Reduction of chromium(vi) to chromium(v) by rat liver cytosolic and microsomal fractions: Is dt-diaphorase involved?

Abstract

Incubation of rat liver cytosolic or microsomal fractions with chromium(VI) led to a dramatic decrease in chromlum(VI) mutagenicity, as determined by the Ames Salmonella assay using the TA100 tester strain. The cytosol-dependent decrease in chromium(VI) mutagenicity was found to be counteracted in the presence of dicumarol, an inhibitor of the cytosolic enzyme NAD(P)H:quinone oxidoreductase (DT-diaphorase). In order to determine whether DT-diaphorase is a significant factor in enzymatic reduction of chromium(VI) in rat liver tissue, cytosolic and microsomal fractions were analyzed for NAD(P)H-dependent chromium(VI) reductase activity leading to chromium(V) formation by using electron paramagnetic resonance (EPR) spectroscopy. Reaction of chromlum(VI) with NADH or NADPH in the presence of either cytosolic or microsomal fractions led to the formation of stable chromium(V)-NAD(P)H complexes. When glucose 6-phosphate (G6P) was present in the reaction as part of a NADPH-generating system, stable chromium(V)-G6P complexes were formed in addition to the chromium(V)-NAD(P)H complexes. The chromium(V) complexes had g values of 1.980-1.982 and superhyperfine splitting constants of 0.8-0.9 characteristic of bis(diol)oxochromium(V) complexes. Inhibition of 90% of the cytosolic DT-diaphorase activity by dicumarol led to only partial (20-22%) inhibition of chromium(V) formation. Visible and EPR spectroscopic studies showed that purified DT-diaphorase had no detectable chromlum(VI) reductase activity and did not catalyze formation of chromium(V). Inhibition of 69% of microsomal aryl hydrocarbon hydroxylase activity by ketoconazole led to partial (10%) inhibition of chromluin(V) formation. These results indicate that intracellular NAD(P)H-dependent enzymatic reduction of chromium(VI) in rat liver cannot be attributed to the activity of any one enzyme in the cytosolic or microsomal fractions. DT-dlaphorase appears to play an indirect role in decreasing chromium(VI}-induced mutagenicity in Salmonella, possibly through interaction with other redox active cellular components. The involvement of diols such as sugars and pyridlne nucleotides in stabilizing intracellularly generated chromium(V) is discussed. © 1992 Oxford University Press.