Effect of Genetic Variation on the Formation of Hepatocarcinogenic Metabolites of Trichloroethylene Using Chloral Hydrate Studies (an Interdisciplinary Approach)

Trichloroethylene (TCE) is listed with the EPA as a suspected human carcinogen and is ubiquitous in the environment (US EPA 1992). Chloral hydrate (CH) is a sedative drug and a cytochrome P450 (CYP 450)-derived metabolite of TCE (Klaassen 2001). Chloral hydrate is metabolized in the liver to the rodent hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH), and to the non-carcinogenic metabolite trichloroethanol (TCEOH) by alcohol dehydrogenase (ADH) (Klaassen 2001). Both ALDH and ADH are polymorphic in humans which can presumably predict the disposition of chloral hydrate into the carcinogenic vs. non-carcinogenic pathways. The likelihood is then raised that subpopulations of humans will produce greater amounts of TCA relative to TCEOH and hence has greater risk of developing liver tumors after TCE exposure. To determine the variability of chloral hydrate metabolism in humans, the compound (0.05-2.0 mM) is added to human hepatocyte suspensions obtained from commercial sources. Incubations are carried out for 10 min at 37°C, with reactions stopped by the addition of an esterizer solution. The formation of TCA and TCEOH is then measured using headspace gas chromatography with electron-capture detection. The evolution of these metabolites was found to be highly variable with the Vmax means of µTCEOH = 26.32 ± 65.66 and µTCA = 16.79 ± 44.94. Prior to determining human hepatocyte chloral hydrate metabolism, the inter- and intra-individual variability in metabolism of chloral hydrate was determined using rat and mouse liver homogenates from in-bred, genetically equivalent animals. Finally, the calculations are involved in predicting risk using the results of the pharmacokinetics and genetic typing. We believe that chloral hydrate metabolism will show variability among humans, exhibiting sensitive genotypes that have specific kinetics which can be extrapolated to trichloroethylene sensitivity. A prudent approach to this problem merges pharmacology, genetics, and quantitative techniques. The best risk predictors for a high Vmax of both TCEOH and TCA metabolism is the ALDH2 genotype, followed by the ADH3 genotypes. We also suspect the ADH2 genotype would be an important covariate in the model; however none of our samples were of the atypical form. A more diverse and larger sample group would provide support of this study\u27s findings. Here we report a framework to determine risk which incorporates genetic typing

Application of Cryopreserved Human Hepatocytes in Trichloroethylene Risk Assessment: Relative Disposition of Chloral Hydrate to Trichloroacetate and Trichloroethanol

BACKGROUND: Trichloroethylene (TCE) is a suspected human carcinogen and a common ground-water contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. METHODS: The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. RESULTS: There was large interindividual variation in the V(max) values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in V(max) values among individuals, disposition of CH into the two competing pathways was relatively constant. CONCLUSIONS: These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver