A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review.

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

Covariation of human microsomal protein per gram of liver with age: absence of influence of operator and sample storage may justify interlaboratory data pooling.

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80 degrees C (23.5 +/- 1.2 mg g(-1)) and those determined using fresh tissue (21.9 +/- 0.3 mg g(-1)). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 +/- 1, 19 +/- 2, and 24 +/- 1 mg g(-1); p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 +/- 1, 30 +/- 5, and 31 +/- 4 mg g(-1)). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g(-1) [95% confidence interval for the geometric mean (95% CI mean(geo)): 37-43 mg g(-1) at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g(-1) at 65 years; 95% CI mean(geo): 27-32 mg g(-1)). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. II: Effects on conjugating enzymes.

The effects of long-term caloric restriction on the hepatic phase II drug metabolizing enzymes were investigated in the male Fischer 344 rat. Rats that had been restricted to 60% of their pair-fed control consumption from 14 weeks post-partum exhibited altered conjugating enzyme activities at 22 months. Caloric restriction significantly reduced the age-related decrease in glutathione-S-transferase activity towards 1,2-dichloro-4-nitrobenzene, but did not significantly alter the age-related changes in UDP-glucuronyltransferase or sulfotransferase activities towards hydroxysteroids. Caloric restriction appeared to increase hepatic microsomal UDP-glucuronyltransferase activity toward bilirubin and gamma-glutamyltranspeptidase activities. These observations suggest that caloric restriction has multiple effects on the hepatic phase II drug metabolizing enzymes in the rat. Such effects may alter hepatic metabolism and activation or detoxification of drugs and carcinogens.

Metabolism of trichloroethylene.

A major focus in the study of metabolism and disposition of trichloroethylene (TCE) is to identify metabolites that can be used reliably to assess flux through the various pathways of TCE metabolism and to identify those metabolites that are causally associated with toxic responses. Another important issue involves delineation of sex- and species-dependent differences in biotransformation pathways. Defining these differences can play an important role in the utility of laboratory animal data for understanding the pharmacokinetics and pharmacodynamics of TCE in humans. Sex-, species-, and strain-dependent differences in absorption and distribution of TCE may play some role in explaining differences in metabolism and susceptibility to toxicity from TCE exposure. The majority of differences in susceptibility, however, are likely due to sex-, species-, and strain-dependent differences in activities of the various enzymes that can metabolize TCE and its subsequent metabolites. An additional factor that plays a role in human health risk assessment for TCE is the high degree of variability in the activity of certain enzymes. TCE undergoes metabolism by two major pathways, cytochrome P450 (P450)-dependent oxidation and conjugation with glutathione (GSH). Key P450-derived metabolites of TCE that have been associated with specific target organs, such as the liver and lungs, include chloral hydrate, trichloroacetate, and dichloroacetate. Metabolites derived from the GSH conjugate of TCE, in contrast, have been associated with the kidney as a target organ. Specifically, metabolism of the cysteine conjugate of TCE by the cysteine conjugate ss-lyase generates a reactive metabolite that is nephrotoxic and may be nephrocarcinogenic. Although the P450 pathway is a higher activity and higher affinity pathway than the GSH conjugation pathway, one should not automatically conclude that the latter pathway is only important at very high doses. A synthesis of this information is then presented to assess how experimental data, from either animals or from (italic)in vitro (/italic)studies, can be extrapolated to humans for risk assessment. (italic)Key words(/italic): conjugate beta-lyase, cysteine glutathione, cytochrome P450, glutathione (italic)S(/italic)-transferases, metabolism, sex dependence, species dependence, tissue dependence, trichloroethylene.

Evaluating human variability in chemical risk assessment: hazard identification and dose-response assessment for noncancer oral toxicity of trichloroethylene.

