0.01-1 mM DCVC for 15 min decreased active transport, with complete inhibition at 1 mM. This was accompanied by a 50% decrease in non-protein sulfhydryl concentration. The addition of GSH (6 mM) simultaneously with DCVC completely prevented any decrease in active transport. The addition of GSH (6 mM) to tubules in which active transport was inhibited by DCVC reversed the inhibition to 80% of control. Similar enhancement of active transport occurred when tubules isolated 1 h after in vivo exposure to DCVC at 20-100 mg kg-1 were incubated with GSH (6 mM). Preincubation of renal tubules with GSH (5-15 mM) made them more refractory to the DCVC-induced decreased PAH and TEA transport. The inhibition of active transport by DCVC is enhanced if the tubular non-protein sulfhydryl is first lowered by diethyl maleate or glycidol. Thus, the tubular GSH concentration appears to be an integral component in regulating the alterations in active transport caused by DCVC.
• Hassall, C. D., Gandolfi, A. J., & Brendel, K. (1983). Correlation of the in vivo and in vitro renal toxicity of S-(1,2-dichlorovinyl)-L-cysteine. Drug and chemical toxicology, 6(6), 507-20.
  More info

  The major site at which vinyl cysteine conjugates exert nephrotoxicity is the proximal tubule. Since this is the site of all active anion and cation transport, tubule transport integrity was used to assess nephrotoxicity. Tubules were isolated from young rabbits to study the in vivo and in vitro nephrotoxicity of the conjugate, dichlorovinyl cysteine (DCVC). In vivo exposure to DCVC caused necrosis in the pars recta region of the proximal tubules (20-100 mg/kg ip) and a dose-dependent decrease in tubular active transport. Addition of DCVC to the perfused kidney and tubule suspensions resulted in similar decreases in tubular organic ion transport. At 0.01 mM DCVC, transport was similar to controls while 1 mM DCVC completely inhibited active accumulation of the organic ions. Thus kidney tubule active transport is similarly inhibited in vivo and in vitro by DCVC indicating that bioactivation of DCVC and inhibition of active transport occur directly in the renal tubule.
• MacDonald, J. R., Gandolfi, A. J., & Sipes, I. G. (1983). Cystamine treatment of chemically induced liver injury. Developments in toxicology and environmental science, 11, 463-6.
• Bentley, J. B., Borel, J. D., Vaughan, R. W., & Gandolfi, A. J. (1982). Weight, pseudocholinesterase activity, and succinylcholine requirement. Anesthesiology, 57(1), 48-9.
• Bentley, J. B., Vaughan, R. W., Gandolfi, A. J., & Cork, R. C. (1982). Halothane biotransformation in obese and nonobese patients. Anesthesiology, 57(2), 94-7.
  More info

  Serum levels of inorganic fluoride, trifluoroacetic acid, and bromide ion were measured at various time intervals following two hours of halothane anesthesia in 17 morbidly obese and eight nonobese patients. Ionic fluoride, a marker of reductive halothane metabolism, increased in the obese but not the nonobese patients. This is of concern since reductive halothane metabolism is associated with hepatoxicity in animals. In addition, serum bromide levels were higher after 48 h in the obese patients compared to the nonobese patients (mean +/- SE, 1,311 +/- 114 vs. 787 +/- 115 microM, P less than 0.01). Sedative levels of bromide were not attained in any patient. Peak trifluoroacetic acid levels were similar in the two patient groups. Sex age, medication intake, and smoking history had no influence on the halothane metabolite levels found in this study.
• Borel, J. D., Bentley, J. B., Vaughan, R. W., & Gandolfi, A. J. (1982). Enflurane blood-gas solubility: influence of weight and hemoglobin. Anesthesia and analgesia, 61(12), 1006-9.
  More info

  The blood-gas partition coefficient of enflurane was measured in nine nonobese and eight morbidity obese patients and correlated with weight, body mass index, and blood hemoglobin. The enflurane blood-gas partition coefficient was lower in the obese patients than in nonobese patients (mean +/- SEM: 2.03 +/- 0.02 versus 1.76 +/- 0.03, respectively, p less than 0.025). There was a negative correlation between enflurane blood solubility and both body mass index and weight (r = 0.59 and -0.55, respectively, p less than 0.01). A positive correlation was found between hemoglobin and the enflurane blood-gas partition coefficient (r = 0.69, p less than 0.01). Equilibrium between inspired and alveolar enflurane concentration should be faster in morbidity obese and anemic patients than in healthy, nonobese patients.
• DiRenzo, A. B., Gandolfi, A. J., & Sipes, I. G. (1982). Microsomal bioactivation and covalent binding of aliphatic halides to DNA. Toxicology letters, 11(3-4), 243-52.
  More info

