Cellular and molecular mechanisms of 4-vinylcyclohexene-diepoxide induced ovotoxicity in rats

Abstract

4-vinylcyclohexene diepoxide (VCD) is an environmental xenobiotic formed as a by-product in the manufacture of rubber and therefore potential human exposure is likely. VCD destroys half of the small pre-antral (25-100 μm) follicles in ovaries of rats following 15 days of dosing. The overall goal of this research, was to determine the mode and earliest time for identification of follicular destruction and examine the specificity of the response for 25-100 μm follicles. The particular involvement of protein synthesis and gene expression in this ovotoxic response was also examined. After daily dosing with VCD (80 mg/kg), the rate of protein synthesis in 25-100 μm follicles was inhibited following 3, 6, and 10 hr of in vitro incubation with VCD; whereas, the inhibition in the rate of protein synthesis at 3 hr in 25-100 μm follicles from untreated animals was reversed at 6 and 10 hr. Furthermore, follicular viability was compromised to a greater extent in 25-100 μm follicles from dosed versus untreated animals. Following 10 days of daily dosing with VCD, there was an increase in random DNA fragmentation in 25-100 μm follicles; however, there was not a reduction in the numbers of primordial and primary (25-100 μm) follicles. Morphological analysis showed changes characteristic of an apoptotic-like form of cell death in oocytes and granulosa cells of primordial and primary follicles 4 hr following 10 days of daily dosing. There was an increase in levels of mRNA for bax, manganese superoxide dismutase (MnSOD) and microsomal epoxide hydrolase (mEH) in 25-100 μm follicles following 10 days dosing with VCD, but the increase was not observed in large pre-antral (100-250 μm) follicles or liver. However, decreases in levels of mRNA for bax in liver and mEH in 100-250 μm follicles were observed. These results suggest that repeated dosing makes 25-100 μm follicles more susceptible to VCD-induced cellular changes and that VCD-induces an apoptotic-like form of cell death which is mediated through changes in levels of expression of genes associated with death of the follicular cells.