Human variability can be addressed during each stage in the risk assessment of chemicals causing noncancer toxicities. Noncancer toxicities arising from oral exposure to trichloroethylene (TCE) are used in this paper as a case study for exploring strategies for identifying and incorporating information about human variability in the chemical specific hazard identification and dose-response assessment steps. Toxicity testing in laboratory rodents is the most commonly used method for hazard identification. By using animal models for sensitive populations, such as developing fetuses, testing can identify some potentially sensitive populations. A large variety of reproductive and developmental studies with TCE were reviewed. The results were mostly negative and the limited positive findings generally occurred at doses similar to those causing liver and kidney toxicity. Physiologically based pharmacokinetic modeling using Monte Carlo simulation is one method for evaluating human variability in the dose-response assessment. Three strategies for obtaining data describing this variability for TCE are discussed: (1) using in vivo human pharmacokinetic data for TCE and its metabolites, (2) studying metabolism in vitro, and (3) identifying the responsible enzymes and their variability. A review of important steps in the metabolic pathways for TCE describes known metabolic variabilities including genetic polymorphisms, enzyme induction, and disease states. A significant problem for incorporating data on pharmacokinetic variability is a lack of information on how it relates to alterations in toxicity. Response modeling is still largely limited to empirical methods due to the lack of knowledge about toxicodynamic processes. Empirical methods, such as reduction of the No-Observed-Adverse-Effect-Level or a Benchmark Dose by uncertainty factors, incorporate human variability only qualitatively by use of an uncertainty factor. As improved data and methods for biologically based dose-response assessment become available, use of quantitative information about variability will increase in the risk assessment of chemicals.

Potential health effects of drinking water disinfection by-products using quantitative structure toxicity relationship.

Disinfection by-products (DBPs) are produced as a result of disinfecting water using various treatment methods. Over the years, chlorine has remained the most popular disinfecting agent due to its ability to kill pathogens. However, in 1974, it was discovered that the superchlorination of drinking water resulted in the production of chloroform and other trihalomethanes. Since then hundreds of additional DBPs have been identified, including haloacetic acids and haloacetonitriles with very little or no toxicological data available, thus necessitating the use of additional methods for hazard estimation. Quantitative Structure Toxicity Relationship (QSTR) is one such method and utilizes a computer-based technology to predict the toxicity of a chemical solely from its molecular attributes. The current research was conducted utilizing the TOPKAT/QSTR software package which is comprised of robust, cross-validated QSTR models for assessing mutagenicity, rodent carcinogenicity (female/male; rat/mouse), developmental toxicity, skin sensitization, lowest-observed-adverse-effect level (LOAEL), fathead minnow LC(50), rat oral LD(50) and Daphia magna EC(50). A total of 252 DBPs were analyzed for the likelihood that they would produce tumors and developmental effects using the carcinogenicity and developmental toxicity submodels of TOPKAT. The model predictions were evaluated to identify generalizations between the functional groups (e.g. alcohols, acids, etc.) and specific toxic endpoints. Developmental toxicity was identified as an endpoint common to the majority of aliphatic mono- and dicarboxylic acids, aliphatic halogenated and non-halogenated ketones, and aliphatic haloacetonitriles. In the case of the carcinogenicity submodels, most aliphatic aldehydes were identified as carcinogens only in the female mouse submodel. The majority of the aliphatic and aromatic dicarboxylic acids were identified as carcinogens in the female rat submodel. All other functional groups examined were largely predicted as non-carcinogens in all the cancer submodels (i.e. male/female rats and mice). The QSTR results should aid in the prioritization for evaluation of toxic endpoints in the absence of in vivo bioassays.

Fetomaternal kinetics of 14C-trimethyltin.

The mechanism of trimethyltin (TMT)-induced neuropathology remains unknown but likely relates to its time-course in the nervous system. To determine the pharmacokinetic profile of TMT in the fetomaternal unit, pregnant rats were injected on gestational day (GD) 17 with 7.0 mg/kg TMT plus 10 uCi 14C-TMT ip. Whole blood and plasma, cerebrum (CBR), cerebellum (CBE), and brainstem (BS) were sampled from mother and fetus/pup (f/p). Whole body saline perfusions of mother and f/p were performed independently to remove 14C contaminated blood. Whole blood total radioactivity showed f/p levels to be below maternal levels: at 4 hr, 42.6%, 24 hr, 54.2%, 96 hr, 64.8%. Litters were cross-fostered (CF) at birth with those from untreated mothers. Two weeks after treatment (postnatal day 10), pups exposed in utero (n=13, 2 litters) had whole blood 14C levels 13.9% that of their mothers. Untreated pups CF to treated mothers showed a blood concentration 1.8% of the dosed mother, indicating that TMT is transferred in milk. Radioactivity in maternal whole blood at 2 weeks was 49 +/- 11% (SEM) of peak (1 hr) levels. Only 2.5 +/- 0.2% of maternal whole blood radioactivity was present in plasma. 14C in urine accounted for 17.5 +/- 1.5% of the dose at 2 weeks (n=4). Both maternal and fetal brains contained about 2-4% as much radioactivity as found in maternal and fetal whole blood, respectively. TMT crosses the placenta, enters fetal blood and attains fetal brain levels that are equal to those found in maternal brain.