  Studies were carried out on the in vitro covalent binding of a series of 14C-labeled aliphatic halides to calf thymus DNA following bioactivation by hepatic microsomes isolated from phenobarbital-treated rats. Six compounds were shown to exhibit binding to DNA of greater than 0.3 nmol/mg DNA (1,2-dibromoethane, bromotrichloromethane, trichloroethylene, carbon tetrachloride, chloroform, and 1,1,2-trichloroethane). Covalent binding of the aliphatic halides to the nucleic acids was confirmed by sedimentation of the DNA-organohalogen adduct in a cesium chloride gradient and Sephadex LH-20 chromatography of the nucleosides released by enzymatic hydrolysis.
• DiRenzo, A. B., Gandolfi, A. J., Sipes, I. G., & McDougal, J. N. (1982). Effect of senescence on the bioactivation of aliphatic halides. Research communications in chemical pathology and pharmacology, 36(3), 493-502.
  More info

  Since the toxicity of xenobiotics is often related to their metabolism, this study was concerned with the relationship between aging and the bioactivation and covalent binding of certain aliphatic halides to microsomal protein and lipid. Hepatic microsomes were isolated from control and phenobarbital induced young (4 mo), adult (11 mo) and senescent (27 mo) Fischer 344 rats. Bioactivation and subsequent covalent binding were studied with 14C-labeled 1,2-dibromoethane, carbon tetrachloride, trichloroethylene, and 1, 1, 2-trichloroethane. With control rats, levels of binding increased slightly between 4 and 11 mo; however the values decreased 50-75% in the 27 mo group compared to the 11 mo group. No significant differences were noted between phenobarbital induced groups with regards to bioactivation of the aliphatic halides and their covalent binding to proteins and lipids. As an explanation for the decreased bioactivation ability in the senescent rats, they were also found to have significantly less hepatic cytochrome P-450 (-35%), NADPH cytochrome C reductase (-31%), and ethyl-morphine N-demethylase activity (-43%) when compared to the 11 mo age group. These differences were reduced when comparisons were made between the various age groups of phenobarbital induced animals. This suppression may indicate a decreased potential for the expression of toxicities requiring bioactivation.
• MacDonald, J. R., Gandolfi, A. J., & Sipes, I. G. (1982). Acetone potentiation of 1,1,2-trichloroethane hepatotoxicity. Toxicology letters, 13(1-2), 57-69.
  More info

  Acetone potentiation of 1,1,2-trichloroethane (TCEA) hepatotoxicity was found to be strongly dose-dependent for both acetone and TCEA. An oral dose of 0.5 ml of acetone/kg was the most effective potentiating pretreatment when administered 16 h prior to TCEA. Surprisingly, higher doses of acetone did not potentiate the hepatotoxicity of TCEA, but may have decreased the severity of this toxicity. Acetone potentiation of hepatotoxicity was most pronounced at or near the hepatotoxic threshold for TCEA. While acetone treatment alone did not affect hepatic glutathione (GSH) stores the most consistent effect produced by acetone pretreatment was a significant potentiation of TCEA-induced depletion of hepatic GSH. These findings suggest that critical doses of acetone may enhance the subsequent hepatotoxicity of TCEA via mechanisms that affect the interaction of TCEA and hepatic GSH stores.
• MacDonald, J. R., Gandolfi, A. J., & Sipes, I. G. (1982). Covalent binding of 14C-1,1,2-trichloroethane to hepatic proteins following acetone pretreatment. Drug and chemical toxicology, 5(3), 233-47.
  More info

  We have recently reported that pretreatment of rats with acetone potentiates both the hepatic glutathione (GSH) depletion and subsequent hepatotoxicity caused by 1,1,2-trichloroethane (TCEA). To determine if acetone treatment enhances the bioactivation of TCEA, the covalent binding of 14C-TCEA to tissue proteins was assessed both in vivo and in vitro. Male, Sprague-Dawley rats were treated with acetone (0.5 ml/kg; po), fasted 15 hr prior to dosing with 14C-TCEA (1.2 mmole/kg; ip), and killed 4 hr later. Overnight fasting alone resulted in a six fold increase in covalent binding of 14C-TCEA to hepatic proteins compared to non-fasted rats. Acetone pretreatment, however, did not cause an increase in binding of 14C-TCEA 4 hr after dosing compared to fasted controls, although it did produce a 30% further decrease in hepatic GSH. When microsomes from acetone treated rats were incubated with 14C-TCEA, covalent binding to protein was significantly increased (35%) over using microsomes from fasted control rats. The covalent binding of 14C-TCEA to microsomal protein was inhibited 80% by the addition of GSH (1 mM). The data suggest that potentiation of TCEA hepatotoxicity by acetone may result in part, from alterations of TCEA bioactivation and hepatic GSH concentrations.
• Bentley, J. B., Vaughan, R. W., Cork, R. C., & Gandolfi, A. J. (1981). Does evidence of reductive halothane biotransformation correlate with hepatic binding of metabolites in obese patients?. Anesthesia and analgesia, 60(8), 548-51.
  More info

