published_or_final_version / Civil and Structural Engineering / Master / Master of Philosophy

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4-Vinylcyclohexene (VCH), an industrial chemical, causes destruction of small pre-antral follicles (F1) in mice. Previous studies suggested that VCH is hepatically bioactivated to its ovotoxic metabolite, vinylcyclohexene diepoxide (VCD), by the cytochrome P450 enzymes, likely Cyp 2E1, Cyp 2A, and Cyp 2B. Additionally, microsomal epoxide hydrolase (mEH) likely participates in detoxifying the epoxide metabolites. The role of ovarian metabolism (bioactivation/detoxification) of VCH and its metabolites is not known. The hypothesis of this dissertation research was that ovarian metabolism contributes to VCH-induced ovotoxicity. These studies investigated whether the mouse ovary expresses (a) mRNA, (b) total protein, and (c) functional protein for several metabolic enzymes (Cyp 2E1, Cyp 2A, Cyp 2B, mEH), as well as, (d) the effects of VCH/VCD dosing on these parameters. Female B6C3F1 mice were dosed (i.p.) daily (15 d) with VCH (7.4 mmol/kg) or VCD (0.57 mmol/kg). Ovaries were removed and enzymatically digested and sorted into specific ovarian fractions (F1, small preantral; F2, large preantral; F3, antral; Int, interstitial cells) for mRNA analysis by realtime PCR, fixed for immunohistochemistry by confocal microscopy, or homogenized for functional assays. Basal expression was detected for mRNA encoding Cyp 2E1, Cyp 2A, Cyp 2B, and mEH in all ovarian fractions. In vivo dosing with VCH/VCD differentially altered expression, as expression increased for all enzymes in targeted F1 follicles. All enzymes were also distributed throughout the ovary, with high immunostaining intensity in the Int. In vivo dosing with VCH/VCD also affected protein distribution. Utilizing model substrates, catalytic activity was evaluated in ovarian fractions or whole ovaries. Basal activity was detected for Cyp 2E1, Cyp 2B, and mEH, while VCH dosing only induced activities for Cyp 2E1 and mEH. Taken together, the ovary has the metabolic capacity to be involved in metabolic reactions. Bioactivation is likely via Cyp 2E1 in the Int. cells. Although the relative contribution of ovarian metabolism in VCH-induced ovotoxicity is not known, the ovary likely plays a greater role in detoxification, due to greater levels of mEH activity. Thus, the ovary may provide a metabolic contribution to mediating the effects of ovotoxicants.

Environmental arsenic exposure is associated with human cancers of the skin, lung, kidney, and bladder. Mechanisms of arsenic toxicity and carcinogenicity, however, remain poorly studied and merit further investigation. The present studies address the effects of arsenic metabolism on toxicity, under the hypothesis that arsenic metabolism results in both detoxication and bioactivation. Inorganic arsenic and methylated metabolites were tested for toxicity in cultured cells, demonstrating that both detoxication and bioactivation occurs with metabolism, dependent upon methylation and valence state. Monomethylated MMAIII , the most toxic metabolite in cultured cells was also a potent inhibitor of pyruvate dehydrogenase and was more lethal than arsenite in hamsters, illustrating its role as an arsenic bioactivation product. The lung is an established target of arsenic exposure. Arsenic also crosses placenta during pregnancy, reaching the developing fetus. Given this evidence, the present studies investigate the ability of arsenic to target the developing lung following in utero exposure to low doses of arsenic during fetal development. Fetal rats were exposed to 500 mug/L arsenic via maternal drinking water, from conception to embryonic day eighteen. In order to assess toxicogenetic alterations in the developing lung, subtractive hybridization was used to create a cDNA library of arsenic-induced differential gene expression. This library consisted of 326 clones that were subsequently spotted on a cDNA microarray, including those involved in lung development and in formation of the extracellular matrix. In order to model effects of arsenic on gene expression in the developing lung, microarrays were conducted utilizing cultured lung cells dosed with four sub-cytotoxic doses of arsenic for up to fourteen days. These arrays showed that arsenic modulates a decreasing number of genes over the time course and that genes are primarily upregulated following short exposures. Selected array and subtracted library gene expression was also evaluated by quantitative real time PCR and western immunoblotting. Additional microarrays were conducted with 500 ppb arsenic treated fetal lung tissue using a commercial cDNA microarray, revealing perturbations in cellular proliferation and angiogenesis genes in vivo. Collectively, these studies indicate that lung development can be perturbed by gestational arsenic exposure.

Isoniazid (INH) is an antimicrobial used around the world in the treatment and chemoprophylaxis of tuberculosis. Hepatotoxicity is a well-recognized adverse effect of INH therapy. Metabolites of INH, namely hydrazine (HD) and acetylhydrazine (AcHD), are believed to be responsible for this hepatotoxicity. Studies were initiated to test the hypothesis that HD and not AcHD administration results in alterations in hepatic lipid homeostasis. In adult male C57B1/6J mice doses up to 300 mg AcHD/kg, p.o. did not produce liver damage. In contrast, exposure to HD resulted in time- and dose-dependent decreases in plasma cholesterol as well as lipid accumulation leading to liver damage. Hepatic gene expression profiles were determined after administration of HD or AcHD (100 mg/kg, p.o.). The expression of genes involved in lipid synthesis, transport and metabolism, as well as genes associated with necrosis were altered by HD In contrast, AcHD produced fewer changes and did not result in the differential expression of genes involved in lipid accumulation or necrosis. Several of the genes changed by HD exposure are regulated by PPARalpha. The involvement of PPARalpha in HD-mediated steatosis was investigated in PPARalpha-deficient mice. Administration of HD to these mice induced greater hepatic lipid accumulation and macrovesicular degeneration than did its administration to wild-type mice. This is consistent with the role of PPARalpha in removing lipids from liver. The pathology and the microarray data suggest that the PPARalpha-deficient mice are less capable of meeting the demands of HD-mediated increases in hepatic lipid presentation. In the wild-type animals, PPARalpha is activated in response to HD-induced increases in fatty acids. Consequently, these mice are better able to compensate for the lipid accumulation. This is the first demonstration of a critical role for PPARalpha in response to HD-induced steatosis.