QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.010538v1 34/12/2020    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kim, D. Articles by Ghanayem, B. I. Search for Related Content PubMed PubMed Citation Articles by Kim, D. Articles by Ghanayem, B. I.

Comparative Metabolism and Disposition of Trichloroethylene in Cyp2e1–/–and Wild-Type Mice

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

(Received April 13, 2006; accepted September 6, 2006)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Trichloroethylene (TCE)1 is an important environmental contaminant, a well established rodent carcinogen, and a "probable human carcinogen". Metabolism of TCE occurs primarily via cytochrome P450 (P450)-dependent oxidation. In vitro studies suggested that CYP2E1 is the principal high-affinity enzyme responsible for TCE metabolism. The objective of the present work is to more directly assess the role of CYP2E1 in the metabolism and disposition of 1,2-¹⁴C-TCE administered at 250 or 1000 mg/kg (gavage) using Cyp2e1–/–[knockout (KO)] versus wild-type (WT) mice. After dosing, animals were individually placed in glass metabolism cages that allowed the collection of expired air, urine, and feces. Exhalation of TCE-derived ¹⁴CO₂ increased in a dose-dependent manner in mice of both genotypes and was significantly higher in WT versus KO mice. A significantly greater percentage of the dose was exhaled in KO versus WT mice as organic volatiles (mainly as TCE). Urinary excretion was the major route of TCE metabolism in WT mice, and the percentage of dose eliminated in urine was significantly higher at the 250 versus 1000 mg/kg dose. Furthermore, urinary excretion and CO₂ exhalation significantly decreased in KO versus WT mice. Pretreatment with 1-aminobenzotriazole clearly inhibited TCE metabolism as evident from increased exhalation of parent TCE, and decreased urinary excretion and CO₂ exhalation in mice of both genotypes. In conclusion, these data showed that whereas CYP2E1 plays an important role in TCE metabolism and disposition, other P450s also play a significant role and may explain earlier results showing that TCE causes lung damage in KO and WT mice.

TCE metabolism occurs via conjugation with glutathione via glutathione S-transferase and oxidation via the cytochrome P450 (P450) enzymes (Lash et al., 2000). glutathione S-transferase-dependent conjugation of TCE is a minor pathway and occurs in the liver and kidney. Subsequent metabolism via the ß-lyase enzyme leads to the conversion of S-(1,2-dichlorovinyl)-L-cysteine to S-1,2-dichlorovinyl thiol, which is implicated in TCE-induced nephrotoxicity in rats (Elfarra et al., 1986; Lash et al., 2001). Although TCE conjugation is distinguishable from its oxidation in terms of the metabolites produced and target organ specificity, this pathway is not considered quantitatively significant. However, at elevated exposure levels of TCE when the high-affinity oxidative pathway is saturated, the role of conjugation may become more significant (Lash et al., 1998).

P450-dependent oxidative metabolism of TCE occurs primarily in the liver and in other organs, such as lung, kidney, and reproductive tract. Although different isozymes (CYP1A1/2, CYP2B1/2, CYP2C11/6, and CYP2E1) have been identified as playing a role in TCE metabolism (Nakajima et al., 1993), CYP2E1 is regarded as a major high-affinity isozyme in the oxidation of TCE. Recent reports showed that CYP2E1, CYP2F, and CYP2B1 are the main enzymes involved in the pulmonary bioactivation of TCE (Forkert et al., 2005, 2006). The initial proposed step in the oxidation of this chemical is the conversion to TCE epoxide (Fig. 1), which may react with DNA or proteins (Anders and Jakobson, 1985). Alternatively, it was proposed that an oxygenated TCE-O-P450 adduct could contribute to the formation of chloral (Fig. 1) (Miller and Guengerich, 1982). Although the oxidation of TCE has not been fully characterized, it has been presumed that it proceeds via epoxide formation which may subsequently lead to the formation of CO, CO₂, dichloroacetic acid (DCA), (mono)chloroacetic acid (MCA), and another intermediate (TCE-O-P450) that may undergo chloride migration to produce chloral, followed by oxidation and reduction to trichloroacetic acid (TCA) and trichloroethanol (TCOH), respectively (Fig. 1). Metabolism is thought to play a critical role in TCE-induced toxicity in various organs. Chloral is thought to contribute to the induction of mouse lung tumors (Green, 2000). TCA and DCA were also shown to cause liver tumors in rats and mice through induction of peroxisome proliferation in hepatocytes (Herren-Freund et al., 1987). Spermatoxicity studies in rats showed that DCA causes delayed spermiation and morphological abnormalities in residual bodies and testes (Linder et al., 1997). Furthermore, it was reported that P450-dependent formation of TCE reactive metabolites and the subsequent formation of dichloroacetyl protein adducts were associated with pulmonary (Forkert et al., 2006) and reproductive (DuTeaux et al., 2003) toxicities. Kidney damage might be attributed to TCOH or to metabolites originating from TCE conjugation with glutathione (Elfarra et al., 1986; Green et al., 2003).