  Covalent binding of fluorinated anesthetic metabolites was measured using intraoperative liver biopsies obtained from 48 morbidly obese patients randomly assigned to receive N2O-O2 combined with either fentanyl, enflurane, or halothane. No difference in binding was found between anesthetic groups. In addition, preoperative and postoperative levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) did not differ among three groups of patients. However, hepatic organic fluoride binding significantly correlated with peak serum ionic fluoride in patients given halothane (p 0.001, r = 0.68). Thus, the fluorinated metabolites binding assay is a reliable index of reductive halothane metabolism. Possible application of this assay to aid in the diagnosis of postoperative liver dysfunction is suggested.
• Bonhaus, D. W., & Gandolfi, A. J. (1981). Conjugation and bioactivation of chlorotrifluoroethylene. Life sciences, 29(23), 2399-405.
• Gandolfi, A. J., Nagle, R. B., Soltis, J. J., & Plescia, F. H. (1981). Nephrotoxicity of halogenated vinyl cysteine compounds. Research communications in chemical pathology and pharmacology, 33(2), 249-61.
  More info

  S-(1,2-dichlorovinyl) cysteine (DCVC), is a potent nephrotoxin. In order to determine if other vinyl cysteine conjugates were nephrotoxic, halogenated vinyl cysteines, HVC-1 and HVC-2, were prepared from chlorotrifluoroethylene (CTFE), a fluorocarbon monomer, or chlorotifluoroethylene, a metabolite of halothane, respectively. Three days after receiving DCVC (5-10 mg/kg), CD-1 mice developed focal renal tubular necrosis. Mice treated with HVC-1 or HVC-2 (5-10 mg/kg) also developed renal necrosis by 3 days post exposure. HVC-1 was not as potent as DCVC with the necrosis limited to the pars recta. At equivalent doses HVC-2 caused less necrosis of the pars recta than HVC-1. The degree of nephrotoxicity by all three compounds exhibited a dose-response from 1-25 mg/kg. Doses greater than 25 mg/kg were often lethal within 3 days and the mice had a complete zonal necrosis of the renal cortex and a two-fold increase in kidney weight. Structural analogues, S-(chlorethyl) or S-(hydroxyethyl) cysteine, did not cause renal necrosis in mice at doses up to 200 mg/kg. These studies indicate that the enzymes reportedly responsible for converting DCVC to a nephrotoxic intermediate will also bioactivate other halogenated vinyl cysteines.
• Potter, C. L., Gandolfi, A. J., Nagle, R., & Clayton, J. W. (1981). Effects of inhaled chlorotrifluoroethylene and hexafluoropropene on the rat kidney. Toxicology and applied pharmacology, 59(3), 431-40.
• Sipes, I. G., & Gandolfi, A. J. (1981). Role of reactive intermediates in halothane associated liver injury. Advances in experimental medicine and biology, 136 Pt A, 603-18.
• Gandolfi, A. J., White, R. D., Sipes, I. G., & Pohl, L. R. (1980). Bioactivation and covalent binding of halothane in vitro: studies with [3H]- and [14C]halothane. The Journal of pharmacology and experimental therapeutics, 214(3), 721-5.
  More info

