Annual Meeting of the North American Society for Pediatric Gastroenterology and Nutrition; Orlando, October 22-24, 1998
OXIDANT STRESS, APOPTOSIS AND NECROSIS IN ISOLATED RAT HEPATOCYTES EXPOSED TO BILE ACIDS
Hepatocytes undergo apoptosis and necrosis in response to toxic bile acids. Bile acids cause the generation of oxidant stress in hepatocytes, however, the role of oxidant stress in the induction of apoptosis by bile acids is unknown. The objective of this study were to determine the apoptosis response of isolated hepatocytes to hydrophobic and hydrophilic bile acids and its relationship to the generation of oxidant stress. Freshly isolated adult rat hepatocytes were incubated in suspension in Krebs-Henseleit buffer containing 0.2%BSA, and then graded concentrations (0-500µM) of a hydrophobic bile acid, glycohenodeoxycholic acid (GCDC), or a hydrophilic bile acid, glycocholic acid (GCA), were added to the incubations for 4 hrs. Cells were assayed hourly for the generation of intracellular hydroperoxides by fluorescence of dichlorofluorescin diacetate (DCF-DA), apoptosis (fluorescent microscopic changes of nuclei by DAPI fluorescent staining), and necrosis by LDH release. Within 15 min. exposure of cells to 25-500 µM GCDC, significant generation of hydroperoxides could be detected compared to control cells. DCF-DA fluorescence continued to increase linearly for 4 hours, even at only 25 µM GCDC. Apoptosis was induced at all concentrations of GCDC, starting at 1 hr and in a concentration-dependent fashion. The hydrophilic GCA led to smaller, but significant, increases in DCF-DA fluorescence and apoptosis at concentrations of 100-500 µM and increasing over the 4 hrs. For both bile acids, the percent apoptotic cells correlated strongly with the values for DCF-DA fluorescence (at 2 hrs, r=0.70 and p<0.001; at 4 hrs, r=0.77, p<0.001) LDH release increased over time for GCDC concentrations of 200-500 µM and correlated very strongly with the severity of apoptosis (r=0.91, p<0.001). GCA-induced LDH release was 50% of that induced by GCDC at 200-500 µM. Conclusions: Hepatocytes generate oxidant stress within minutes when exposed to very low levels of hydrophobic and higher levels of hydrophilic bile acids, which appears related to the subsequent development of apoptosis and LDH release. These data support a role for oxidant stress and apoptosis in cholestasis.
PLENARY SESSION III© 1998 Lippincott Williams & Wilkins, Inc.