HEAT SHOCK IN TOXICITY ASSAYS

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DESCRIPTION: (Adapted from the Investigator's Abstract) Experiments conducted in the previous period demonstrated that hepatotoxicant exposure induces heat shock proteins (Hsps) in mouse liver and human hepatocyte-derived HepG2 cells and provide correlative evidence that Hsp accumulation protects against toxicant damage. The objective of this proposal is to confirm that Hsps are protective and test mechanistic hypotheses that could explain why and how Hsps mitigate hepatotoxicant damage. The proposed experiments should also reveal which particular Hsp(s) produces the protective effect. Hypotheses to be tested include the possibilities that Hsp over-expression inhibits formation of reactive metabolites of toxicants, alters the protein targets adducted by reactive metabolites, promotes the maintenance and reacquisition of a native conformation of toxicant-damaged proteins, enhances proteolytic degradation of toxicant-damaged proteins, or prevents oxidation of non-protein thiols and/or thiol oxidation of proteins. The basic approach taken to over- or underexpress Hsps involves adenoviral delivery into HepG2 cells and into mouse liver of positive- and negative-dominant mutants of heat shock transcription factor 1 (HSF1). These HSF1 mutants are capable of drastically enhancing expression of all major Hsps in the absence of toxicant stress and to inhibit toxicant stress-induction of Hsp synthesis, respectively. To identify the Hsp(s) responsible for observed effects, experiments will be conducted using cells infected with adenoviral vehicles delivering Hsps instead of HSF1 mutants.