  To determine if the hydrogen atom of halothane (CF3CHBrCl) is retained on the reactive intermediates that covalently bind to microsomal lipids and protein, [3H]halothane and [14C]halothane were incubated with rat hepatic microsomes and a NADPH generating system. Both [3H]- and [14C]halothane were bioactivated and bound to a greater degree when incubations were performed in a N2 atmosphere rather than an O2 atmosphere. Binding of [3H]- and [14C]halothane equivalents was significanty enhanced when heaptic microsomes from phenobarbital- or Aroclor 1254-treated rats were used in the incubations. Omission of NADPH or incubation with CO was inhibitory to the binding of both [3H]- and [14C]halothane. The apparent kinetic constants for binding or halothane equivalents, Km and Vmax, indicate a significantly higher Km but lower Vmax for the formation and/or binding of 3H-binding equivalents. The results indicate tht halothane is primarily bioactivated under conditions that promote its reductive metabolism and that this reactive metabolism does not involve cleavage of the carbon-hydrogen bond of halothane. Differences in binding under N2 and O2 as well as between [3H]- and [14C]halothane suggest that multiple reactive intermediates may form during the biotransformation of halothane.
• Sipes, I. G., & Gandolfi, A. J. (1980). In vitro comparative bioactivation of aliphatic halogenated hydrocarbons. Developments in toxicology and environmental science, 8, 501-6.
• Sipes, I. G., Gandolfi, A. J., Pohl, L. R., Krishna, G., & Brown, B. R. (1980). Comparison of the biotransformation and hepatotoxicity of halothane and deuterated halothane. The Journal of pharmacology and experimental therapeutics, 214(3), 716-20.
  More info

  To investigate the effect of deuterium substitution on the biotransformation and hepatotoxicity of halothane, male, phenobarbital-pretreated rats were exposed for 2 hr to 1% halothane or deuterated halothane (d-halothane) delivered in 14% O2-85% N2. The exposures were performed at mildly hypoxic conditions (14% O2) since it was previously established that the decreased oxygen tension promotes both the reductive metabolism of halothane and halothane-induced liver injury. At the end of anesthesia or at 24 hr, the rats were sarificed so that blood, liver and urine samples could be obtained for measurement of metabolites and assessment of liver damage. Deuterium substitution did not affect the levels of reductive metabolites of halothane (fluoride, CF3CH2Cl and CF2CHCl) nor did it alter the degree of hepatotoxicity as assessed by serum glutamic-pyruvic transaminase levels and morphological examination. The levels of oxidative metabolites (CF3COOH and bromide) were significantly reduced at the end of anesthesia and at 24 hr. It is concluded that halothane-induced hepatotoxicity is initiated by reactive intermediates formed during its reductive metabolism and that cleavage of the C-H bond is not involved in this pathway. The oxidative biotransformation of halothane proceeds by an oxygen insertion reaction at the C-H bond. Thus, the increased stability of the C-D bond explains the reduction in oxidative metabolities observed after exposure to d-halothane.
• Soltis, J. J., & Gandolfi, A. J. (1980). Detection of fluorinated anesthetic metabolities by sodium fusion. Anesthesia and analgesia, 59(1), 61-4.
  More info

  A method is reported which utilizes a sodium fusion reaction to decompose organofluorine compounds and yield ionic fluoride which may then be quantified with a specific ion electrode. The method is simple, quick, sensitive, and applicable to fluorinated anesthetic metabolities.
• Blitt, C. D., Brown, B. R., Wright, B. J., Gandolfi, A. J., & Sipes, I. G. (1979). Pulmonary biotransformation of methoxyflurane: an in-vitro study in the rabbit. Anesthesiology, 51(6), 528-31.
• Tinker, J. H., Gandolfi, A. J., & Van Dyke, R. A. (1976). Elevation of plasma bromide levels in patients following halothane anesthesia: Time correlation with total halothane dosage. Anesthesiology, 44(3), 194-6.
  More info

  Plasma bromide concentrations of 25 patients were determined before and after halothane anesthesia. A high correlation (r greater than .70) between exposure to halothane in MAC-hours and plasma bromide levels 24, 48 and 72 hours later was found. Peak bromide levels occurred 48-72 hours after anesthesia in 16 patients (64 per cent), and ranged from 52 mug/ml (0.65 mEq/l) to 180 mug/mo (2.25 mEq/l). Bromide levels remained elevated for prolonged periods (at least 22 days in some patients). Possible sedative or psychoactive effects of increased plasma bromide levels are discussed.
• Gandolfi, A. J., Nakue, H. S., & Buhler, D. R. (1974). Effect of hexachlorophene on hepatic drug-metabolizing enzymes in the rat. Biochemical pharmacology, 23(14), 1997-2003.
• Gandolfi, A. J., & Van Dyke, R. A. (1973). Dechlorination of chloroethane with a reconstituted liver microsomal system. Biochemical and biophysical research communications, 53(3), 687-92.
• Bottini, A. T., Dev, V., Dowden, B. F., & Gandolfi, A. J. (1969). Structure-activity relationships of ethylenimines. 8. Optically active methyl-substituted ethylenimines. Journal of medicinal chemistry, 12(2), 332-3.