IMMUNOCHEMICAL DETECTION OF HEPATIC COCAINE-PROTEIN ADDUCTS IN MICE

Cocaine is capable of producing hepatic necrosis in laboratory animals and humans. Studies in mice indicate that N-oxidative metabolism of c ocaine is required for hepatotoxicity and have suggested that toxicity may result from the adduct;ion of proteins by cocaine-reactive metabo lites. To aid in identifying protein targets for cocaine-reactive meta bolites, an antibody was raised in rabbits immunized with cocaine link ed via the tropane nitrogen to a carrier protein (bovine serum albumin ). Hepatic proteins from cocaine-treated mice (ICR males, 50 mg of coc aine/kg of body weight, ip) and saline-treated controls were prepared from whole liver homogenate or following subcellular fractionation, an d Western blot analyses of hepatic proteins using this antibody were c onducted following one-and two-dimensional SDS-PAGE. Analysis of liver homogenate from cocaine-treated mice revealed major protein targets w ith approximate molecular masses of 20 kDa (pI = 6.0), 44 kDa (two pro teins with pI's of 5.0 and 7.0), 52-54 kDa (pI = 4.5), and 64 kDa (pi = 5.5). These specific protein targets were shown to be localized in t he mitochondria and microsomes. Several minor bands of immunoreactivit y were also seen in mice treated with cocaine, but not in saline-treat ed controls. Pretreatment of mice with the P450 inhibitor SKF 525A dim inished or eliminated the formation of these cocaine-protein adducts. Liver sections from cocaine-treated mice immunostained using the antib ody indicated the presence of cocaine-adducted proteins in the centril obular and midzonal regions of the lobule, corresponding to areas of h epatocyte swelling and necrosis. This study indicates that reactive me tabolites from cocaine bind to discrete proteins in specific regions o f the liver, consistent with a role for protein adduction in cocaine h epatotoxicity.