Involvement of CYP 2E1 enzyme in ovotoxicity caused by 4-vinylcyclohexene and its metabolites

Abstract

4-Vinylcyclohexene (VCH) is bioactivated by hepatic CYP 2A and 2B to a monoepoxide (VCM) and subsequently to an ovotoxic diepoxide metabolite (VCD). Studies suggest that the ovary can directly bioactivate VCH via CYP 2E1. The current study was designed to evaluate the role of ovarian CYP 2E1 in VCM-induced ovotoxicity. Postnatal day 4 B6C3F(1) and CYP 2E1 wild-type (+/+) and null (-/-) mouse ovaries were cultured (15 days) with VCD (30 microM), 1,2-VCM (125-1000 microM), or vehicle. Twenty-eight days female CYP 2E1 +/+ and -/- mice were dosed daily (15 days; ip) with VCH, 1,2-VCM, VCD or vehicle. Following culture or in vivo dosing, ovaries were histologically evaluated. In culture, VCD decreased (p<0.05) primordial and primary follicles in ovaries from all three groups of mice. 1,2-VCM decreased (p<0.05) primordial follicles in B6C3F(1) and CYP 2E1 +/+ ovaries, but not in CYP 2E1 -/- ovaries in culture. 1,2-VCM did not affect primary follicles in any group of mouse ovaries. Conversely, following in vivo dosing, primordial and primary follicles were reduced (p<0.05) by VCD and VCM in CYP2E1 +/+ and -/-, and by VCH in +/+ mice. The data demonstrate that, whereas in vitro ovarian bioactivation of VCM requires CYP 2E1 enzyme, in vivo CYP 2E1 plays a minimal role. Thus, the findings support that hepatic metabolism dominates the contribution made by the ovary in bioactivation of VCM to its ovotoxic metabolite, VCD. This study also demonstrates the use of a novel ovarian culture system to evaluate ovary-specific metabolism of xenobiotics.

Figure 1. Proposed scheme for hepatic bioactivation of VCH

The parent compound, VCH, is bioactivated by cytochrome P450 (CYP 450) to form either 1,2 or 7,8-monoepoxide metabolites. These intermediate metabolites further undergo bioactivation by CYP 450 enzymes to form the ultimate ovotoxic diepoxide metabolite (VCD).

Figure 2. VCD - induced ovotoxicity in various strains of mouse ovaries in culture

Ovaries from PND4 B6C3F₁, CYP 2E1 +/+, or CYP 2E1 -/- mice were cultured with medium control or 30μM VCD for 15d. Following incubation, ovaries were collected, and processed for histological evaluation as described in materials and methods. Healthy (A) primordial, and (B) primary follicles were classified and counted. Values are mean ± SE total follicles counted/ovary, n=5; different letters differ (p<0.05) from one another within each follicle type.

Figure 3. Involvement of CYP 2E1 in 1,2-VCM - induced ovotoxicity in ovarian cultures

Ovaries from PND4 B6C3F₁, CYP 2E1 +/+ or -/- mice were cultured with medium control or 1,2-VCM (125, 250, 500, 750, or 1000 μM) for 15d. Following incubation, ovaries were collected, and processed for histological evaluation as described in materials and methods. Healthy (A) primordial, and (B) primary follicles were classified and counted. Values are mean ± SE total follicles counted/ovary, n=5; different letters differ (p<0.05) from one another within each follicle type.

Figure 4. Effect of in vitro VCM and VCD exposure on ovarian morphology

Ovaries from PND4 CYP 2E1 +/+ (A,C and E) and -/- (B,D and F) mice were cultured with control medium (A and B), 1,2-VCM (1000 μM; C and D), or 30μM VCD (E and F) for 15d. Following incubation, ovaries were collected, and processed for histological evaluation as described in materials and methods. All images were captured with a 40X objective lens. Bar represents 20μm.

Figure 5. Involvement of CYP 2E1 in VCH – induced ovotoxicity

Female CYP 2E1 +/+ and -/- mice were treated with repeated daily doses (i.p.) of sesame oil (control), VCH (7.4mmol/kg/d), 1,2-VCM (2.74mmol/kg/d), or VCD (0.57mmol/kg/d) for 15d. 4h following the final dose ovaries were collected, and processed for histological evaluation as described in materials and methods. Healthy (A) primordial, (B) primary, (C) secondary, and (D) antral follicles were classified and counted. Values are mean ± SE total follicles counted/ovary, n=5; different letters differ (p<0.05) from one another within each follicle type.