Covalent binding of trichloroethylene to proteins in human and rat hepatocytes.

The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte cultures were exposed to vaporized trichloroethylene at 0-10,000 parts per million for up to 2 h. Immunochemical detection of three major dose- and time-dependent trichloroethylene protein adducts at 50, 52 and 100 kDa was demonstrated in the rat hepatocytes, while a single, distinctively different 47 kDa adduct was detected in human hepatocytes. The 50 kDa adduct in rat hepatocytes was found to comigrate on SDS-PAGE with cytochrome P450 2E1 (CYP2E1), while the adduct found in humans did not comigrate with CYP2E1. These data show that reactive metabolites of trichloroethylene can be formed in human and rat hepatocytes and bind covalently to discrete hepatic proteins, and suggests that in rats, but not humans, that one of the targets is CYP2E1.

The disposition of 14C-trimethyltin in the pregnant rat and fetus.

Trimethyltin (TMT) is a potent neurotoxicant. For unknown reasons, age at exposure to TMT may dramatically influence the severity of TMT-induced neuropathology. We have demonstrated previously that radiolabel derived from [14C]-TMT given to pregnant dams on gestational day (GD) 17 is found in fetal brain and blood. The present study was designed to determine the distribution of radiolabel derived from [14C]-TMT to brain and other tissue in fetuses from dams dosed on either GD 12 or 17 with 7.0 mg/kg TMT chloride. Radioactivity in GD 12 and GD 17 maternal whole blood peaked 1 hour after IP treatment. Whole blood elimination half-lives were 12-15 days. Peak radiolabel concentrations in GD 12 maternal and fetal brain were only 11-30% of those from GD 17 animals, however, peak fetal brain concentrations of radiolabel were not different from their respective maternal brain concentrations. Radiolabel concentrations in liver, kidney, and adrenal of GD 17 dams were higher than those in corresponding GD 12 tissues. Combined urinary and fecal elimination of radiolabel for two weeks after dosing accounted for 31 and 22% of the GD 12 and 17 doses, respectively. It appears that gestational age influences the distribution and elimination of TMT in the rat.

Chloral hydrate formation in the Japanese medaka minnow.

Trichloroethylene (TRI) is a common groundwater contaminant that has been shown to be tumorigenic and toxic in laboratory animals. The toxicity of TRI is increased by inducing the production of cytochrome P-450-dependent metabolites. Cytochrome P450 (CYP) 2E1 metabolizes TRI in mammals; however, this isoform of CYP2E1 does not appear to be expressed in fish. Medaka microsomal protein containing CYP was exposed to TRI and extracted with ethyl acetate. The extract was analyzed using gas chromatography (liquid injection) with an electron capture detector and separately using mass spectrometry. The formation of chloral hydrate, a precursor of toxic metabolites, was confirmed following exposure of hepatic microsomes of the medaka to TRI. These results indicate that medaka catalyze the first step in the formation of toxic metabolites and CYP forms in addition to CYP2E1 which catalyzes this reaction in fish.

The role of mouse intestinal microflora in the metabolism of trichloroethylene, an in vivo study.

1. Both trichloroethylene and its metabolite, dichloroacetic acid, produce liver tumors peroxisome proliferation and other adverse cellular alterations in rodents. 2. The hepatic mechanism by which dichloroacetic acid is formed is not conclusively demonstrated, but pharmacokinetic models have successfully associated its formation with trichloroacetic acid as immediate precursor. 3. Previous investigations have shown that dichloroacetic acid is formed from trichloroacetic acid by gut microflora isolated in vitro. 4. To determine the impact of gut microflora on dichloroacetic acid formation from a trichloroethylene dose in vivo, we developed a procedure which reduced gut microflora some 3 orders of magnitude below published levels. 5. The administration of trichloroethylene to control mice and to mice whose gut was practically sterile resulted in equivalent concentrations of dichloroacetic acid and other metabolites in blood and liver, but significantly different content of these metabolites in cecum contents. 6. These data indicate that gut microflora contribute minimally, if at all, to the formation of circulating dichloroacetic acid under these conditions.