View larger version (22K): [in this window] [in a new window]   FIG. 1. A proposed scheme of TCE metabolism in rodents.

Metabolism of TCE is influenced by many factors, including gender, tissue, and dose (Lash et al., 2000; Rhomberg, 2000) and is closely related to the expression and distribution of enzymes involved in the oxidation and glutathione conjugation pathways. Therefore, understanding the enzymatic basis of TCE metabolism is critical for scrutinizing the mechanisms of action and the human risks of this chemical. In the current work, the metabolism and disposition of TCE were investigated to assess the role of CYP2E1 and other P450s using Cyp2e1–/–and wild-type mice. Doses were selected for the current work because similar TCE doses were reported to cause cancer and lung injury in mice and because of the fact that TCE metabolism and toxicity were well investigated using similar doses (Clewell and Andersen, 2004; Forkert et al., 2005, 2006).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals. Trichloroethylene-1,2-¹⁴C, specific activity 5.4 mCi/mmol, was purchased from Sigma-Aldrich (St. Louis, MO). The radiochemical purity (99+%) was determined using high performance liquid chromatography (HPLC). Tween 80, TCE, TCA, DCA, MCA, TCOH, chloral hydrate, 1-aminobenzotriazole (ABT), and ß-glucuronidase (type VII-A, 25,000 units) were obtained from Sigma-Aldrich. Ethanol was obtained from Pharmaco (Trinidad, WI), and ethanolamine and ethylene glycol monomethyl ether were obtained from Fisher (Fair Lawn, NJ). HPLC-grade water and acetonitrile were purchased from Caledon Laboratories Ltd. (Georgetown, ON, Canada).

Animals and Dose Preparation. Male Cyp2e1+/+ (wild-type, WT) and Cyp2e1–/–(Cyp2e1 knockout, KO) mice, 3 to 4 months old and ranging in weight from 21 to 36 g, were first produced at the National Cancer Institute, Bethesda, MD (Lee et al., 1996) and were re-derived and bred at Charles River Laboratories (Wilmington, DE) as described previously (Hoffler et al., 2003). Dosing solutions were freshly prepared before administration by diluting 1,2-¹⁴C-TCE with unlabeled TCE in 1% aqueous Tween 80 solution. TCE was administered by gavage at 250 or 1000 mg/kg to deliver 40 to 300 µCi/kg in a dose volume of 10 ml/kg. Pretreatment with ABT at 50 mg/2.5 ml saline/kg (i.p.) was performed 1 h before TCE administration. Food and water were available ad libitum throughout the experiments. All animal care and procedures were carried out according to the National Institutes of Health guidelines (U.S. Department of Health and Human Services, 1985).

Disposition Experiments. Disposition studies were basically similar to methods described by Ghanayem et al. (1999) and Hoffler et al. (2003) with minor modifications. Groups of WT and KO mice were treated in groups as follows: 1) WT and KO mice (n = 4) received a single dose of 250 mg 1,2-¹⁴C-TCE/kg by gavage and were euthanized 24 h later; 2) WT and KO mice received a single dose of 1000 mg TCE/kg by gavage and were euthanized 24 h (n = 7) or 48 h (n = 4) later; and 3) WT and KO mice (n = 8) were administered 50 mg ABT/kg (i.p.) followed 1 h later by a single dose of 1000 mg TCE/kg by gavage and were euthanized 24 h after TCE administration. Immediately after TCE administration, mice were housed in individual glass metabolism cages (Wyse Glass Specialties, Inc. Freeland, MI) to separately collect urine, feces, and exhaled radioactivity over 24 or 48 h. CO₂ was trapped using ethanolamine/ethylene glycol monomethyl ether (3:7) trapping solutions, and organic volatiles were trapped using charcoal traps. Tissue, fecal, and charcoal samples were combusted using a sample oxidizer and counted. All other steps and procedures were followed as described previously (Ghanayem et al., 1999; Hoffler et al., 2003).

