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Background: Both trichloroethylene (TCE) and tetrachloroethylene (PCE) are high-priority chemicals subject to numerous human health risk evaluations by a range of agencies. Metabolism of TCE and PCE determines their ultimate toxicity; important uncertainties exist in quantitative characterization of metabolism to genotoxic moieties through glutathione (GSH) conjugation and species differences therein., Objectives: This study aimed to address these uncertainties using novel in vitro liver models, interspecies comparison, and a sensitive assay for quantification of GSH conjugates of TCE and PCE, S -(1,2-dichlorovinyl)glutathione (DCVG) and S -(1,2,2-trichlorovinyl) glutathione (TCVG), respectively., Methods: Liver in vitro models used herein were suspension, 2-D culture, and micropatterned coculture (MPCC) with primary human, rat, and mouse hepatocytes, as well as human induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep)., Results: We found that, although efficiency of metabolism varied among models, consistent with known differences in their metabolic capacity, formation rates of DCVG and TCVG generally followed the patterns human ≥ rat ≥ mouse , and primary hepatocytes > iHep . Data derived from MPCC were most consistent with estimates from physiologically based pharmacokinetic models calibrated to in vivo data., Discussion: For TCE, the new data provided additional empirical support for inclusion of GSH conjugation-mediated kidney effects as critical for the derivation of noncancer toxicity values. For PCE, the data reduced previous uncertainties regarding the extent of TCVG formation in humans; this information was used to update several candidate kidney-specific noncancer toxicity values. Overall, MPCC-derived data provided physiologically relevant estimates of GSH-mediated metabolism of TCE and PCE to reduce uncertainties in interspecies extrapolation that constrained previous risk evaluations, thereby increasing the precision of risk characterizations of these high-priority toxicants. https://doi.org/10.1289/EHP12006.

Quantification of interindividual variability is a continuing challenge in risk assessment, particularly for compounds with complex metabolism and multi-organ toxicity. Toxicokinetic variability for perchloroethylene (perc) was previously characterized across 3 mouse strains and in 1 mouse strain with various degrees of liver steatosis. To further characterize the role of genetic variability in toxicokinetics of perc, we applied Bayesian population physiologically based pharmacokinetic (PBPK) modeling to the data on perc and metabolites in blood/plasma and tissues of male mice from 45 inbred strains from the Collaborative Cross (CC) mouse population. After identifying the most influential PBPK parameters based on global sensitivity analysis, we fit the model with a hierarchical Bayesian population analysis using Markov chain Monte Carlo simulation. We found that the data from 3 commonly used strains were not representative of the full range of variability in perc and metabolite blood/plasma and tissue concentrations across the CC population. Using interstrain variability as a surrogate for human interindividual variability, we calculated dose-dependent, chemical-, and tissue-specific toxicokinetic variability factors (TKVFs) as candidate science-based replacements for the default uncertainty factor for human toxicokinetic variability of 100.5. We found that toxicokinetic variability factors for glutathione conjugation metabolites of perc showed the greatest variability, often exceeding the default, whereas those for oxidative metabolites and perc itself were generally less than the default. Overall, we demonstrate how a combination of a population-based mouse model such as the CC with Bayesian population PBPK modeling can reduce uncertainty in human toxicokinetic variability and increase accuracy and precision in quantitative risk assessment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Nonalcoholic fatty liver disease (NAFLD), a major cause of chronic liver disease in the Western countries with increasing prevalence worldwide, may substantially affect chemical toxicokinetics and thereby modulate chemical toxicity., Objectives: This study aims to use physiologically-based pharmacokinetic (PBPK) modeling to characterize the impact of NAFLD on toxicokinetics of perchloroethylene (perc)., Methods: Quantitative measures of physiological and biochemical changes associated with the presence of NAFLD induced by high-fat or methionine/choline-deficient diets in C57B1/6 J mice are incorporated into a previously developed PBPK model for perc and its oxidative and conjugative metabolites. Impacts on liver fat and volume, as well as blood:air and liver:air partition coefficients, are incorporated into the model. Hierarchical Bayesian population analysis using Markov chain Monte Carlo simulation is conducted to characterize uncertainty, as well as disease-induced variability in toxicokinetics., Results: NAFLD has a major effect on toxicokinetics of perc, with greater oxidative and lower conjugative metabolism as compared to healthy mice. The NAFLD-updated PBPK model accurately predicts in vivo metabolism of perc through oxidative and conjugative pathways in all tissues across disease states and strains, but underestimated parent compound concentrations in blood and liver of NAFLD mice., Conclusions: We demonstrate the application of PBPK modeling to predict the effects of pre-existing disease conditions as a variability factor in perc metabolism. These results suggest that non-genetic factors such as diet and pre-existing disease can be as influential as genetic factors in altering toxicokinetics of perc, and thus are likely contribute substantially to population variation in its adverse effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Interindividual variability in susceptibility remains poorly characterized for environmental chemicals such as tetrachloroethylene (PERC). Development of population-based experimental models provide a potential approach to fill this critical need in human health risk assessment., Objectives: In this study, we aimed to better characterize the contribution of glutathione (GSH) conjugation to kidney toxicity of PERC and the degree of associated interindividual toxicokinetic (TK) and toxicodynamic (TD) variability by using the Collaborative Cross (CC) mouse population., Methods: Male mice from 45 strains were intragastrically dosed with PERC ([Formula: see text]) or vehicle (5% Alkamuls EL-620 in saline), and time-course samples were collected for up to 24 h. Population variability in TK of S-(1,2,2-trichlorovinyl)GSH (TCVG), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (NAcTCVC) was quantified in serum, liver, and kidney, and analyzed using a toxicokinetic model. Effects of PERC on kidney weight, fatty acid metabolism-associated genes [ Acot1 (Acyl-CoA thioesterase 1), Fabp1 (fatty acid-binding protein 1), and Ehhadh (enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase)], and a marker of proximal tubular injury [KIM-1 (kidney injury molecule-1)/Hepatitis A virus cellular receptor 1 ( Havcr1)] were evaluated. Finally, quantitative data on interstrain variability in both formation of GSH conjugation metabolites of PERC and its kidney effects was used to calculate adjustment factors for the interindividual variability in both TK and TD., Results: Mice treated with PERC had significantly lower kidney weight, higher kidney-to-body weight (BW) ratio, and higher expression of fatty acid metabolism-associated genes ( Acot1, Fabp1, and Ehhadh) and a marker of proximal tubular injury (KIM-1/ Havcr1). Liver levels of TCVG were significantly correlated with KIM-1/ Havcr1 in kidney, consistent with kidney injury being associated with GSH conjugation. We found that the default uncertainty factor for human variability may be marginally adequate to protect 95%, but not more, of the population for kidney toxicity mediated by PERC., Discussion: Overall, this study demonstrates the utility of the CC mouse population in characterizing metabolism-toxicity interactions and quantifying interindividual variability. Further refinement of the characterization of interindividual variability can be accomplished by incorporating these data into in silico population models both for TK (such as a physiologically based pharmacokinetic model), as well as for toxicodynamic responses. https://doi.org/10.1289/EHP5105.

Accounting for genetic and other (eg, underlying disease states) factors that may lead to inter-individual variability in susceptibility to xenobiotic-induced injury is a challenge in human health assessments. A previous study demonstrated that nonalcoholic fatty liver disease (NAFLD), one of the common underlying disease states, enhances tetrachloroethylene (PERC)-associated hepatotoxicity in mice. Interestingly, NAFLD resulted in a decrease in metabolism of PERC to nephrotoxic glutathione conjugates; we therefore hypothesized that NAFLD would protect against PERC-associated nephrotoxicity. Male C57BL/6J mice were fed a low-fat (LFD), high-fat (31% fat, HFD), or high-fat methionine/choline/folate-deficient (31% fat, MCD) diets. After 8 weeks mice were administered either a single dose of PERC (300 mg/kg i.g.) and euthanized at 1-36 h post dose, or five daily doses of PERC (300 mg/kg/d i.g.) and euthanized 4 h after last dose. Relative to LFD-fed mice, HFD- or MCD-fed mice exhibited decreased PERC concentrations and increased trichloroacetate (TCA) in kidneys. S-(1,2,2-trichlorovinyl)glutathione (TCVG), S-(1,2,2-trichlorovinyl)-l-cysteine (TCVC), and N-acetyl-S-(1,2,2,-trichlorovinyl)-l-cysteine (NAcTCVC) were also significantly lower in kidney and urine of HFD- or MCD-fed mice compared with LFD-fed mice. Despite differences in levels of nephrotoxic PERC metabolites in kidney, LFD- and MCD-fed mice demonstrated similar degree of nephrotoxicity. However, HFD-fed mice were less sensitive to PERC-induced nephrotoxicity. Thus, whereas both MCD- and HFD-induced fatty liver reduced the delivered dose of nephrotoxic PERC metabolites to the kidney, only HFD was protective against PERC-induced nephrotoxicity, possibly due to greater toxicodynamic sensitivity induced by methyl and choline deficiency. These results therefore demonstrate that pre-existing disease conditions can lead to a complex interplay of toxicokinetic and toxicodynamic changes that modulate susceptibility to the toxicity of xenobiotics.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar chemicals that are metabolized through oxidation and glutathione conjugation pathways. Both chemicals have been shown to elicit liver and kidney toxicity in rodents and humans; however, TCE has been studied much more extensively in terms of both metabolism and toxicity. Despite their qualitative similarities, quantitative comparison of tissue- and strain-specific metabolism of TCE and PCE has not been performed. To fill this gap, we conducted a comparative toxicokinetic study where equimolar single oral doses of TCE (800 mg/kg) or PCE (1000 mg/kg) were administered to male mice of C57BL/6J, B6C3F1/J, and NZW/LacJ strains. Samples of liver, kidney, serum, brain, and lung were obtained for up to 36 h after dosing. For each tissue, concentrations of parent compounds, as well as their oxidative and glutathione conjugation metabolites were measured and concentration-time profiles constructed. A multi-compartment toxicokinetic model was developed to quantitatively compare TCE and PCE metabolism. As expected, the flux through oxidation metabolism pathway predominated over that through conjugation across all mouse strains examined, it