Tumor necrosis factor receptor 1 (TNFR1) activation in hepatocytes can trigger apoptotic or inflammatory signaling. The factors that determine which signaling pathway dominates are not clear and are thought to relate to the efficiency of death-inducing signaling complex (DISC) formation. However, the steps involved in DISC formation in hepatocytes are poorly understood. In characterizing DISC formation within cultured hepatocytes, we demonstrated that TNF-␣ exposure leads to the rapid formation of a DISC involving TNF-␣, the TNFR-associated death domain adaptor molecule (TRADD), the Fas-associated death domain adaptor molecule (FADD), caspase-8, TNFR-associated factor 2 (TRAF2), and receptor-interacting protein (RIP). The inclusion of the sensitizing agent actinomycin D both accelerated and amplified the appearance of the DISC. Notably, TNFR1 along with some DISC components also appeared within mitochondria within 30 minutes. Whereas TNFR1 consistently co-localized with the TRADD, FADD, the caspase-8, and TRAF2 in the cytosolic fraction, TNFR1 in the mitochondria was associated only with caspase-8 after TNF-␣ exposure. Similar observations were made in vivo using TNF-␣ with D-galactosamine. Actinomycin D alone also enhanced the appearance of DISC components in both cytosol and the mitochondria. Thus the DISC that includes TNFR1 forms in the cytosol of hepatocytes under both survival and pro-apoptotic conditions. The observations also suggest that TNF-␣-mediated signaling includes the translocation of TNFR1 to mitochondria.
The present study was done to determine the effect of trolox C, a hydrophilic analogue of vitamin E, on hepatic injury, especially the alteration in cytochrome P-450 (CYP)-dependent drug metabolism during ischemia and reperfusion (I/R). Rats were subjected to 60 min of hepatic ischemia and 5 h of reperfusion. Rats were treated intravenously with trolox C (2.5 mg/kg) or vehide (PBS, pH 7.4), 5 min before reperfusion. Serum alanine aminotransferase and lipid peroxidation levels were markedly increased after I/R. This increase was significantly suppressed by trolox C. Cytochrome P-450 content was decreased after I/R but was restored by trolox C. There were no significant differences in ethoxyresorufin O-dealkylase (CYP 1A1) and methoxyresorufin O-dealkylase (CYP 1A2) activities among any of the experimental groups. Pentoxyresorufin O-dealkylase (CYP 2B1) activity was decreased and aniline p-hydroxylase (CYP 2E1) activity was increased after I/R. Both these changes were prevented by trolox C. Our findings suggest that trolox C reduces hepatocellular damage as indicated by abnormalities in microsomal drug-metabolizing function during I/R, and that this protection is, in part, caused by decreased lipid peroxidation.
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