Chem. Res. Toxicol. 1995, 8, 963-969
963
Ovarian Toxicity of 4-Vinylcyclohexene and Related Olefins in B6C3F1 Mice: Role of Diepoxides Julie K. Doerr, Stephen B. Hooser,+Bill J. Smith,* and I. Glenn Sipes* Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721 Received March 6, 1995@ 4-Vinylcyclohexene (VCH) is a n ovarian toxicant in mice. Studies have established that bioactivation of VCH to epoxides is required for its ovotoxicity, with vinylcyclohexene diepoxide being the most potent epoxide of VCH in terms of follicular depletion. To determine the role of the diepoxide in the ovarian toxicity of VCH and related compounds, a structure-activity study was conducted. Following administration (ip) of VCH for 30 days, a significant depletion of ovarian follicles was observed. No alteration of small ovarian follicle counts occurred following treatment with structural analogues of VCH (vinylcyclohexane, ethylcyclohexene, and cyclohexene) t h a t contain only a single unsaturated site. These VCH analogues were converted to monoepoxides both in vitro and in vivo. In addition, when the monoepoxide forms of the VCH analogues were administered to mice, they were not ovotoxic. These results indicate that vinylcyclohexene diepoxide may be the ultimate ovotoxic metabolite of VCH. A diepoxide was also shown to be critical for butadiene- and isoprene-induced follicular loss. Butadiene monoepoxide, butadiene diepoxide, and isoprene were ovotoxic. I n contrast, the monoepoxide, epoxybutane, was not ovotoxic. The ovotoxicity of these compounds correlated with their chemical reactivity a s assessed by alkylation of nicotinamide. Vinylcyclohexene diepoxide and butadiene diepoxide had a 3.5- to 10-fold higher chemical reactivity as compared to their monoepoxide precursors and structurally related monoepoxides. Thus, a relationship exists between chemical reactivity and ovotoxicity. Only those compounds which are metabolized to a diepoxide or a r e a diepoxide were ovotoxic. The formation of these diepoxide metabolites may in t u r n be linked to the ovarian toxicity and carcinogenicity of these olefins.
Introduction 4-Vinylcyclohexene (VCH)I is produced as a byproduct in butadiene processing. In addition, it is utilized as an intermediate in chemical production, as well as in the manufacture of flame retardants, insecticides, plasticizers, and antioxidants. This industrial chemical is of toxicological significance, as animal studies have revealed that its toxic effects specifically target the ovary. Depletion of oocytes contained in pre-antral follicles, premature ovarian failure, and the development of ovarian neoplasms have been observed in B6C3F1 mice ( I -3). An 85% depletion of small pre-antral follicles (primordial) occurred following administration of VCH for 30 days (7.5 mmolkg/day, ip). At 360 days these animals' ovaries were void of oocytes at any stage of development and, thus, were acyclic (cessation of estrous cyclicity) as compared to the vehicle control group (2). Follicular loss and the succeeding hormonal alterations are believed to be associated with the carcinogenic effects of VCH (2). In a two-year study conducted by NTP, a n increased
* To whom correspondence and reprint requests should be addressed at the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, 1703 E. Mabel, Tucson, AZ 85721. Telephone: 520-626-7123. FAX: 520-626-2466. ' Present address: Purdue University Animal Disease Diagnostic Laboratory, 1175 ADDL, West Lafayette, IN 47907-1175. i Present address: Procter and Gamble Pharmaceuticals, Drug Safety Assessment, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, OH 452394707, @Abstractpublished in Advance ACS Abstracts, July 15, 1995. Abbreviations: VCWvinylcyclohexene, 1,Z-VCHEIvinylcyclohexene 1,2-monoepoxide, 7,8-VCHEIvinylcyclohexene 7,8-monoepoxide, VCDI vinylcyclohexene diepoxide, ECHEIethylcyclohexene, ECHE OWethylcyclohexene oxide, VCHNvinylcyclohexane, VCHA OWvinylcyclohexane oxide, CHE/cyclohexene, CHE OWcyclohexene oxide, BDI butadiene, BMOhutadiene monoepoxide, BDEhutadiene diepoxide, EPBIepoxybutane, ISO/isoprene, IS0 OWZ-methyl-2-vinyloxirane.
