Approaches for detecting an exposure to a potentially toxic substance vary with the chemical under consideration and with the types of exposure (duration, level of exposure, route of exposure). Previously used biomarkers have varied in complexity from detection of the parent compound or a primary metabolite in the urine to detecting an adduct (mainly to hemoglobin or albumin) in blood. Urinary metabolites tend to be relatively easily measured but suffer from the standpoint that samples must be collected relatively soon after exposure. Hemoglobin adducts have the advantage of being long-lived and thus adducts tend to accumulate after continuous low level exposure. In both cases however, it is difficult to link the presence of a metabolite in the urine or an adduct in the blood stream with a specific toxicity. The work proposed in this application is to develop a biomarker(s) for a group of compounds that undergo metabolic activation to produce cell selective injury to pulmonary bronchiolar Clara cells. These compounds include naphthalene, nitronaphthalene, and, if time permits, several chlorinated ethylenes. These compounds, as well as close structural analogs, have been identified frequently in hazardous waste sites. The proposed work is based on the finding that naphthalene is metabolized to reactive intermediates that selectively arylate two proteins (15-16 kDa) in target cell populations and on preliminary data suggesting that arylation of these proteins may play a critical role in the cell injury that ensues after naphthalene administration. Development of a biomarker(s) that is tightly correlated with the intracellular targets for the toxicant, should allow discrimination of exposures that are above and below the threshold for toxicity. A variety of biochemical and morphologic approaches will be applied to this problem. A system for the isolation and short term culture of dissected pulmonary airways will be used to determine the formation of adducted macromolecules in target cell populations as well as the fate of the adducts. In all cases, quantitative morphometry will be used to assess toxicity and this will be related to adduct formation to carefully define the relationship between interaction at specific cellular targets and cell injury. Adducts will be identified by determining N-terminal sequences of the adducted protein and tryptic digests as well as from cDNA. The chemical nature of the adducts will be determined by tandem mass spectrometry and by in vitro incubations of the suspected proximate metabolites with protein target(s). The disposition kinetics of the adducted proteins will be assessed to determine appropriate sampling site (can adducts or primary decomposition products be detected in urine?, blood?) and to determine the half life of these products. Multiple route, multiple dose exposures in animals will be used to establish appropriate conditions for use of the biomarkers developed in the project.
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