Application of in vitro biotransformation data and pharmacokinetic modeling to risk assessment.

The adverse biological effects of toxic substances are dependent upon the exposure concentration and the duration of exposure. Pharmacokinetic models can quantitatively relate the external concentration of a toxicant in the environment to the internal dose of the toxicant in the target tissues of an exposed organism. The exposure concentration of a toxic substance is usually not the same as the concentration of the active form of the toxicant that reaches the target tissues following absorption, distribution, and biotransformation of the parent toxicant. Biotransformation modulates the biological activity of chemicals through bioactivation and detoxication pathways. Many toxicants require biotransformation to exert their adverse biological effects. Considerable species differences in biotransformation and other pharmacokinetic processes can make extrapolation of toxicity data from laboratory animals to humans problematic. Additionally, interindividual differences in biotransformation among human populations with diverse genetics and lifestyles can lead to considerable variability in the bioactivation of toxic chemicals. Compartmental pharmacokinetic models of animals and humans are needed to understand the quantitative relationships between chemical exposure and target tissue dose as well as animal to human differences and interindividual differences in human populations. The data-based compartmental pharmacokinetic models widely used in clinical pharmacology have little utility for human health risk assessment because they cannot extrapolate across dose route or species. Physiologically based pharmacokinetic (PBPK) models allow such extrapolations because they are based on anatomy, physiology, and biochemistry. In PBPK models, the compartments represent organs or groups of organs and the flows between compartments are actual blood flows. The concentration of a toxicant in a target tissue is a function of the solubility of the toxicant in blood and tissues (partition coefficients), blood flow into the tissue, metabolism of the toxicant in the tissue, and blood flow out of the tissue. The appropriate degree of biochemical detail can be added to the PBPK models as needed. Comparison of model simulations with experimental data provides a means of hypothesis testing and model refinement. In vitro biotransformation data from studies with isolated liver cells or subcellular fractions from animals or humans can be extrapolated to the intact organism based upon protein content or cell number. In vitro biotransformation studies with human liver preparations can provide quantitative data on human interindividual differences in chemical bioactivation. These in vitro data must be integrated into physiological models to understand the true impact of interindividual differences in chemical biotransformation on the target organ bioactivation of chemical contaminants in air and drinking water.

Physiologically based pharmacokinetic modeling of the lactational transfer of methylmercury.

The developmental neurotoxicity of methylmercury (MeHg) in humans has been described following catastrophic events in Minamata Bay, Japan and in Iraq, and following the exposure to lower doses elsewhere in the world. The most common route of MeHg exposure in humans is through the intake of contaminated food, especially fish. Although the precautions against the ingestion of potentially contaminated food during pregnancy are well recognized, precautions against the ingestion of MeHg during lactation are not so uniformly recognized. However, the continued development of the central nervous system during the early postnatal period serves to prolong the period during which this critical system is susceptible to the toxic insult of MeHg. Because no direct method is available to quantitatively assess the lactational transfer of MeHg to humans, a computer-aided simulation method was developed. An available gestational physiologically based pharmacokinetic model was refined and expanded to include parameters and algorithms specific for the elimination of MeHg in breast milk. The predictions of the completed model were compared with experimental data obtained from rodents, and the model parameters were allometrically scaled to humans. Finally, the model was validated by comparing its predictions against the available clinical data for MeHg distribution and elimination in mothers and their nursing infants. This model incorporated current and previous maternal exposures to MeHg to accurately predict the kinetics of MeHg excretion in breast milk and the daily intake by the nursing infant. This model may be used to quantify MeHg intake by the nursing infant, under different rates of maternal MeHg ingestion.

Interindividual variance of cytochrome P450 forms in human hepatic microsomes: correlation of individual forms with xenobiotic metabolism and implications in risk assessment.

Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reactions of drugs and other xenobiotics and differences in their expression may directly produce interindividual differences in susceptibility to compounds whose toxicity is modulated by these enzymes. To rapidly quantify CYP450 forms in human hepatic microsomes, we developed, and applied, an ELISA to 40 samples of microsomes from adult human organ donors. The procedure was reliable and the results were reproducible within normal limits. Protein content for CYP1A, CYP2E1, and CYP3A positively correlated with suitable marker activities. CYP1A, CYP2B, CYP2C6, CYP2C11, CYP2E1, and CYP3A protein content demonstrated 36-, 13-, 11-, 2-, 12-, and 22-fold differences between the highest and lowest samples and the values were normally distributed. Of the forms examined, CYP3A was expressed in the highest amount and it was the only form whose content was correlated with total CYP450 content. Content of other forms was independent of total CYP450. We further determined the contribution of specific forms to the biotransformation of trichloroethylene as a model substrate. CYP2E1 was strongly correlated with chloral hydrate formation from trichloroethylene; CYP2B displayed the strongest correlation with trichloroethanol formation. These data describing the expression and distribution of these forms in human microsomes can be used to extrapolate in vitro derived metabolic rates for toxicologically important reactions, when form selectivity and specific activity are known. This approach may be applied to refine estimates of human interindividual differences in susceptibility for application in human health risk assessment.

Cytochrome P450-dependent metabolism of trichloroethylene: interindividual differences in humans.

Trichloroethylene (TRI) is an industrial solvent with a history of use in anesthesia, and is a common groundwater contaminant. Cytochrome P450 (CYP)-dependent metabolism of TRI produces chloral hydrate (CH) and is rate limiting in the ultimate production of trichloro- and/or dichloroacetic acid from TRI. Exposure of rodents to TRI results in lung and liver tumors (mice) and nephrotoxicity (rats). The toxicity is exacerbated by pretreatment of mice with CYP inducers. We report significant variability in TRI metabolism in a sample of 23 human hepatic microsomal samples and demonstrate the dependence of TRI metabolism on CYP2E1. K(m) values in this limited sample population are not normally distributed. We have correlated microsomal CH formation with the activity toward routine CYP2E1 substrates and with immunologically detectable CYP2E1 protein. Further, TRI metabolism in microsomes from lymphoblastoid cell lines expressing CYP2E1, CYP1A1, CYP1A2, or CYP3A4 indicated minimal involvement of the latter forms, with CYP2E1 catalyzing more than 60% of total microsomal TRI metabolism. These results indicate that humans are not uniform in their capacity for CYP-dependent metabolism of TRI and increased CYP2E1 activity may increase susceptibility to TRI-induced toxicity in the human.

Toxicant-induced alterations in two-dimensional electrophoretic patterns of hepatic and renal stress proteins.

Recent studies in this laboratory and by others suggest that two-dimensional polyacrylamide gel electrophoresis of proteins (2-DE) possesses significant utility in the detection of chemical toxicity and in providing information regarding toxic mechanism. After having identified a set of specific heat-shock and glucose-regulated proteins whose expression in rodent liver and kidney is highly conserved and constitutive, we compared the effect of in vivo exposure to perfluoro-n-octanoic acid and perfluoro-n-decanoic acid on their expression. The following stress proteins were identified, their x, y coordinate positions mapped, and abundance statistically analyzed and compared: hsp32, hsp60, hsc70, hsp70, hsp90, grp75, grp94, protein disulfide isomerase (PDI), and ER60. We report here that the stress response to perfluorocarboxylic acids is tissue-, toxicant-, and stress protein class-specific and dose-related. Furthermore, because nearly all of the proteins studied were constitutively expressed at detectable levels in both liver and kidney, the 2-DE stress protein pattern may be suitable to future toxicologic screening applications.

Application of QSTRs in the selection of a surrogate toxicity value for a chemical of concern.

