Effect of CYP2E1 gene deletion in mice on expression of microsomal epoxide hydrolase in response to VCD exposure

Abstract

Females are born with a finite number of primordial follicles. 4-Vinylcyclohexene diepoxide (VCD) is a metabolite formed by epoxidation of 4-vinylcyclohexene (VCH) via its two monoepoxides 1,2- and 7,8-4-vinylcyclohexene monoepoxide (VCM). VCD specifically destroys small preantral (primordial and small primary) follicles in the rodent ovary. The phase I enzyme, cytochrome P450 isoform 2E1 (CYP2E1) is involved in ovarian metabolism of VCM to VCD. Further, microsomal epoxide hydrolase (mEH) can detoxify VCD to an inactive tetrol (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). This study evaluated the effects of VCD-induced ovotoxicity on mEH in CYP2E1+/+ and -/- mice (129S(1)/SvImJ background strain) using a postnatal day 4 mouse whole ovary culture system. The hypothesis of our study is that there is a relationship between CYP2E1 and mEH gene expression in the mouse ovary. Relative to control, VCD exposure caused follicle loss (p < 0.05) in ovaries from both genotypes; however, after 15 days, this loss was greater (p < 0.05) in CYP2E1+/+ ovaries. In a time course (2-15 days), relative to control, VCD (5 microM) caused an increase (p < 0.05) in mEH mRNA by 0.5-fold (day 10) and 1.84-fold (day 15) in CYP2E1-/- but not +/+ ovaries. 7,12-Dimethylbenz[a]anthracene (DMBA) also destroys ovarian follicles but, unlike VCD, is bioactivated by mEH to an ovotoxic 3,4-diol-1,2-epoxide metabolite. Incubation of ovaries in increasing concentrations of DMBA (0.5-1 microM, 15 days) resulted in greater (p < 0.05) follicle loss in CYP2E1-/-, relative to +/+ ovaries. With greater mEH (CYP2E1-/-), increased follicle loss with DMBA (bioactivation) and decreased follicle loss with VCD (detoxification) support that ovarian expression of CYP2E1 and mEH may be linked.

FIG. 1.

Metabolism of VCH and DMBA. (A) VCH is bioactivated by CYP450 isoforms to form either 1,2- or 7,8-monoepoxide (VCM) metabolites and, subsequently, VCD. The action of mEH metabolizes VCD to an inactive tetrol metabolite. Adapted from Keller et al. (1997). (B) DMBA is bioactivated by CYP450 isoforms to a DMBA-3,4-epoxide and, subsequently, hydrolyzed by mEH to DMBA-3,4-diol. Further activation by CYP450 isoforms results in formation of the bioactive metabolite, DMBA-3,4-diol-1,2-epoxide. Adapted from Miyata et al. (1999).

FIG. 2.

Effect of CYP2E1 expression on ovotoxicity with VCD. Ovaries from PND4 CYP2E1+/+ and −/− mice were incubated with control medium or increasing concentrations of VCD (5–30μM) for 15 days. Following incubation, ovaries were collected and processed for histological evaluation as described in the Materials and Methods section. (A) Primordial and (B) small primary follicles were classified and counted. Values are mean ± SE total follicles counted per ovary, n = 4–9; values with different letters are significantly different at p < 0.05.

FIG. 3.

Effect of CYP2E1 expression on VCD-induced ovotoxicity. Ovaries from PND4 CYP2E1+/+ and −/− mice were incubated with control medium or 5μM VCD for 2, 5, 10, or 15 days. Following incubation, ovaries were collected and processed for histological evaluation as described in the Materials and Methods section. (A) Primordial and (B) small primary follicles were classified and counted. Values are mean ± SE total follicles counted per ovary, n = 3; *different (p < 0.05) from control.

FIG. 4.

Effect of VCD on ovarian mEH mRNA. Ovaries from PND4 CYP2E1+/+ and −/− mice were incubated with control medium or 5μM VCD for 2, 5, 10, or 15 days. Following incubation, total RNA was isolated as described in the Materials and Methods section. One microgram total RNA was reverse transcribed to cDNA and analyzed for mEH and β-actin mRNA by RT-PCR. Values are mean fold change ± SE; n = 3 (8–10 ovaries per pool). *Different (p < 0.05) from control.

FIG. 5.

Effect of VCD on mEH protein. (A) Representative Western blotting of adult B6C3F₁ mouse ovary protein incubated with anti-mEH antibody. Ovaries from PND4 CYP2E1+/+ (B, C) and CYP2E1−/− (D, E) mice were incubated with control medium (B, D) or 5μM VCD (C, E) for 15 days. Following culture, ovaries were processed for immunofluoresence and confocal microscopy as described in the Materials and Methods section. All images were captured with a ×40 objective lens. Red staining = Cy5-labeled anti-mEH antibody. Green staining = YoYo1 genomic DNA. (F) Merged genomic stain and mEH protein stain. (G) Immunonegative stain (Cy5 and YoYo1 without anti-mEH antibody). (H) Quantification of mEH staining intensity, relative to controls, in small preantral follicles. Broken arrows indicate primordial follicles. Solid arrows indicate small primary follicles. Scale bar = 25μM.

FIG. 6.

Effect of VCD on hepatic mEH mRNA. PND28 CYP2E1+/+ and −/− mice were dosed daily with sesame oil (control) or VCD (0.57 mmol/kg/day; ip) for 15 days. Liver was removed and total RNA was isolated as described in the Materials and Methods section. One microgram total RNA was reverse transcribed to cDNA and analyzed for mEH and β-actin mRNA by RT-PCR. Values are mean fold change ± SE; n = 3.

FIG. 7.

Effect of CYP2E1 expression on ovotoxicity with DMBA. Ovaries from PND4 CYP2E1+/+ and −/− mice were incubated with control medium or increasing concentrations of DMBA (0.5–1μM) for 15 days. Following incubation, ovaries were collected and processed for histological evaluation as described in the Materials and Methods section. (A) Primordial and (B) small primary follicles were classified and counted. Values are mean ± SE total follicles counted per ovary, n = 4–10; values with different letters are significantly different at p < 0.05.