| Abstrakt: |
Isolated human hepatocytes exhibited time-, trichloroethylene (Tri) concentration-, and cell concentration-dependent formation of S-(1, 2-dichlorovinyl)glutathione (DCVG) in incubations in sealed flasks with 25 to 10,000 ppm Tri in the headspace, corresponding to 0.011 to 4.4 mM in hepatocytes. Maximal formation of DCVG (22.5 +/- 8.3 nmol/120 min per 10(6) cells) occurred with 500 ppm Tri. Time-, protein concentration-, and both Tri and GSH concentration-dependent formation of DCVG were observed in liver and kidney subcellular fractions. Two kinetically distinct systems were observed in both cytosol and microsomes from pooled liver samples, whereas only one system was observed in subcellular fractions from pooled kidney samples. Liver cytosol exhibited apparent Km values (microM Tri) of 333 and 22.7 and Vmax values (nmol DCVG formed/min per mg protein) of 8.77 and 4.27; liver microsomes exhibited apparent Km values of 250 and 29.4 and Vmax values of 3.10 and 1.42; kidney cytosol and microsomes exhibited apparent Km values of 26.3 and 167, respectively, and Vmax values of 0.81 and 6.29, respectively. DCVG formation in samples of liver cytosol and microsomes from 20 individual donors exhibited a 6.5-fold variation in microsomes but only a 2.4-fold variation in cytosol. In coincubations of pooled liver cytosol and microsomes, addition of an NADPH-regenerating system produced marked inhibition of DCVG formation, but addition of GSH had no effect on cytochrome P-450-catalyzed formation of chloral hydrate. These results indicate that both human kidney and liver have significant capacity to catalyze DCVG formation, indicating that the initial step of the GSH-dependent pathway is not limiting in the formation of nephrotoxic and nephrocarcinogenic metabolites. |
