Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated-dibenzo- p-dioxins (PCDDs), biphenyls (PCBs) and dibenzofurans (PCDFs), and related compounds represent a diverse group of widespread environmental contaminants, many of which are toxic and persistent in the environment. Exposure to and bioaccumulation of HAHs have been observed to produce a wide variety of species and tissue-specific toxic and biological effects, such as tumor promotion, lethality, birth defects, hepatotoxicity, immunotoxicity, dermal toxicity, alterations in endocrine homeostasis and induction of numerous enzymes (1,2). Although exposure to specific HAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD,dioxin), can produce these wide variety of effects at low concentrations, the induction of cytochrome P45O1A1 by HAHs is one response that is highly conserved across species and is used as the model system for analysis of the mechanism of action of HAHs. Induction of P45O1A1 is mediated by a soluble intracellular protein (the Ah receptor (AhR)) which binds the HAH specifically and with high affinity. After binding, HAH:AhR complexes are converted into their DNA binding form, they subsequently accumulate within the nucleus and activate gene transcription through a high affinity interaction with a specific DNA sequence (dioxin responsive enhancers) upstream of the P45O1A1 gene. In addition to mediating the induction of P45O1A1, structure-activity relationship studies have demonstrated a strong correlation between the ability of a chemical to bind to the AhR and its ability to produce toxicity, implicating the AhR in mediating the toxicity of HAHs. Thus, many, if not all, of the toxic and biological responses to HAHs currently appear to mediated by the AhR. The overall goal of this proposal is to use several mechanistic aspects of this AhR- dependent system to develop bioassay systems for the detection of HAHs. Given the high degree of correlation between the ability of a chemical to bind to the AhR, its ability to induce AhR transformation and DNA binding and gene expression, we will develop, optimize and utilize two AhR DNA binding assays (gel retardation analysis and a novel membrane filtration assay) as bioassays for detection of dioxin-like chemicals. In addition, we will stably transfect several HAH-inducible expression vectors which contains a luciferase or alkaline phosphatase reporter gene, into HAH- responsive human and mouse cell lines. Exposure of these recombinant cell lines to dioxin-like HAHs will result in the induction of expression of the reporter gene to a level proportional to the HAH dose. The DNA binding and gene expression bioassay systems will be characterized, calibrated and validated using known HAH standards, HAH mixtures and unknown sample extracts containing complex mixtures of HAHs. The ability of these bioassay/biomarker systems to accurately predict the TCDD-TBQs of complex HAH mixtures present in sample extracts from various biotic and abiotic matrices will be evaluated by direct comparison to the concentration of HAHs in these samples, as determined by instrumental analysis. These studies will not only produce several new sensitive bioassay/biomarker systems for detection and monitoring of HAHs but they will also be useful for toxicant identification evaluation and will provide new avenues for examining the effects of bioactive HAHs in man and animals.
NIH ExPorter
NIH ExPorter