Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes.
Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.
Borthiry
Griselda R
GR
Department of Pharmacology and Toxicology.
Antholine
William E
WE
Department of Biophysics.
Myers
Judith M
JM
Department of Pharmacology and Toxicology.
Myers
Charles R
CR
Department of Pharmacology and Toxicology.|Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA (phone: +1-414-456 8593; fax: +1-414-456 6545).
D017608:Chromium Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D015786:Cytochromes b5 / Q000737:chemistry*; D053903:DNA Breaks, Double-Stranded; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D017665:Hydroxyl Radical / Q000138:chemical synthesis / Q000737:chemistry; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D010084:Oxidation-Reduction; D011510:Proteolipids / Q000737:chemistry*; D013481:Superoxides / Q000138:chemical synthesis / Q000737:chemistry; D013997:Time Factors
D017608:Chromium Compounds; D004269:DNA, Bacterial; D011510:Proteolipids; C020485:proteoliposomes; D013481:Superoxides; D017665:Hydroxyl Radical; D015786:Cytochromes b5; D009251:NADPH-Ferrihemoprotein Reductase