As part of the EPA's mission to protect the environment, chemicals of concern (CoCs) at Superfund or other hazardous waste sites are cleaned up based on their potential toxicity to humans and the surrounding ecosystem. Oftentimes, there is a lack of experimental toxicity data to assess the health effects for a CoC in the literature. This research describes a method using Quantitative Structure Toxicity Relationships (QSTRs) for identifying a surrogate chemical for any given CoC. The toxicity data of the surrogate chemical can then be used to rank hazardous waste-site chemicals prior to cleanup decisions. A commercial QSTR model, TOPKAT, was used to establish structural and descriptor similarity between the CoC and the compounds in the QSTR model database using the Oral Rat Chronic LOAEL model. All database chemicals within a similarity distance of < or = 0.200 from the CoC are considered as potential surrogates. If the CoC fails to satisfy model considerations for the LOAEL model, no surrogate is suggested. Potential surrogates that have toxicity data on Integrated Risk Information System (IRIS), Health Effects Assessment Summary Tables (HEAST), or National Center for Environmental Assessment (NCEA) provisional toxicity value list become candidate surrogates. If more than one candidate surrogate is identified, the chemical with the most conservative RfD is suggested as the surrogate. The procedure was applied to determine an appropriate surrogate for dichlorobenzophenone (DCBP), a metabolite of chlorobenzilate, dichlorodiphenyltrichloroethane, and dicofol. Forty-seven potential surrogates were identified that were within the similarity distance of < or = 0.200, of which only five chemicals had an RfD on IRIS, HEAST, or on the NCEA provisional toxicity value list. Among the five potential surrogates, chlorobenzilate with an RfD of 2 x 10(-2) mg/kg-day was chosen as a surrogate for DCBP as it had the most conservative toxicity value. This compared well with surrogate selection using available metabolic information for DCBP and its metabolites or parent compounds in the literature and the provisional toxicity value of 3 x 10(-2) mg/kg-day that NCEA developed using a subchronic study.

Dichloroacetic acid: metabolism in cytosol.

Dichloroacetic acid (DCA) arises from the chlorination of drinking water and the metabolism of trichloroethylene (TRI) and is used therapeutically. The toxicity of TRI exposure is dependent on metabolism, and DCA has been proposed to be one contributor to this toxicity. Beyond the identification of some metabolites of DCA and some pharmacokinetic studies, little is known about the tissue distribution and enzymology of DCA metabolism. We present data that indicate that DCA degradation occurs primarily in the cytosol. Low molecular weight components of cytosol are required for the reaction, including nicotinamide cofactor and glutathione (GSH). GSH plays a role in the removal of DCA from cytosol, although not through transferase-mediated conjugation. In rat cytosol, the KM is approximately 0.3 mM, and the apparent Vmax approximates 12 nmoles/min/mg cytosolic protein. These results set DCA apart from other chlorinated compounds that are metabolized by the cytochrome P450 enzyme family.

Glutathione conjugation of trichloroethylene in human liver and kidney: kinetics and individual variation.

Isolated human hepatocytes exhibited time-, trichloroethylene (Tri) concentration-, and cell concentration-dependent formation of S-(1, 2-dichlorovinyl)glutathione (DCVG) in incubations in sealed flasks with 25 to 10,000 ppm Tri in the headspace, corresponding to 0.011 to 4.4 mM in hepatocytes. Maximal formation of DCVG (22.5 +/- 8.3 nmol/120 min per 10(6) cells) occurred with 500 ppm Tri. Time-, protein concentration-, and both Tri and GSH concentration-dependent formation of DCVG were observed in liver and kidney subcellular fractions. Two kinetically distinct systems were observed in both cytosol and microsomes from pooled liver samples, whereas only one system was observed in subcellular fractions from pooled kidney samples. Liver cytosol exhibited apparent Km values (microM Tri) of 333 and 22.7 and Vmax values (nmol DCVG formed/min per mg protein) of 8.77 and 4.27; liver microsomes exhibited apparent Km values of 250 and 29.4 and Vmax values of 3.10 and 1.42; kidney cytosol and microsomes exhibited apparent Km values of 26.3 and 167, respectively, and Vmax values of 0.81 and 6.29, respectively. DCVG formation in samples of liver cytosol and microsomes from 20 individual donors exhibited a 6.5-fold variation in microsomes but only a 2.4-fold variation in cytosol. In coincubations of pooled liver cytosol and microsomes, addition of an NADPH-regenerating system produced marked inhibition of DCVG formation, but addition of GSH had no effect on cytochrome P-450-catalyzed formation of chloral hydrate. These results indicate that both human kidney and liver have significant capacity to catalyze DCVG formation, indicating that the initial step of the GSH-dependent pathway is not limiting in the formation of nephrotoxic and nephrocarcinogenic metabolites.