HPLC Analysis of Urine, Hydrolyzed Urine, and Charcoal Trap Extracts. Metabolites in urine and exhaled organic volatiles retained in the charcoal traps were extracted, separated, and identified using a Waters HPLC System (Waters Corp., Milford, MA) connected to a UV detector and an online 515T Radiomatic flow scintillation analyzer (PerkinElmer Life and Analytical Sciences, Boston, MA). The activated charcoal (150 mg) taken after breaking the glass traps was extracted three times with acetonitrile (200 µl each). The extract was injected into an HPLC system equipped with a Microsorb MV column (C₁₈, 4.6 x 250 mm; Rainin Instruments Co., Woburn, MA) with a mobile phase of a 50:50 mixture of 0.1% phosphoric acid in water and acetonitrile for 10 min at a flow rate of 0.4 ml/min, then a linear gradient to 100% acetonitrile over 15 min, where it was held for 35 min. The extract was further analyzed by NMR (300 MHz; Varian Inc., Palo Alto, CA). Urine samples were syringe-filtered through a 0.22-µm filter and directly injected into the HPLC system using an Aminex HPX-87H column (Bio-Rad Laboratories, Richmond, CA) maintained at 40°C with 0.008 M H₂SO₄ at a flow rate of 0.7 ml/min. Characterization of the urinary metabolites was based on the method described by Green and Prout (1985) and was modified for this study. Each urine sample was incubated with ß-glucuronidase (2000 units/ml urine) in 0.1 M phosphate buffer (pH 7.0) at 37°C for 4 h. The hydrolyzed urine samples were filtered and analyzed by the HPLC method described above. The metabolite profile of the hydrolyzed samples was compared with that of the same urine samples incubated without ß-glucuronidase in phosphate buffer. All peaks were detected using the online Radiomatic and UV detectors at 210 nm.

Macromolecular Binding of TCE-Derived Radioactivity in the Liver. Fractionation of liver homogenates to determine binding was based on the method described by Ghanayem et al. (1987) and was modified for this study. Liver specimens (weighing 200 mg each) from mice (n = 3 each, performed in duplicates) administered 250 mg TCE/kg were homogenized in 400 µlof 0.25 M cold sucrose using an Omni µH homogenizer (Omni International, Marietta, GA). To the homogenates, 400 µl of 10% TCA was added, mixed, and placed in a refrigerator overnight to facilitate the precipitation of proteins. The homogenates were centrifuged at 10,000 rpm for 10 min, and the supernatants were transferred into a preweighed vial. Precipitants were washed twice with 5% TCA, and the supernatants were combined to the previous solution (unbound radioactivity). The pellets were extracted with ethanol and a mixture of ethanol and ethyl ether (3:1), and the radioactivity contents of the supernatants represented the lipid-bound radioactivity. To the pellets, 10% NaCl was added and boiled in a water bath for 20 min to prepare the supernatants that contained the nucleic acid-bound radioactivity. Precipitates were digested overnight using 1.0 N NaOH. Triplicate samples of each prepared fraction (50 µl) were mixed with EcoLume (MP Biomedicals, Solon, OH), and the radioactivity was determined using scintillation counting. The TCE-derived radioactivity in the pellet represented the protein binding. HPLC analysis of the unbound radioactivity (soluble fraction) was performed using the same method described above to analyze the urine.

Statistical Analysis. All values are presented as the mean ± standard error. Group mean comparisons were conducted using Student's t test. Values were considered statistically significant at p 0.05.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Exhalation of TCE-derived organic volatiles (presented as cumulative percentage of dose) in WT and KO mice as a function of dose and time. TCE was administered at 250 or 1000 mg/kg (A) and 1000 mg/kg with or without ABT pretreatment (B). Values are the mean ± standard error of 4 to 11 mice. a denotes values in KO mice that are significantly different from values in similarly treated WT mice at p 0.05. b denotes values in mice treated with 1000 mg TCE/kg that are significantly different from values in mice of the same genotype that received 250 mg TCE/kg at p 0.05. c denotes values in mice treated with ABT + 1000 mg TCE/kg that are significantly different from values in mice that received 1000 mg TCE/kg without ABT at p 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

View larger version (17K): [in this window] [in a new window]   FIG. 3. Representative HPLC radiochromatograms of charcoal trap extracts collected at 12 h after TCE administration.

Exhalation of TCE-Derived ¹⁴CO₂. Exhalation of ¹⁴C-TCE-derived ¹⁴CO₂ continued over the 48-h monitoring period and was dose-dependent in mice of both genotypes (Fig. 4). WT and KO mice treated with 250 mg TCE/kg exhibited a decrease in the percentage of dose exhaled as ¹⁴CO₂ in comparison to mice administered 1000 mg TCE/kg (Fig. 4A). Furthermore, there was a significantly greater percentage of the TCE dose exhaled as ¹⁴CO₂ in WT versus KO mice (Fig. 4A; Table 1). When WT and KO mice were pretreated with ABT, 1 h before treatment with 1000 mg TCE/kg, the percentage of dose exhaled as ¹⁴CO₂ drastically decreased (Fig. 4B). ABT pretreatment apparently rendered WT and KO mice metabolically similar, and CO₂ exhalation accounted for 4% of the dose.