is 1,200-3,800 fold higher for TCE and 26-34 fold higher for PCE. However, the flux through glutathione conjugation, albeit a minor metabolic pathway, was 21-fold higher for PCE as compared to TCE. The degree of inter-strain variability was greatest for oxidative metabolites in TCE-treated and for glutathione conjugation metabolites in PCE-treated mice. This study provides critical data for quantitative comparisons of TCE and PCE metabolism, and may explain the differences in organ-specific toxicity between these structurally similar chemicals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar olefins that can cause liver and kidney toxicity. Adverse effects of these chemicals are associated with metabolism to oxidative and glutathione conjugation moieties. It is thought that CYP2E1 is crucial to the oxidative metabolism of TCE and PCE, and may also play a role in formation of nephrotoxic metabolites; however, inter-species and inter-individual differences in contribution of CYP2E1 to metabolism and toxicity are not well understood. Therefore, the role of CYP2E1 in metabolism and toxic effects of TCE and PCE was investigated using male and female wild-type [129S1/SvlmJ], Cyp2e1(-/-), and humanized Cyp2e1 [hCYP2E1] mice. To fill in existing gaps in our knowledge, we conducted a toxicokinetic study of TCE (600 mg/kg, single dose, i.g.) and a subacute study of PCE (500 mg/kg/day, 5 days, i.g.) in 3 strains. Liver and kidney tissues were subject to profiling of oxidative and glutathione conjugation metabolites of TCE and PCE, as well as toxicity endpoints. The amounts of trichloroacetic acid formed in the liver was hCYP2E1≈ 129S1/SvlmJ > Cyp2e1(-/-) for both TCE and PCE; levels in males were about 2-fold higher than in females. Interestingly, 2- to 3-fold higher levels of conjugation metabolites were observed in TCE-treated Cyp2e1(-/-) mice. PCE induced lipid accumulation only in liver of 129S1/SvlmJ mice. In the kidney, PCE exposure resulted in acute proximal tubule injury in both sexes in all strains (hCYP2E1 ≈ 129S1/SvlmJ > Cyp2e1(-/-)). In conclusion, our results demonstrate that CYP2E1 is an important, but not exclusive actor in the oxidative metabolism and toxicity of TCE and PCE.

Background: Perchloroethylene (perc) induced target organ toxicity has been associated with tissue-specific metabolic pathways. Previous physiologically-based pharmacokinetic (PBPK) modeling of perc accurately predicted oxidative metabolites but suggested the need to better characterize glutathione (GSH) conjugation as well as toxicokinetic uncertainty and variability., Objectives: We updated the previously published "harmonized" perc PBPK model in mice to better characterize GSH conjugation metabolism as well as the uncertainty and variability of perc toxicokinetics., Methods: The updated PBPK model includes expanded models for perc and its oxidative metabolite trichloroacetic acid (TCA), and physiologically-based sub-models for conjugative metabolites. Previously compiled mouse kinetic data in B6C3F1 and Swiss-Webster mice were augmented to include data from a recent study in male C57BL/6J mice that measured perc and metabolites in serum and multiple tissues. Hierarchical Bayesian population analysis using Markov chain Monte Carlo was conducted to characterize uncertainty and inter-strain variability in perc metabolism., Results: The updated model fit the data as well or better than the previously published "harmonized" PBPK model. Tissue dosimetry for both oxidative and conjugative metabolites was successfully predicted across the three strains of mice, with estimated residuals errors of 2-fold for majority of data. Inter-strain variability across three strains was evident for oxidative metabolism; GSH conjugation data were only available for one strain., Conclusions: This updated PBPK model fills a critical data gap in quantitative risk assessment by predicting the internal dosimetry of perc and its oxidative and GSH conjugation metabolites and lays the groundwork for future studies to better characterize toxicokinetic variability., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent pathological liver condition in developed countries. NAFLD results in severe alterations in liver function, including xenobiotic metabolism. Perchloroethylene (PERC) is a ubiquitous environmental pollutant, a known hepatotoxicant in rodents, and a probable human carcinogen. It is known that PERC disposition and metabolism are affected by NAFLD in mice; here, we examined how NAFLD changes PERC-associated liver effects. Male C57Bl6/J mice were fed a low-fat diet (LFD), high-fat diet (HFD), or methionine/folate/choline-deficient diet (MCD) to model a healthy liver, or mild and severe forms of NAFLD, respectively. After 8 weeks on diets, mice were orally administered PERC (300 mg/kg/day) or vehicle (5% aqueous Alkamuls-EL620) for 5 days. PERC-induced liver effects were exacerbated in both NAFLD groups. PERC exposure was associated with up-regulation of genes involved in xenobiotic, lipid, and glutathione metabolism, and down-regulation of the complement and coagulation cascades, regardless of the diet. Interestingly, HFD-fed mice, not MCD-fed mice, were generally more sensitive to PERC-induced liver effects. This was indicated by histopathology and transcriptional responses, where induction of genes associated with cell cycle and inflammation were prominent. Liver effects positively correlated with diet-specific differences in liver concentrations of PERC. We conclude that NAFLD alters the toxicodynamics of PERC and that NAFLD is a susceptibility factor that should be considered in future risk management decisions for PERC and other chlorinated solvents., (Published by Oxford University Press on behalf of the Society of Toxicology 2017. This work is written by US Government employees and is in the public domain in the US.)

