Many hazardous waste sites contain complex mixtures of halogenated aromatic hydrocarbons (HAHs) of the class that includes the polychlorinated- and polybrominated-biphenyls and polychlorinated dibenzo-p-dioxins and dibenzofurans. These persistent, toxic compounds can migrate off-site through surface and ground water flow and once in the surrounding environment they can be readily bioaccumulated and biomagnified. Although exposure to specific HAHs can result in a wide variety of species-specific toxic and biological effects at low concentrations, the induction of cytochrome P450IA1 by HAHs is one response that is highly conserved across species. Induction of P450IA1 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 accumulate within the nucleus and activate gene transcription through an interaction with specific DNA sequences (dioxin responsive enhancers) upstream of the P450IA1 gene. Studies of structure-activity relationship also implicate the AhR in mediating the toxicity of HAHs. Thus, many, if not all, of the toxic and biological responses to HAHs are mediated by the AhR. The overall goal of this proposal is to use several aspects of this mechanism to develop bioassay/biomarker systems for the detection of HAHs. We will stably transfect an HAH-inducible expression vector, which contains an alkaline phosphatase or luciferase reporter gene, into HAH-responsive human, rat and fish cells. Exposure of bioactive HAHs to these cells will induce expression of the reporter gene to a level proportional to the HAH dose. This bioassay system will be characterized, calibrated and validated using known HAH standards, HAH mixtures and unknown sample extracts containing complex mixtures of HAHs. Gel retardation analysis will be used to develop a sensitive assay system for quantitating the relative amount of HAH-bound ahR complex in peripheral lymphocytes of animals exposed to HAHs in vivo. We will also examine the molecular mechanism of action of HAHs in fishes in order to determine if HAHs act in a manner analogous to that described in mammalian systems. In this way we can evaluate the utility of fishes as potential sentinel species for monitoring for HAH contamination and its relationship to potential adverse effects in human populations. The ability of these bioassay/biomarker systems to accurately predict the TCDD-TEQs of complex HAH mixtures present in sample extracts from various biotic and abiotic matrices will be evaluated by direct comparison to the absolute 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 will provide new avenues for examining the effects of bioactive HAHs in man and animals.
Showing the most recent 10 out of 417 publications
