Addition of DNA to Cr^VI and Cytochrome b₅ Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and Cr^III Complexes

Abstract

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 ), and the generation of Cr V , superoxide , 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, , and plasmid DNA, intense EPR signals at g = 5.7and 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.

Role of Protein–Protein Interactions in Cytochrome P450-Mediated Drug Metabolism and Toxicity

“…108 Indeed, using a reconstituted system containing both lipid and CPR, b5 has been shown to generate superoxide and hydroxyl radicals in the presence of Cr(VI), leading to double-stranded DNA breaks in vitro. 109,110 The fact that CPR was needed to observe the effect suggests the importance of the interaction between b5 and CPR to generate these ROS. Additionally, ROS generated by b5 have been demonstrated to increase lipid peroxidation in reconstituted biological membranes, which may in turn lead to cellular damage and death.…”

Quinic acid and hypervalent chromium: a spectroscopic and kinetic study

“…9A. Using these experimental data and eqn (12), the bimolecular rate constant k 5H could be calculated. A quadratic dependence was revealed by plotting k 5H vs. [H + ] (Fig.…”

“…1 Although it is not doubted that Cr VI induces cancer, [2][3][4][5][6][7][8] there is still a discussion regarding the species most probably responsible for cell damage and the mechanism(s) involved. [9][10][11][12] Cr VI itself cannot react with DNA in vitro or with isolated nuclei. On the other hand, when reducing agents are present in the medium, it causes an extensive diversity of DNA damage, which includes damage to Cr-DNA complex, DNA-protein crosslinks, and apurinic-apyrimidinic sites as well as oxidative damage.…”