CHARACTERIZATION OF NOVEL BUTADIENE-INDUCED PUTATIVE p53 TARGET GENES

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DESCRIPTION (provided by applicant): Butadiene (BD) is a high production volume chemical that is currently regulated as a hazardous air pollutant. This environmental chemical is a known mutagen and human carcinogen, and possesses a wide range of hematopoietic, reproductive, and neurological toxicity, which includes bone marrow depletion, ovarian, spleen and thymus atrophy. Although the toxic effects of BD are known to be mediated through its active metabolites, the cellular and molecular mechanisms responsible for BD's toxic effects are not completely understood. Elucidation of the molecular mechanisms responsible for BD toxicity is a necessary pre-requisite to the development of therapeutic and intervention protocols for the management of BD exposure, and this is the long-term goal of this application. Butadiene's most potent active metabolite, diepoxybutane (DEB), has previously been reported to induce p53-regulated apoptosis in human lymphocytes. Although the tumor suppressor p53 protein is a multifunctional transcription factor that mediates apoptosis in response to various forms of stress, the mechanisms of p53 action and p53 signaling are known to be cell type and damage specific. Efforts towards the understanding of the mechanisms of p53 action in DEB-exposed lymphocytes have led to the discovery of novel DEB-induced putative p53-target genes. The objective of this proposal, therefore, is to determine how p53 regulates the expression of these novel DEB-induced putative p53-target genes, and determine the functional aspects of this regulation in DEB-exposed human lymphocytes. The central hypothesis to be tested is that the novel DEB-induced putative p53-target genes are regulated by p53 at the transcriptional and/or post-transcriptional level to signal and/or execute DEB-induced p53-dependent apoptosis in exposed lymphocytes. This project will utilize high-throughput approaches involving small interfering RNA, chromatin immunoprecipitation-quantitative PCR assays, reporter gene assays, polymerase chain reaction arrays, primary lymphocytes, as well as genetically-paired p53-deficient and proficient cell lines to test the central hypothesis through two specific aims: (1). Determine how p53 regulates the expression of the novel DEB-induced putative p53-target genes in DEB-exposed human lymphocytes. (2). Determine the role of the novel DEB-induced putative p53-target genes in the cellular and molecular toxicity of DEB within exposed human lymphocytes. At the completion of these studies, our expectation is that novel DEB-induced direct and/or indirect p53 targets will be identified, and their functions in DEB-exposed cells will be deduced. Collectively, this information is important because it will lead to a better understanding, of not only the toxicity of butadiene at the cellular and molecular level, but also the role of p53 in that process. This knowledge will have an important vertical impact on butadiene risk assessment as well as better means of therapeutic intervention after butadiene exposure and carcinogenesis. PUBLIC HEALTH RELEVANCE: The research proposed in this application is important because it will lead to a better understanding on novel mechanisms of p53 signaling and action as they occur in lymphocytes that have been exposed to the environmental toxicant butadiene, a mutagen and human carcinogen. This knowledge will have an important vertical impact on butadiene risk assessment, as well as on the design of better means of therapeutic intervention after butadiene exposure.