Polarity of hepatic glutathione and glutathione S-conjugate efflux, and intraorgan mercapturic acid formation in the skate

Abstract

Mechanisms of hepatic glutathione and glutathione S-conjugate efflux were investigated in isolated hepatocytes and perfused liver of the little skate ( Raja erinacea). Glutathione was released by isolated skate hepatocytes at a rate of 0.12 ± 0.03 nmol · hr −1 · (mg protein) −1. In the perfused liver, glutathione concentrations in bile were high (∼0.7mM) compared to hepatic tissue levels (0.61 ± 0.11 μmol · g −1). During the first hour of perfusion, the biliary glutathione excretion rate was 3 nmol · hr −1 · (g liver) −1, whereas glutathione accumulated in the recirculating perfusate at a rate of only 1.5nmol · hr −1 · (g liver) −1. Release of glutathione by isolated hepatocytes and perfused liver was not affected by the addition of acivicin, an inhibitor of γ-glutamyltransferase (EC 2.3.2.2), to cell suspension medium or liver perfusate. 1-Chloro-2,4-dinitrobenzene (CDNB) was taken up by isolated hepatocytes, conjugated to glutathione, and released as 5-(2,4-dinitrophenyl) (DNP)-glutathione. After infusion of 0.5 μmol CDNB in perfused liver, S-DNP-glutathione was concentrated in bile (0.5 mM) and was associated with choleresis. S-DNP-Conjugates of cysteinylglycine, cysteine and N-acetylcysteine, were also found in bile, suggesting intrahepatic breakdown of S-DNP-glutathione and subsequent acetylation of the resulting cysteine conjugate to form the mercapturic acid, S-DNP- N-acetylcysteine. This mercapturic acid accounted for 31% of the total S-DNP-conjugates collected in bile. In contrast, neither S-DNP-glutathione nor other S-DNP-conjugates were detected in the perfusate (< 0.5 μM). These findings demonstrate that biliary excretion is the predominant route for efflux of glutathione and a glutathione S-conjugate from skate liver. The results also identify an intrahepatic pathway for mercapturic acid biosynthesis facilitated by biliary glutathione S-conjugate excretion.