View larger version (20K): [in this window] [in a new window]   FIG. 4. Exhalation of TCE-derived CO2 (presented as cumulative percentage of dose) in WT and KO mice as a function of dose and time. TCE was administered at 250 or 1000 mg/kg (A) and 1000 mg/kg with or without ABT pretreatment (B). Values are the mean ± standard error of 4 to 11 mice. a denotes values in KO mice that are significantly different from values in similarly treated WT mice at p 0.05. b denotes values in mice treated with 1000 mg TCE/kg that are significantly different from values in mice of the same genotype that received 250 mg TCE/kg at p 0.05. c denotes values in mice treated with ABT + 1000 mg TCE/kg that are significantly different from values in mice that received 1000 mg TCE/kg without ABT at p 0.05.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Representative HPLC radiochromatograms of authentic standards (A), urinary metabolites at the 1000 mg TCE/kg dose (B), and urinary metabolites after incubation with ß-glucuronidase (C). Metabolite 1, trichloroacetic acid; 2, trichloroethanol-glucuronide; 3, unknown; and 4, trichloroethanol.

Based on HPLC analysis and coelution with authentic standard (¹⁴C-TCA), the metabolite eluting at 7 min was tentatively identified as TCA and accounted for 14% of the total urinary metabolites excreted in WT and KO mice that received the high dose (Fig. 5; Table 2). However, significant reduction of TCA excretion (calculated as percentage of dose) in the urine was found in KO versus WT mice treated with either dose of TCE (Table 2). The major TCE metabolite eluting at 12 min was characterized as TCOH-glucuronide, which is in agreement with an earlier report by Green and Prout (1985). Urine was incubated with ß-glucuronidase to convert TCOH-glucuronide to free TCOH and analyzed using HPLC (Fig. 5). After the urine of WT or KO mice was incubated with ß-glucuronidase, the major peak eluting at 12 min disappeared and a new major peak emerged at 36 min, which is consistent with the retention time of authentic TCOH (Fig. 5). This finding clearly confirmed that this major metabolite was TCOH-glucuronide. The TCA peak was not affected by incubation with ß-glucuronidase, nor did any new peak appear in the chromatogram. Although the minor peak that eluted at 15 min was not identified, it can be concluded that it is neither DCA (retention time =9 min) nor MCA (retention time =17 min) (Fig. 5). Interestingly, when 250 mg TCE/kg was administered to mice, a small peak corresponding to free TCOH was detected in the urine. At the 1000 mg TCE/kg dose, the urinary metabolite profiles showed differences in mice of both genotypes as a function of time (Table 2). The proportion of TCA in the urine of mice of both genotypes collected between 24 and 48 h versus 0 and 24 h increased significantly, whereas that of TCOH-glucuronide declined. At the 250 mg TCE/kg dose, the proportion of TCA was significantly higher in WT versus KO mice (Table 2). In contrast, the proportion of TCA was significantly higher in KO versus WT mice pretreated with ABT (Table 2).

TCE-Derived Radioactivity in Feces, Blood, and Tissues. Fecal excretion of TCE-derived radioactivity accounted for 2 to 5% of the dose in WT and KO mice (Table 1). Pretreatment with ABT caused a significant decrease in the excretion of TCE-derived radioactivity in the feces and accounted for 2 and 1% of the dose in WT and KO mice, respectively (Table 1). TCE-derived radioactivity was widely distributed in the tissues of WT and KO mice and retention was dose-dependent, with the highest levels found in the forestomach, liver, and kidney of mice of both genotypes (Table 3). Twenty-four hours after exposure to 1000 mg TCE/kg, tissues collected from WT mice retained higher levels of TCE-derived radioactivity than tissues from KO mice. Overall, ABT pretreatment caused a notable decrease in the concentration of TCE-derived radioactivity in tissues of mice of both genotypes (Table 3).

Macromolecular Binding of TCE-Derived Radioactivity in the Liver. Characterization of the macromolecular binding in the liver of treated mice showed that 79 and 72% of the total TCE-derived radioactivity was bound to liver macromolecules of WT and KO, respectively (Table 4). Binding of TCE-derived radioactivity to lipids and nucleic acids was comparatively minimal and was similar in KO and WT mice (Table 4). Most interesting, however, is the fact that 61 and 68% of the total TCE-derived radioactivity was bound to the protein fraction of the liver homogenates in KO and WT mice, respectively (Table 4). The concentration of radioactivity in the unbound fraction (supernatant) was low and, therefore, HPLC analysis showed no distinct peaks, and identification of the soluble metabolites was inconclusive.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
TCE is a chlorinated solvent that is heavily used in the preparation of consumer and industrial products (Gist and Burg, 1995; ATSDR, 1997). Although the uses of TCE have diminished because of its toxicity, TCE remains a common environmental contaminant (ATSDR, 1997). Bioactivation of TCE is considered a prerequisite for the development of TCE-induced toxicity and carcinogenicity. Oxidation via P450s is considered the primary pathway of TCE metabolism, and metabolites formed via this pathway are implicated in the pulmonary and reproductive toxicities as well as the carcinogenicity of this chemical (IARC, 1995; DuTeaux et al., 2003; Forkert et al., 2006). Earlier studies suggested that whereas CYP2E1 is the main high-affinity enzyme responsible for TCE oxidation, other P450s, including CYP2F, CYP2B1/2, CYP1A1/2, and CYP2C11, are also involved (Nakajima et al., 1992; Lipscomb et al., 1997; Forkert et al., 2005, 2006). Using KO mice, the current study was undertaken to assess the enzymatic basis of TCE metabolism and disposition in vivo.

