Diffusion of reactive metabolites out of hepatocytes: Studies with bromobenzene

We have developed a simple experimental technique which allows the determination of the relative rates of intracellular inactivation of chemically reactive metabolites and their diffusion out of isolated rat hepatocytes. By using bromobenzene as a model compound we have demonstrated that bromobenzene-3,4-oxide generated with hepatocytes is sufficiently stable to leave the endoplasmic reticulum in which it is formed, traverse the cytoplasm and cross the cell membrane to the external environment. The addition of varying amounts of protein, which serves as an external sink to trap the epoxide as a covalently bound adduct, permits the calculation of the relative rates at which the epoxide is inactivated within the cells and diffuses out of the cells. As much as 35% of bromobenzene-3,4-oxide is capable of leaving hepatocytes and being trapped as covalently bound adduct to glutathione (GSH)-transferase B. The extensive diffusion of bromobenzene-3,4-oxide may play an important role in the intercellular toxicity of this compound within the liver and perhaps may contribute to extrahepatic toxicity. The addition of GSH-transferase B to isolated hepatocyte suspension caused a decrease in the formation of the 3,4-dihydrodiol, p-bromophenol and o- and p-bromophenol glucuronides, an increase in the formation of bromobenzene GSH conjugates, but did not affect intracellular covalent binding. Kinetic analyses of the data revealed that, in the absence of GSH-transferase B, nearly all of the bromobenzenes GSH conjugates are formed within hepatocytes as the epoxide is formed, whereas rearrangement of bromobenzene-3,4-oxide to p-bromophenol and hydration to bromobenzene-3,4-dihydrodiol occurs almost exclusively outside the hepatocytes. The experimental technique and kinetic analyses described herein should be applicable to the study of the fate of many chemically reactive intermediates.