Monday, August 2, 2004
10:30 AM–12:30 PM
(92) METHOXYCHLOR METABOLITES INDUCE CHANGES IN GROWTH, ATRESIA AND CYTOCHROME P450 EXPRESSION IN MOUSE OVARIAN ANTRAL FOLLICLES.
Miller, Kimberly1, Borgeest, Christina1, Flaws, Jodi1, 1 Program in Toxicology and Dept of Epidemiology and Preventive Medicine, Baltimore, MD
ABSTRACT- The organochlorine pesticide methoxychlor (MXC) is a reproductive toxicant that targets the mammalian ovary. Previous in vivo studies indicate that antral follicles are primary targets of MXC in the mouse ovary, while less mature follicles are unaffected by MXC exposure. Toxic endpoints include increased antral follicle atresia and increased ovarian Bax protein levels. Cytochrome P450 (CYP) enzymes metabolize MXC to polar compounds such as mono-OH MXC and bis-OH MXC (HPTE), which may be more toxic than MXC. Thus, this work tested the hypothesis that MXC metabolites are responsible for antral follicle-specific toxicity, and that ovarian CYP enzymes mediate MXC metabolism to cause antral follicle toxicity. Using in vitro follicle culture, antral follicles isolated from immature mouse ovaries were exposed to MXC (0.01-100g/mL), mono-OH MXC (0.01-10g/mL) or HPTE (0.01-10g/mL) for 96hrs. Follicle diameters were measured at 24hr intervals. After culture, follicles were processed for morphological analysis of atresia. Immunohistochemistry was also performed on in vitro cultured follicles as well as ovaries from MXC-exposed mice (32mg/kg/day MXC for 20 days) to evaluate expression of MXC metabolizing enzymes CYP3A4 and CYP2C19. MXC significantly inhibited antral follicle growth in vitro compared to controls at 10 and 100g/mL (27% and 23% respectively, p≤0.001), and increased atresia over controls at 100g/mL (45%, p≤0.04). Mono-OH MXC (10g/mL) also inhibited antral follicle growth (20%, p≤0.001) and increased atresia (38%, p≤0.05). Further, HPTE (10g/mL) slightly inhibited follicle growth (15%, p≤0.02), but did not increase atresia (p=0.68). CYP3A4 and CYP2C19 were present in the ovaries of in vivo treated mice, and were induced in corpora lutea, surface epithelium and antral/preovulatory follicles by MXC exposure. These isoforms were also present in cultured antral follicles following MXC exposure. These data suggest that MXC metabolites inhibit follicle growth and induce atresia, and that CYPs present in the ovary may metabolize MXC to toxic metabolites, which subsequently cause antral follicle toxicity. (Supported by NIH HD38955, T32 ES07263-13 and a Colgate-Palmolive Fellowship).
KEY WORDS: antral follicles, cytochrome P450, methoxychlor, ovary