Earlier studies (Green and Prout, 1985; Prout et al., 1985; Larson and Bull, 1992; Fisher, 2000; Keys et al., 2003) demonstrated that TCE is rapidly absorbed and metabolized in animals and humans. In agreement, current work confirmed that after gavage administration, TCE was rapidly absorbed and distributed to all major tissues. Excretion of TCE-derived radioactivity occurred primarily in the urine, expired air (as parent compound and CO₂), and feces. The importance of CYP2E1 in TCE metabolism was evident when comparing the exhalation of organic volatiles, which showed that a significantly greater percentage of the dose was exhaled in KO versus WT mice. This indicated that in the absence of CYP2E1, TCE metabolism is inhibited, resulting in retention of parent compound, which is subsequently eliminated via exhalation. Increased exhalation of the parent compound in KO versus WT mice was also associated with significant decreases in CO₂ exhalation and urinary excretion of TCE-derived metabolites in KO mice. In an earlier study, Prout et al. (1985) reported that the major route of excretion of TCE-derived radioactivity was in the urine and there was no significant effect of dose on the urinary excretion in mice (57, 53, and 52% of dose at 10, 500, and 1000 mg TCE/kg, respectively). In contrast, our study showed that urinary elimination is both dose- and genotype-dependent. Urinary excretion was the primary route of TCE metabolism at the 250 mg/kg dose and the percentage of dose excreted in the urine of KO and WT mice had significantly declined at the high dose. This finding suggested that enzymes involved in the production of TCE metabolites that are subsequently excreted in the urine might be saturated at the high dose.

Recently, the catalytic efficiencies (V_max/K_m) were determined using recombinant rat CYP2E1 (0.79), CYP2F4 (0.27), and CYP2B1 (0.07), mouse CYP2F2 (0.11), and human CYP2E1 (0.02) in TCE metabolism to chloral hydrate (Forkert et al., 2005). Subsequently, it was reported that TCE causes bronchiolar Clara cell injury in WT and KO mice in association with dichloroacetyl protein adduct formation (Forkert et al., 2006). It was suggested that TCE oxidation to chloral hydrate in murine lungs was mediated by CYP2E1 and other P450s including CYP2F2. In an attempt to assess the involvement of P450s other than CYP2E1, WT and KO mice were pretreated with ABT (a universal P450 inhibitor). Generally, ABT pretreatment rendered mice of both genotypes metabolically similar. The drastic increase in TCE exhalation in KO mice pretreated with ABT versus KO mice untreated with ABT was attributed to the contribution of P450s other than CYP2E1. Furthermore, the decline in the exhalation of TCE-derived CO₂ and excretion of TCE-derived urinary metabolites in ABT-pretreated KO mice may be attributed to P450s other than CYP2E1. Collectively, this work as well as earlier reports demonstrated that multiple P450s are responsible for TCE metabolism. However, the contribution of various P450s may be dependent on the organ in question and may also be affected by the absence of CYP2E1 in the KO mice. In comparison to WT mice, inhibition of TCE metabolism in the absence of CYP2E1 in KO mice may lead to an increase in the substrate (TCE) concentration. Hence, the metabolic contribution of other P450s may change because of the increase in the substrate (TCE) concentration in KO mice. However, it remains to be confirmed whether the metabolic activities of CYP2F and CYP2B enzymes increase at high blood TCE levels in KO mice. Furthermore, TCE exhalation over an extended period in conjunction with the presence of P450s including CYP2E1, CYP2F, and CYP2B1 in the respiratory tract (Forkert et al., 2005, 2006) may lead to the in situ metabolism of TCE to reactive intermediates, which may explain the pulmonary toxicity of this chemical.

