MECHANISMS OF ARYL HYDROCARBON INDUCED OVOTOXICITY

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Data from epidemiological and animal studies suggest that polycyclic aromatic hydrocarbons (PAHs) cause ovarian follicular degeneration; however, the mechanisms by which PAHs destroy female germ cells are unknown. Recent studies have demonstrated the existence of an intracellular binding protein for PAHs termed the aryl hydrocarbon receptor complex (AHRC). Since the AHRC functions as a transcriptional regulator of genes following ligand binding, the actions of PAHs may be directly mediated via altered gene expression. Additionally, PAHs are metabolized by many cells including those of the ovary, to reactive intermediates that can disrupt the oxidative state of the cell. As such, PAHs may also cause follicular destruction indirectly by pathways associated with oxidative stress. Recent data support a fundamental role for apoptosis, and proteins encoded by several recently characterized "cell death genes," in the normal attrition of ovarian germ cells and follicles. Of particular interest is the gene, bax, which encodes a death-susceptibility factor that appears to be required for ovarian cells to die. The bax gene promoter contains transcriptional response elements activated by oxidative stress, as well as a consensus aryl hydrocarbon response element (AHRE), suggesting that PAHs can induce bax expression by both direct (AHRE) and indirect (oxidative stress) mechanisms. Therefore, it is hypothesized that PAH-induced germ cell loss is mediated via apoptosis, and that this apoptosis occurs via activation of direct (PAH-AHRC acting via the AHRE) and indirect (PAH metabolism and oxidative strees) pathways leading to enhanced bax expression. To address this hypothesis, the following specific aims are proposed: 1) to characterize the dose- and time-dependent effects of PAHs on apoptosis in germ cells and follicular granulosa cells of fetal and post-natal mouse ovaries; 2) to determine if bax gene expression is increased in germ cells and granulosa cells exposed to PAHs, and if this response and the induction of apoptosis are dependent upon PAH-AHRC interaction; 3) to evaluate if oxidative stress resulting from PAH metabolism is involved in PAH-induced ovotoxicity; 4) to use a BAX loss-of-function mouse model to determine if functional BAX protein is required for the ovotoxic effects of PAHs; and 5) to evaluate if overexpression of BAX antagonist (the product of the bcl-2 survival gene) in mouse oocytes conveys protection from PAH-induced apoptosis. Using state-of-the-art molecular biological approaches, the long-range goal of these studies is to decipher the intracellular effector pathways that mediate the deleterious actions of PAHs in ovarian germ cells (oocytes) and granulosa cells as a means to identify novel sites of intervention for precenting toxicant-induced tissue damage.