Retrieval of the mrp2 Gene Encoded Conjugate Export Pump from the Canalicular Membrane Contributes to Cholestasis Induced by tert-Butyl Hydroperoxide and Chloro-Dinitrobenzene

Abstract

Oxidative stress is known to induce cholestasis, but the underlying mechanisms are poorly understood. In this study we have characterized the short-term effects of tert-butyl hydroperoxide (t-BOOH)- and 1-chloro-2,4-dinitrobenzene (CDNB) on the mrp2 gene encoded canalicular export pump (Mrp2). The effects of t-BOOH and CDNB on bile formation, tissue GSH levels and subcellular 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 levels by more than 90% and an almost complete cessation of bile flow. As revealed by confocal laser scanning microscopy, this cholestasis was accompanied by a loss of immunoreactive MRP2 from the canalicular membrane and its appearance inside the hepatocytes in putative intracellular vesicles. On the other hand, the intracellular distribution of dipeptidyl peptidase IV (DPPIV), another canalicular protein, and of zonula occludens associated polypeptide (ZO-1) remained unaffected, indicating selectivity of the Mrp2 retrieval pattern. Both, t-BOOH and CDNB induced a rapid net K+ efflux from the liver and a significant decrease of liver cell hydration. We conclude that severe glutathione depletion induces cholestasis by a retrieval of Mrp2, but not of DPPIV from the canalicular membrane. The underlying mechanism is unclear; however, a decrease in liver cell hydration, which occurs under these conditions, may contribute to this effect.