Chlorinated acetic acids derived from TCE are considered a major source of CO₂ exhalation in treated mice (Fig. 1) (Green and Prout, 1985; Larson and Bull, 1992). CYP2E1 was reported to be involved in the conversion of chloral to TCA (Ni et al., 1996). This finding infers that the absence of CYP2E1 should lead to less TCA formation in KO versus WT mice, which was corroborated by the fact that the proportion of TCA in the urine of KO and WT mice accounted for 8 versus 17%, respectively, at the low dose of TCE. Interestingly, the percentage of TCA in the urine increased in KO mice pretreated with ABT, which may imply that TCA accumulation could occur in mice devoid of CYP2E1. Furthermore, although there was no direct evidence that CYP2E1 is responsible for the metabolism of TCA and/or DCA, relevant studies revealed that dechlorination of 1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)-ethane (Kitamura et al., 2002) and debromination of ethylene dibromide (Doty et al., 2000) may be mediated by CYP2E1. It is therefore possible that CYP2E1 may be involved in TCA metabolism.

In comparing the urinary metabolite profiles in WT and KO mice, the proportion of various metabolites changed as a function of time. The TCA proportion of total urinary metabolites increased and TCOH-glucuronide decreased in the urine of mice of both genotypes collected between 24 and 48 h versus urine collected between 0 and 24 h. This change in the urinary metabolite profiles might be due to enterohepatic recirculation of TCOH-glucuronide that is transformed to TCOH and then to TCA, which results in retaining TCA in the blood (Stenner et al., 1997). The long half-life of TCE-derived TCA could provide an explanation for CO₂ formation over a 48-h period after TCE administration and may contribute to the hepatocarcinogenicity of TCE through proliferation of hepatic peroxisomes in rodents (Herren-Freund et al., 1987).

The pattern of distribution of TCE-derived radioactivity showed that CYP2E1-mediated metabolism might be linked to retention of TCE-derived radioactivity in tissues. In comparison to WT mice, KO mice exhaled a significantly greater portion of the administered TCE unchanged and excreted less TCE-derived metabolites in the urine. However, the concentrations of TCE-derived radioactivity in tissues of KO mice were not distinctly different from those of WT mice. Interestingly, current work showed that most of the TCE-derived radioactivity in the liver of mice of both genotypes was bound to macromolecules, and protein binding accounted for 60 to 70% of the total binding. This finding is in agreement with a recent report that demonstrated significant formation of dichloroacetyl lysine protein adducts in the lungs of mice of both genotypes (Forkert et al., 2006). Current work also demonstrated that the total bound and the protein-bound TCE-derived radioactivity were significantly higher in WT versus KO mice. Collectively, these data confirmed that in addition to CYP2E1, other P450s play an important role in TCE metabolism to reactive intermediates and subsequent formation of protein adducts.

In conclusion, the current in vivo study confirmed that whereas CYP2E1 plays an important role in the metabolism and disposition of TCE administered by gavage at 250 or 1000 mg TCE/kg, other P450s perform a significant function as well. Although this conclusion is derived from studies conducted at doses that are higher than the reported K_m value for this chemical, the contribution of various P450 isoforms to the metabolism of TCE may vary at lower doses. In the absence of CYP2E1, inhibition of TCE metabolism occurs, leading to increased substrate (TCE) concentrations. It is possible that other P450s may metabolize TCE at an enhanced rate and compensate for the loss of CYP2E1 in KO animals. This premise is in agreement with the fact that TCE caused comparatively similar bronchiolar Clara cell damage in both KO and WT mice. It is also in agreement with the finding that substantial macromolecular binding of TCE-derived metabolites occurs in mice of both genotypes. Further investigation of the role of CYP2E1 in the metabolism of TCE metabolites such as TCA and DCA will be required to further understand the role of various P450s in TCE metabolism and toxicity.

	Acknowledgments

 
We thank Drs. Tom Burka, Ling-Jen Ferguson, and Undi Hoffler for the thoughtful review of the manuscript.

	Footnotes

 
This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.010538.

ABBREVIATIONS: TCE, trichloroethylene; TCA, trichloroacetic acid; DCA, dichloroacetic acid; MCA, (mono)chloroacetic acid; TCOH, trichloroethanol; P450, cytochrome P450; KO, knockout (Cyp2e1–/–mice); WT, wild-type (Cyp2e1+/+ mice); ABT, 1-aminobenzotriazole; HPLC, high performance liquid chromatography.

Address correspondence to: Dr. Burhan I. Ghanayem, National Institute of Environmental Health Sciences, P.O. Box 12233 MD B3-10, 111 Alexander Dr., Research Triangle Park, NC 27709. E-mail: ghanayem{at}niehs.nih.gov

	References

Top Abstract Materials and Methods Results Discussion References

 


Anders MW and Jakobson I (1985) Biotransformation of halogenated solvents. Scand J Work Environ Health 11 (Suppl 1): 23–32.