Lifestyle factors and chronic pathologic states are important contributors to interindividual variability in susceptibility to xenobiotic-induced toxicity. Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent condition that can dramatically affect chemical metabolism. We examined the effect of NAFLD on toxicokinetics of tetrachloroethylene (PERC), a ubiquitous environmental contaminant that requires metabolic activation to induce adverse health effects. Mice (C57Bl/6J, male) were fed a low-fat diet (LFD), high-fat diet (HFD), or methionine/folate/choline-deficient diet (MCD) to model a healthy liver, steatosis, or nonalcoholic steatohepatitis (NASH), respectively. After 8 weeks, mice were orally administered a single dose of PERC (300 mg/kg) or vehicle (aqueous Alkamuls-EL620) and euthanized at various time points (1-36 hours). Levels of PERC and its metabolites were measured in blood/serum, liver, and fat. Effects of diets on liver gene expression and tissue:air partition coefficients were evaluated. We found that hepatic levels of PERC were 6- and 7.6-fold higher in HFD- and MCD-fed mice compared with LFD-fed mice; this was associated with an increased PERC liver:blood partition coefficient. Liver and serum C max for trichloroacetate (TCA) was lower in MCD-fed mice; however, hepatic clearance of TCA was profoundly reduced by HFD or MCD feeding, leading to TCA accumulation. Hepatic mRNA/protein expression and ex vivo activity assays revealed decreased xenobiotic metabolism in HFD- and MCD-, compared with LFD-fed, groups. In conclusion, experimental NAFLD was associated with modulation of xenobiotic disposition and metabolism and increased hepatic exposure to PERC and TCA. Underlying NAFLD may be an important susceptibility factor for PERC-associated hepatotoxicity., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Tetrachloroethylene (perchloroethylene; PERC) is a high-production volume chemical and ubiquitous environmental contaminant that is hazardous to human health. Toxicity attributed to PERC is mediated through oxidative and glutathione (GSH) conjugation metabolites. The conjugation of PERC by glutathione-s-transferase to generate S-(1,2,2-trichlorovinyl) glutathione (TCVG), which is subsequently metabolized to form S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) is of special importance to human health. Specifically, TCVC may be metabolized to N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (NAcTCVC) which is excreted through urine, or to electrophilic metabolites that are nephrotoxic and mutagenic. Little is known regarding toxicokinetics of TCVG, TCVC, and NAcTCVC as analytical methods for simultaneous determination of these metabolites in tissues have not yet been reported. Hence, an ultra-high-performance liquid chromatography electrospray ionization tandem mass spectrometry-based method was developed for analysis of TCVG, TCVC, and NAcTCVC in liver, kidneys, serum, and urine. The method is rapid, sensitive, robust, and selective for detection all three analytes in every tissue examined, with limits of detection (LOD) ranging from 1.8 to 68.2 femtomoles on column, depending on the analyte and tissue matrix. This method was applied to quantify levels of TCVG, TCVC, and NAcTCVC in tissues from mice treated with PERC (10 to 1000 mg/kg, orally) with limits of quantitation (LOQ) of 1-2.5 pmol/g in liver, 1-10 pmol/g in kidney, 1-2.5 pmol/ml in serum, and 2.5-5 pmol/ml in urine. This method is useful for further characterization of the GSH conjugative pathway of PERC in vivo and improved understanding of PERC toxicity.