Mechanisms of Aryl-Hydrocarbon-Induced Ovotoxicity

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DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAH) are released into the environment by fossil fuel combustion. Another primary route of human exposure to PAH is cigarette smoke. Evidence that PAH cause premature ovarian failure has been provided by both epidemiological and animal studies. However, the mechanisms by which PAH damage the ovary have remained obscure for decades. These chemicals are particularly intriguing as there exists an intracellular binding protein for PAN termed the aryl hydrocarbon receptor (AHR). The AHR is a basic helix-loop-helix transcription factor that regulates gene expression following ligand interaction and nuclear translocation. During the previous funding cycle of this grant, the P1 completed experiments to validate the central hypothesis that PAN-mediated activation of the AHR in oocytes induces apoptosis by increasing expression of the gene encoding Bax, a pro-apoptotic member of the bcl-2 gene family. However, several new questions arose during these studies. First, does the AHR function to set the "apoptosis susceptibility" of female germ cells by transcriptional regulation of cell death regulatory genes in addition to bax? Second, is the inability of dioxin, a known AHR ligand, to damage the ovary related to differences in AHR response element (AHRE) flanking sequences in "target" cell death regulatory genes (e.g., bax) that convey specificity for PAH-AHR versus dioxin-AHR interactions? Third, does the induction of bax gene expression in oocytes require an AHR-interacting protein(s) that permits AHR-bax gene promoter interaction? Based on preliminary data presented herein, the PT has hypothesized that PAH-AHR. but not dioxin-AHR, interaction transcriptionally sets the cell death susceptibility "rheostat" in female germ cells to favor apoptosis. This is accomplished via a coactivator(s) expressed in oocytes that facilitates functional interaction between the PAN-activated AHR and cell death regulatory gene promoters possessing AHRE. To test this hypothesis, the following Specific Aims are proposed for continuation of these studies: 1) to determine if expression of the pro-apoptotic bak gene is increased in oocytes exposed to PAN, if PAN-AHR interaction is required for increased transcriptional activity of the bak gene, and if expression of the endogenous bak gene is required for the ovotoxic effects of PAN; 2) to examine if the expression patterns of a number of key apoptosis regulatory genes, known to be expressed in oocytes, are altered by AHR deficiency and/or by PAH-driven AHR activation; 3) to produce a gene expression profile for AHR initiated cell death signaling in oocytes using a microscale gene array technology recently developed for small biological samples; 4) to delineate the role of nucleotide sequences flanking the "core" five-nucleotide AHRE in the bax gene promoter in specifying responses to the AHR activated by PAN versus dioxin; and, 5) to identify AHR-interacting proteins in oocytes, using matrix-assisted laser desorption and ionization/time-of-flight mass spectrometry (MALDI/TOF-MS), that may be involved in specifying cell lineage-selective induction of bax gene transcription by the PAN-activated AHR.