The role of human cytochrome b(5) in microsomal chromium(VI) reduction and resulting DNA damage

The reduction of Cr(VI) by cellular reductants to its lower oxidation states, Cr(V), Cr(IV), and Cr(III), is considered to be an important step in the toxicity and carcinogenicity of Cr(VI) compounds (3). These reactive Cr intermediates could directly mediate the DNA damage associated with Cr(VI) or they could generate various carbon-based radicals (4) or reactive oxygen species (ROS) by Fenton-type reactions (5–8).; The goals of these studies were to determine if human cytochrome b₅ could account for the NADPH- and NADH-dependent reduction of Cr(VI) observed in human liver microsomes.; Proteoliposomes containing recombinant NADPH:P450 reductase and human cytochrome b₅ were made. Using these proteoliposomes, it was demonstrated that cytochrome b5 in combination with NADPH:P450 reductase could account for the NADPH-dependent Cr(VI) reduction rates observed in human liver microsomes. Proteoliposomes containing only NADPH:P450 reductase or cytochrome b₅ exhibited poor Cr(VI) reducing capabilities. In addition, both ferric chloride (FeCl₃) and ferric adenosine-5 ′-diphosphate (FeADP) were shown to stimulate Cr(VI) reduction; this stimulation could be abolished by the addition of deferoxamine (DFX), a specific Fe(III) chelator. The rates of NADPH-dependent reduction of various ferric complexes by proteoliposomes were sufficient to account for the increased Cr(VI) reduction rates seen with the addition of FeCl₃ or FeADP. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy as a transient intermediate formed{09}during the NADPH-dependent Cr(VI) reduction mediated by proteoliposomes containing cytochrome b ₅ and NADPH:P450 reductase. Cr(IV) was also detected by EPR using an indirect measurement. Using the EPR spin trap 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), carbon-based radicals and, in the presence of oxygen, hydroxyl radicals (·OH) were also detected during the NADPH-dependent reduction of Cr(VI) by cytochrome b₅. Finally, the reductive metabolism of Cr(VI) to Cr(III) by cytochrome b₅, via reactive intermediates, was shown to produce a variety of DNA lesions such as single-strand breaks and DNA base modifications including 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-hydroxy-2′-deoxyadenosine (8-OHdA). (Abstract shortened by UMI.)...