Several clinical reports have indicated that some cocaine users experience severe, even fatal, liver damage. The factors that create risk of liver injury from cocaine are unknown. The broad objective of this research is to develop an understanding of the factors and circumstances that result in susceptibility to cocaine hepatotoxicity. Several studies have shown that cocaine is bioactivated to a reactive metabolite in liver, and the formation of this reactive metabolite is highly correlated with toxicity. While this reactive metabolite has been shown to bind to protein irreversibly, the cellular and molecular protein targets for this reactive metabolite have not been identified, nor have functional consequences of protein adduction been determined. Preliminary studies in mice indicate that mitochondria are a preferred target for reactive metabolite binding within the hepatocyte, suggesting that cytotoxicity may result from an early impairment of mitochondrial function. Given the instability of the postulated reactive metabolite for cocaine, localized binding in the mitochondrial further suggests that this organelle may be the site of the terminal oxidation of cocaine to the reactive metabolite. Increased generation of reactive oxygen species, a plausible consequences of mitochondrial damage has been suggested in previous studies as having a role in cocaine hepatotoxicity. The hypothesis to be tested is that cocaine (or more probably, its nitroxide metabolite) is bioactivated in mitochondria to a reactive metabolite which binds to proteins, disrupting mitochondrial function leading to excessive reactive oxygen species generation. The following specific aims will be used to test this hypothesis; 1) Determine the distribution of intracellular binding of cocaine reactive metabolite over time, in mice; 2) Determine whether incubation of isolated mitochondrial with cocaine or its oxidative metabolites results in formation of a reactive metabolite which binds to proteins; 3) Isolated and identify adducted mitochondrial proteins; and 4) Determine the effect of cocaine and oxidative metabolites on mitochondrial reactive oxygen species release. Intracellular distribution experiments will be conducted using immunohistochemistry coupled with electron microscopy, as well as radiometric techniques involving liver tissue subfractions. The formation of reactive metabolites by isolated mitochondria will be evaluated following incubation with radiolabled cocaine and n-oxidative metabolites. Mitochondrial proteins from cocaine- treated mice will be separated by 2D polyacrylamide gel electrophoresis and adducted proteins will be detected by fluorography and antibody binding. Attempts will be made to identify the adducted proteins through N--terminal amino acid sequencing, mass spectrometry, and/or internal amino acid sequencing. The ability of cocaine and n-oxidative metabolites to stimulate reactive oxygen species release from mitochondrial will be examined directly during in vivo and in vitro experiments. The proposed research will link cocaine bioactivation to specific target proteins and cellular responses, creating an improved understanding of the mechanism of cocaine hepatotoxicity. This understanding will be critical in the identification of potential factors that increase the likelihood of liver damage as consequence of cocaine abuse.
