Retrieval of the mrp2 gene encoded conjugate export pump from the canalicular membrane contributes to cholestasis induced by tert-butyl hydroperoxideand chloro-dinitrobenzene

Oxidative stress is known to induce cholestasis, but the underlying mechani sms are poorly understood. In this study we have characterized the short-te rm effects of tert-butyl hydroperoxide (t-BOOH)- and 1-chloro-2,4-dinitrobe nzene (CDNB) on the mrp2 gene encoded canalicular export pump (Mrp2). The e ffects of t-BOOH and CDNB on bile formation, tissue GSH levels and subcellu lar Mrp2 localization were studied in perfused rat liver. Both, t-BOOH (0.5 mM) and CDNB (0.1 mM) induced within 60 min a decrease of hepatic GSH leve ls by more than 90% and an almost complete cessation of bile flow. As revea led by confocal laser scanning microscopy, this cholestasis was accompanied by a loss of immunoreactive MRP2 from the canalicular membrane and its app earance inside the hepatocytes in putative intracellular vesicles. On the o ther hand, the intracellular distribution of dipeptidyl peptidase IV (DPPIV ), another canalicular protein, and of zonula occludens associated polypept ide (ZO-1) remained unaffected, indicating selectivity of the Mrp2 retrieva l pattern. Both, t-BOOH and CDNB induced a rapid net K+ efflux from the liv er and a significant decrease of liver cell hydration. We conclude that sev ere glutathione depletion induces cholestasis by a retrieval of Mrp2, but n ot of DPPIV from the canalicular membrane. The underlying mechanism is uncl ear; however, a decrease in liver cell hydration, which occurs under these conditions, may contribute to this effect.