ATSDR (1997) Toxicological Profile for Trichloroethylene. Agency for Toxic Substances and Disease Registry, Department of Health and Human Services, Atlanta.

Clewell HJ and Andersen ME (2004) Applying mode-of-action and pharmacokinetic considerations in contemporary cancer risk assessments: an example with trichloroethylene. Crit Res Toxicol 34: 385–445.[CrossRef]

Davidson IW and Beliles RP (1991) Consideration of the target organ toxicity of trichloroethylene in terms of metabolite toxicity and pharmacokinetics. Drug Metab Rev 23: 493–599.[Medline]

Doty SL, Shang TQ, Wilson AM, Tangen J, Westergreen AD, Newman LA, Strand SE, and Gordon MP (2000) Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1. Proc Natl Acad Sci USA 97: 6287–6291.[Abstract/Free Full Text]

DuTeaux SB, Berger T, Hess RA, Sartini BL, and Miller MG (2004) Male reproductive toxicity of trichloroethylene: sperm protein oxidation and decreased fertilizing ability. Biol Reprod 70: 1518–1526.[Abstract/Free Full Text]

DuTeaux SB, Hengel MJ, DeGroot DE, Jelks KA, and Miller MG (2003) Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts. Biol Reprod 69: 771–779.[Abstract/Free Full Text]

Elfarra AA, Jakobson I, and Anders MW (1986) Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity. Biochem Pharmacol 35: 283–288.[CrossRef][Medline]

Fisher JW (2000) Physiologically based pharmacokinetic models for trichloroethylene and its oxidative metabolites. Environ Health Perspect 108 (Suppl 2): 265–273.[Medline]

Forkert PG, Baldwin RM, Millen B, Lash LH, Putt DA, Schultz MA, and Collins KS (2005) Pulmonary bioactivation of trichloroethylene to chloral hydrate: relative contributions of CYP2E1, CYP2F, and CYP2B1. Drug Metab Dispos 33: 1429–1437.[Abstract/Free Full Text]

Forkert PG, Millen B, Lash LH, Putt DA, and Ghanayem BI (2006) Pulmonary bronchiolar cytotoxicity and formation of dichloroacetyl lysine protein adducts in mice treated with trichloroethylene. J Pharmacol Exp Ther 316: 520–529.[Abstract/Free Full Text]

Ghanayem BI, Burka LT, and Matthews HB (1987) Ethyl acrylate distribution, macromolecular binding, excretion, and metabolism in male Fisher 344 rats. Fundam Appl Toxicol 9: 389–397.[CrossRef][Medline]

Ghanayem BI, Sanders JM, Chanas B, Burka LT, and Gonzalez FJ (1999) Role of cytochrome P-450 2E1 in methacrylonitrile metabolism and disposition. J Pharmacol Exp Ther 289: 1054–1059.[Abstract/Free Full Text]

Gist GL and Burg JR (1995) Trichloroethylene—a review of the literature from a health effects perspective. Toxicol Ind Health 11: 253–307.[Medline]

Green T (2000) Pulmonary toxicity and carcinogenicity of trichloroethylene: species differences and modes of action. Environ Health Perspect 108 (Suppl 2): 261–264.

Green T, Dow J, Ellis MK, Foster JR, and Odum J (1997) The role of glutathione conjugation in the development of kidney tumours in rats exposed to trichloroethylene. Chem-Biol Interact 105: 99–117.[CrossRef][Medline]

Green T, Dow J, and Foster J (2003) Increased formic acid excretion and the development of kidney toxicity in rats following chronic dosing with trichloroethanol, a major metabolite of trichloroethylene. Toxicology 191: 109–119.[CrossRef][Medline]

Green T and Prout MS (1985) Species differences in response to trichloroethylene. II. Biotransformation in rats and mice. Toxicol Appl Pharmacol 79: 401–411.[CrossRef][Medline]

Herren-Freund SL, Pereira MA, Khoury MD, and Olson G (1987) The carcinogenicity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse liver. Toxicol Appl Pharmacol 90: 183–189.[CrossRef][Medline]

Hoffler U, El-Masri HA, and Ghanayem BI (2003) Cytochrome P450 2E1 (CYP2E1) is the principal enzyme responsible for urethane metabolism: comparative studies using CYP2E1-null and wild-type mice. J Pharmacol Exp Ther 305: 557–564.[Abstract/Free Full Text]

IARC (1995) Trichloroethylene. IARC Monogr Eval Carcinog Risks Hum 63: 558.

