Hepatobiliary effects of tertiary-butylhydroperoxide (tBOOH) in isolated rat hepatocyte couplets

The organic hydroperoxide, tertiary-butylhydroperoxide (tBOOH), causes oxidative damage in a number of cell types. It is used here in an isolated rat hepatocyte couplet preparation to study adverse hepatobiliary effects of peroxidative damage in vitro. At subcytotoxic concentrations (as determined by lactate dehydrogenase release and maintenance of cytoplasmic ATP concentrations) tBOOH caused decreased accumulation of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), in the canalicular vacuole of couplets (a hepatobiliary effect; cholestasis). This was dose dependent in the range 100-200 μM. At the same concentrations it brought about release of preaccumulated CLF, suggesting that its effect was more likely to be on sealing properties of the vacuole than processes of uptake, transcytosis, and secretion. Pretreatment of tBOOH-treated couplets with the antioxidants deferoxamine mesylate (iron chelator) and dimethyl sulfoxide (free radical scavenger) resulted in the prevention of both canalicular vacuolar accumulation (cVA, which assesses canalicular function) and canalicular vacuolar retention (cVR, which assesses the retaining ability of couplets) depression at 100 μm tBOOH but not at higher concentrations. This indicates that the cholestatic effect of tBOOH has a preventable and nonpreventable phase and that free radicals are involved in these processes. By selectively generating the two types of tBOOH radical, peroxyl (tBOO·) and alkoxyl (tBO·), using suitable catalysts, we were able to determine that the peroxyl radical was most probably involved in tBOOH-induced cholestasis. This was further supported by experiments employing specific peroxyl and alkoxyl radical scavengers; only the peroxyl scavenger reduced the effect of tBOOH upon canalicular function under the conditions studied.