Metabolism and Toxicity of Trichloroethylene and Tetrachloroethylene in Cytochrome P450 2E1 Knockout and Humanized Transgenic Mice

Abstract

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.

Figure 1.

Overview of the metabolic pathways of TCE and PCE. Upon absorption, TCE and PCE can either be oxidized via CYPs or conjugated with GSH via GSTs. Abbreviations: TCE, trichloroethylene; PCE, tetrachloroethylene; CH, chloral hydrate; TCOH, trichloroethanol; TCA, trichloroacetate; TCAC, trichloroacetyl chloride; TCOG, trichloro-glucuronide conjugate; DCA, dichloroacetate; DCVG, S-(1, 2-dichlorovinyl)-GSH; DCVC, S-(1, 2-dichlorovinyl)-cysteine; NAcDCVC, N-acetyl-S-(1, 2-dichlorovinyl)-cysteine; TCVG, S-(1, 2, 2-trichlorovinyl)-GSH; TCVC, S-(1, 2, 2-trichlorovinyl)-cysteine; NAcTCVC, N-acetyl-S-(1, 2, 2-trichlorovinyl)-cysteine; GGT, gamma-glutamyl transferase; DP, dipeptidase; NAT, n-acetyl transferase; FMO, flavin monooxygenase; CCBL, cysteine conjugate beta-lyase.

Figure 2.

Schematic representation of study designs. Male and female mice from 3 strains (129S1/SvlmJ wild type, Cyp2e1(−/−) and hCYP2E1) were used in these studies. One study examined toxicokinetics of TCE in a single dose (600 mg/kg, gavage) study where samples were collected for up to 24 h. Second study examined metabolism and toxicity of PCE that was administered for 5 consecutive days (500 mg/kg, gavage); samples were collected 2 h after the last dose. Alkamuls EL-620 (5%, 10 ml/kg) was used as an aqueous emulsion vehicle for administration of both chemicals. Vertical lines indicate time points of sample collection. Down-arrows indicate chemical treatments. Endpoints collected in each study are shown as a bulleted list. Raw data for each animal and endpoint are reported in Supplementary Tables 2 and 3.

Figure 3.

Protein expression of CYP2E1 in liver and kidney of (A) male and (B) female 129S1/SvlmJ, Cyp2e1(−/−), and hCYP2E1 mice at 4 hours after dosing with a single dose of vehicle (V, Alkamuls EL-620) or TCE (T, 600 mg/kg). Representative Western blots and quantitative analysis of CYP2E1 protein expression are shown (n = 4/group). Expression of beta actin (Actb) was used to normalize for protein loading. Data are shown as mean ± SD. Asterisks denote statistical significant difference between groups (1-way ANOVA with Newman-Keuls post hoc test, p < .05).

Figure 4.

Comparative analysis of liver (Li) toxicokinetics of major oxidative (TCA and TCOH), and GSH conjugation (DCVG) metabolites of TCE (600 mg/kg) in (A) male and (B) female 129S1/SvlmJ (SV129), Cyp2e1(−/−), and hCYP2E1 mice. Average kinetic profiles (left panels, strains are identified by symbols as shown in an inset in the top left graph) and AUC (right panels) are shown (n = 2–4 per group per time point, as detailed in Supplementary Table 2).

Figure 5.

Comparative analysis of kidney (Kd) toxicokinetics of oxidative (TCA), and GSH conjugation metabolites (DCVG, DCVC, and NAcDCVC) of TCE (single dose, 600 mg/kg) in (A) male and (B) female 129S1/SvlmJ (SV129), Cyp2e1(−/−), and hCYP2E1 mice. Average kinetic profiles (left panels, strains are identified by symbols as shown in an inset in the top left graph) and AUC (right panels) are shown (n = 2–4 per group per time point, as detailed in Supplementary Table 2).

Figure 6.

Comparative analysis of liver (Li) and kidney (Kd) levels of major oxidative (TCA), and GSH conjugation (DCVG, DCVC, and NAcDCVC for TCE; TCVG, TCVC, and NAcTCVC for PCE) metabolites. Data for (A) TCE (600 mg/kg) and (B) PCE (5 daily doses, 500 mg/kg) in male and female 129S1/SvlmJ (Sv129), Cyp2e1(−/−), and hCYP2E1 mice are shown. Metabolite levels shown are for a 2 h time point after dosing. Data are shown as mean ± SD. Bars with different letters are significantly different (1-way ANOVA with Newman-Keuls post hoc test, p < .05).

Figure 7.

Effects of PCE on liver fat accumulation in male 129S1/SvlmJ (Sv129), Cyp2e1(−/−), and hCYP2E1 mice treated with 5 consecutive daily doses of vehicle (V, Alkamuls EL-620) or PCE (P, 500 mg/kg). (A) Representative images of H&E staining with 200× magnification. Arrows point to fat accumulation in Sv129 mice and glycogen accumulation in hCYP2E1 mice. Triglyceride levels in (B) liver (Li) and (C) serum (Se) are shown as mean ± SD. Bars with letters are significantly different from other groups (1-way ANOVA with Newman-Keuls post hoc test, p < .05).

Figure 8.

Effects of PCE on (A) proximal tubule injury and (B) cell proliferation in kidney of male 129S1/SvlmJ (SV129), Cyp2e1 (−/−), and hCYP2E1 mice treated with 5 consecutive daily doses of vehicle (V, Alkamuls EL-620) or PCE (P, 500 mg/kg). The top figures show the representative images of KIM-1 or BrdU staining with 400× magnification. Quantitative results of proximal tubule injury and cell proliferation are expressed as % positive/total proximal tubules or nuclei, and are summarized in the lower bar charts. Asterisks denote significant difference by paired t test (p < .05).