Production of intracellular 35S-glutathione by rat and human hepatocytes for the quantification of xenobiotic reactive intermediates

The quantification and identification of xenobiotic reactive intermediates is difficult in the absence of highly radiolabeled drug. We have developed a method for identifying these intermediates by measuring the formation of adducts to intracellularly generated radiolabeled glutathione (GSH). Freshly isolated adherent rat and human hepatocytes were incubated overnight in methionine and cystine-free ('thio-free') medium. They were then exposed to 100 muM methionine and 10 muCi S-35-labeled methionine in otherwise thio-free medium to replete Cellular GSH pools with intracellularly generated S-35-labeled GSH. After 3 h, acetaminophen was added as a test compound and the cells were incubated for an additional 24 h. Intracellular GSH and its specific activity were quantified after reaction with monobromobimane followed by HPLC analysis with fluorescence and radiochemical detection. Radiolabeled GSH as detectable at 3 h and maintained high specific activity and physiological concentrations for up to 24 h. Incubation medium from acetaminophen treated and nontreated hepatocytes were analyzed for radiolabeled peaks by HPLC using radiochemical detection, Radiolabeled peaks not present in nontreated hepatocytes were identified as acetaminophen GSH adducts by LC-MS. Formation of acetaminophen S-35-GSH adducts by rat hepatocytes containing endogenously synthesized S-35-GSH was increased with acetaminophen concentrations ranging from 500 to 2 mM. Published by Elsevier Science Ireland Ltd.