ANIT-induced disruption of biliary function in rat hepatocyte couplets

alpha-Naphthylisothiocyanate (ANIT) induces intrahepatic cholestasis in rat s, involving damage to biliary epithdial cells; our study aims to investiga te whether disruption of biliary function in hepatocytes can contribute to early stages of ANIT-induced intrahepatic cholestasis. Isolated rat hepatoc yte couplets were used to investigate biliary function in vitro by canalicu lar vacuolar accumulation (cVA) of a fluorescent bile acid analogue, cholyl -lysyl-fluorescein (CLF), within the canalicular vacuole between the two ce lls. After a 2-h exposure to ANIT, there was a concentration-dependent inhi bition of cVA (cVA-IC50, 25 mu M), but no cytotoxicity (LDH leakage or [ATP ] decline) within this ANIT concentration range. There was no loss of cellu lar [GSH] at low ANIT concentrations, but, at 50 mu M ANIT, a small but sig nificant loss of [GSH] had occurred. Diethylmaleate (DEM) partially deplete d cellular [GSH], but addition of 10 mu M ANIT had no further effect on GSH depletion. Reduction in cVA was seen in DEM-treated cells; addition of ANI T to these cells reduced cVA further, but the magnitude of this further red uction was no greater than that caused by ANIT alone, indicating that gluta thione depletion does not enhance the effect of ANIT. F-actin distribution (by phalloidin-FITC staining) showed an increased frequency of morphologica l change in the canalicular vacuoles but only a small, nonsignificant (0.05 < p < 0.1) increase in proportion of the F-actin in the region of the peri candicular web. The results are in accord with a disruption of hepatocyte c analicular secretion within two h in vitro, at low, non-cytotoxic concentra tions of ANIT, and the possible involvement of a thiocabamoyl-GSH conjugate of ANIT (GS-ANIT) in this effect.