incidence in the occurrence of uncommon ovarian neoplasms, including mixed benign tumors, granulosa cell tumors, and granulosa cell carcinomas, was observed following VCH treatment ( 3 ) . The relevance of VCH-induced ovotoxic effects is apparent when one considers the nature of the female reproductive system. The ovary is unique in that the complete complement of germ cells is present a t fetal development, and the most immature population of cells (primordial follicles) is a t a stage of meiotic arrest and is nondividing (4). Thus, xenobiotics that target the primordial follicular population elicit a n irreversible response, as the follicles cannot replace themselves. Of additional concern is the long latency period prior to manifestation of this irreversible ovarian failure. Cyclicity, hormonal regulation, and fertility are unaltered as long as a pool of primordial follicles are present from which to recruit (2, 5). The processes involved in the chemical-mediated ovotoxicity of VCH are not known. However, it has been determined that bioactivation of the parent compound to epoxide metabolites is required (1). Both monoepand oxides (vinylcyclohexene 1,2-monoepoxide/l,2-VCHE vinylcyclohexene 7,8-monoepoxide/7,8-VCHE), as well as the diepoxide (vinylcyclohexene diepoxideNCD) have been shown to be more potent ovotoxicants than VCH (1). The most potent ovotoxicant was the diepoxide (1). ED50 values revealed that VCD was 13.5 times more potent than VCH, whereas 7,8-VCHE and 1,2-VCHE were only 5-7 times more potent than the parent compound, respectively. Circulating levels of 1,B-VCHE ( 6 ) and VCD2 have been observed in mice following administration of a single dose of VCH (7.5 mmol/kg, ip)
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964 Chem. Res. Toxicol., Vol. 8, No. 7, 1995
and 1,2-VCHE (2.74 mmolkg, ip), respectively. Formation of the VCH epoxides has also been demonstrated in rodent and human hepatic microsomal preparations (68). Although the monoepoxides of VCH have been shown to be ovotoxic, it is not known if these monoepoxides directly target the ovary or if they must be converted to the diepoxide to exert ovotoxicity. Therefore, the objective of the following study was to delineate the role of these epoxides in VCH-induced ovotoxicity. It is hypothesized that VCD, which has been characterized as a more potent ovotoxicant, as well as a direct-acting mutagen and carcinogen (9), is the ultimate ovotoxic metabolite of VCH.
Table 1. Ovotoxicity of VCH and VCH Analogues Foilicie Counts a Compound
J. K. Doerr and I. G. Sipes, manuscript in preparation.
Small
o^
Growing
36f3' (37%f 4%)
Ethylcyclohexene (ECHE)
75
98 f 15
53f6
75
66 f 12
43f8
Vinylcyclohexane (VCHA)
Experimental Procedures Chemicals. Vinylcyclohexane oxide and ethylcyclohexene oxide were custom synthesized in the laboratory of Dr. Eugene Mash of the University of Arizona, Tucson, AZ. Identity and purity (99%) of the compounds were established with NMR and IR. Ethylcyclohexene was a gift of Dr. Gerhard Nowack of Phillips Petroleum Co., Bartlesville, OK. Other test compounds were purchased from Aldrich Chemical Co. (Milwaukee, WI) with a purity of 95-99%. 3,3,3-Trichloropropene oxide was obtained from Chem Service (West Chester, PA). /%NADP+ (sodium salt), glucose 6-phosphate (monosodium salt), and glucose-6-phosphate dehydrogenase (type XV) were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals were of reagent grade. Caution:All test compounds are either potential carcinogens or are carcinogens in animals and should be handled with appropriate precautions. Animals. Twenty-one day old female B6C3F1 mice were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The mice were housed 5 per cage in a biohazard hood, provided food (Harlan Teklad 4% Mousemat Diet, Madison, WI) and water ad libtium, and maintained on a 12 h lighudark cycle in a controlled temperature of 22 & 2 "C. The animals were acclimated to this environment for 7 days prior to use in studies. Structure-Activity Studies. The structure-activity experiments were designed according to previous ovarian toxicity studies conducted in our laboratory ( I ) . Twenty-eight day old mice were administered VCH (positive control), sesame seed oil (vehicle control, 2.5 mL/kg), or test compound ip daily for 30 days ( n = lO/group). Test compounds, as well as their structures and doses administered, are presented in Tables 1-3. Test compounds were administered at either a dose equimolar to that of VCH (7.5 mmol/kg), o r at a lower dose which had been determined from previous studies as the maximum tolerated dose by mice over a 30 day period without significant weight loss. Following day 30, mice were killed by COn inhalation on the first day of diestrus of their cycle. The stage of estrus was determined by vaginal cytology (10). Ovaries were removed, weighed, fixed in Bouin's solution for 24 h, and transferred to 70% ethanol. One ovary was randomly selected and further processed for sectioning. Serial sections were prepared (6 pm) and stained with hematoxylin and eosin. Oocytes contained in small and growing pre-antral follicles were counted microscopically a t every 20th section. Follicular populations were classified by the method of Pedersen and Peters (111,in which a small follicle (primordial) consisted of a n oocyte surrounded by a single layer of noncuboidal granulosa cells. A growing follicle (primary to pre-antral) consisted of a n oocyte surrounded by a single or multiple layers of cuboidal granulosa cells. Oocyte counts a t every 20th section were summed to calculate the oocyte number for each ovary. In Vitro Metabolism Studies. In vitro metabolism was analyzed using hepatic microsomal preparations from 46 day old female mice (16-19 g), Livers were removed, and microsomes were isolated by differential ultracentrifugation as described by Guengerich (12). Protein concentration was de-
Dose (mmollkg)
Cyciohexene (CW
7.5
12fZC (86% f 2%)
16f2' (72% f 4%)
Vinylcyclohexene (VCH) Sesame Seed Oil
90f 14
57f4
Mean follicle counts for each treatment group were calculated by summing the counts determined a t every 20th section per mouse and using these totals to obtain an average zk SE. Percent follicular depletion i SE as compared to the sesame seed oil group. Statistically significant a t p