Keys DA, Bruckner JV, Muralidhara S, and Fisher JW (2003) Tissue dosimetry expansion and cross-validation of rat and mouse physiologically based pharmacokinetic models for trichloroethylene. Toxicol Sci 76: 35–50.[Abstract/Free Full Text]

Kitamura S, Shimizu Y, Shiraga Y, Yoshida M, Sugihara K, and Ohta S (2002) Reductive metabolism of p,p'-DDT and o,p'-DDT by rat liver cytochrome P450. Drug Metab Dispos 30: 113–118.[Abstract/Free Full Text]

Larson JL and Bull RJ (1992) Metabolism and lipoperoxidative activity of trichloroacetate and dichloroacetate in rats and mice. Toxicol Appl Pharmacol 115: 268–277.[CrossRef][Medline]

Lash LH, Fisher JW, Lipscomb JC, and Parker JC (2000) Metabolism of trichloroethylene. Environ Health Perspect 108 (Suppl 2): 177–200.[Medline]

Lash LH, Qian W, Putt DA, Hueni SE, Elfarra AA, Krause RJ, and Parker JC (2001) Renal and hepatic toxicity of trichloroethylene and its glutathione-derived metabolites in rats and mice: sex-, species-, and tissue-dependent differences. J Pharmacol Exp Ther 297: 155–164.[Abstract/Free Full Text]

Lash LH, Qian W, Putt DA, Jacobs K, Elfarra AA, Krause RJ, and Parker JC (1998) Glutathione conjugation of trichloroethylene in rats and mice: sex-, species-, and tissue-dependent differences. Drug Metab Dispos 26: 12–19.[Abstract/Free Full Text]

Lee SS, Buters JT, Pineau T, Fernandez-Salguero P, and Gonzalez FJ (1996) Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem 271: 12063–12067.[Abstract/Free Full Text]

Linder RE, Klinefelter GR, Strader LF, Suarez JD, and Roberts NL (1997) Spermatotoxicity of dichloroacetic acid. Reprod Toxicol 11: 681–688.[CrossRef][Medline]

Lipscomb JC, Garrett CM, and Snawder JE (1997) Cytochrome P450-dependent metabolism of trichloroethylene: interindividual differences in humans. Toxicol Appl Pharmacol 142: 311–318.[CrossRef][Medline]

Miller RE and Guengerich FP (1982) Oxidation of trichloroethylene by liver microsomal cytochrome P-450: evidence for chlorine migration in a transition state not involving trichloroethylene oxide. Biochemistry 21: 1090–1097.[CrossRef][Medline]

Nakajima T, Wang RS, Elovaara E, Park SS, Gelboin HV, and Vainio H (1992) A comparative study on the contribution of cytochrome P450 isozymes to metabolism of benzene, toluene and trichloroethylene in rat liver. Biochem Pharmacol 43: 251–257.[CrossRef][Medline]

Nakajima T, Wang RS, Elovaara E, Park SS, Gelboin HV, and Vainio H (1993) Cytochrome P450-related differences between rats and mice in the metabolism of benzene, toluene and trichloroethylene in liver microsomes. Biochem Pharmacol 45: 1079–1085.[CrossRef][Medline]

Ni YC, Wong TY, Lloyd RV, Heinze TM, Shelton S, Casciano D, Kadlubar FF, and Fu PP (1996) Mouse liver microsomal metabolism of chloral hydrate, trichloroacetic acid, and trichloroethanol leading to induction of lipid peroxidation via a free radical mechanism. Drug Metab Dispos 24: 81–90.[Abstract]

NTP (2002) Trichloroethylene. Rep Carcinog 10: 244–2472.[Medline]

Prout MS, Provan WM, and Green T (1985) Species differences in response to trichloroethylene. I. Pharmacokinetics in rats and mice. Toxicol Appl Pharmacol 79: 389–400.[CrossRef][Medline]

Raaschou-Nielsen O, Hansen J, Thomsen BL, Johansen I, Lipworth L, McLaughlin JK, and Olsen JH (2002) Exposure of Danish workers to trichloroethylene, 1947–1989. Appl Occup Environ Hyg 17: 693–703.[CrossRef][Medline]

Rhomberg LR (2000) Dose-response analyses of the carcinogenic effects of trichloroethylene in experimental animals. Environ Health Perspect 108 (Suppl 2): 343–358.

Stenner RD, Merdink JL, Stevens DK, Springer DL, and Bull RJ (1997) Enterohepatic recirculation of trichloroethanol glucuronide as a significant source of trichloroacetic acid. Metabolites of trichloroethylene. Drug Metab Dispos 25: 529–535.[Abstract/Free Full Text]

Westirck JJ, Mello JW, and Thomas RF (1984) The groundwater supply survey. J Am Water Works Assoc 76: 52.

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.010538v1 34/12/2020    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kim, D. Articles by Ghanayem, B. I. Search for Related Content PubMed PubMed Citation Articles by Kim, D. Articles by Ghanayem, B. I.