Analysis of heat-labile sites generated by reactions of depleted uranium and ascorbate in plasmid DNA

Abstract

The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single-strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate, cisplatin, and chromic chloride were compared with those formed by reaction of uranyl acetate and ascorbate. Uranyl ion in the presence of ascorbate produced U-DNA adducts that converted to SSB on heating. Piperidine, which acted on DNA methylated by methyl methanesulfonate to convert methyl-DNA adducts to SSB, served in the opposite fashion as U-DNA adducts by decreasing the level of SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cisplatin and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect the formation of uranium-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from the presence of free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. The results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding the mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposure in humans.

Figure 1

Effects of heat and piperidine incubations on pBR322 plasmid DNA degradation after exposure to methyl methanesulfonate (MMS). (A) Representative gel illustrating reaction of MMS (0 – 10 mM) with pBR322 DNA (0.2 mM DNA-P, 25 mM ACES, pH 7.4, 37 °C, 30 min) followed by post-treatment exposure to water (30 min, RT) (lanes 1–4), water and heat (30 min, 60 °C) (lanes 5–8) or 30 μM piperidine and heat (30 min, 60 °C) (lanes 9–12). (B) Quantification of DNA degradation as % DNA migrating as Form II for post-treatment exposure to either water (30 min, RT) (open bars); water (30 min, 60 °C) (grey bars); or 30 μM piperidine (30 min, 60 °C) (black bars). Data represent mean ± SEM for n = 5 independent experiments. Statistical significance of the effect of ± heat or ± piperidine was determined by ANOVA (NS not significant, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 2

Effects of heat and piperidine incubations on pBR322 plasmid DNA gel shifts after exposure to cis-platin for 30 min or 24 h. (A) Representative gel illustrating reaction of cis-platin (0 – 8 μM) with pBR322 DNA (0.2 mM DNA-P, 25 mM ACES, pH 7.4, 37 °C, 30 min) followed by post-treatment exposure to water (30 min, RT) (lanes 1–4), water and heat (30 min, 60 °C) (lanes 5–8) or 30 μM piperidine and heat (30 min, 60 °C) (lanes 9–12). (B) Representative gel illustrating reaction of cis-platin (0 – 8 μM) with pBR322 DNA (0.2 mM DNA-P, 25 mM ACES, pH 7.4, 37 °C, 24 h) followed by post-treatment exposure to water (30 min, RT) (lanes 1–4), water and heat (30 min, 60 °C) (lanes 5–8) or 30 μM piperidine and heat (30 min, 60 °C) (lanes 9–12).

Figure 3

Effects of heat and piperidine incubations on pBR322 plasmid DNA gel shifts after exposure to chromic chloride for 24 h. Representative gel illustrating reaction of CrCl₃•6H₂O (0 – 300 μM) with pBR322 DNA (0.2 mM DNA-P, 25.0 mM ACES, pH 6.5, 37 °C, 24 hr) followed by post-treatment exposure to water (30 min, RT) (lanes 1–4), water and heat (30 min, 60 °C) (lanes 5–8) or 30 μM piperidine and heat (30 min, 60 °C) (lanes 9–12).

Figure 4

Effects of heat and piperidine post-treatment incubations on pBR322 plasmid DNA degradation measured as % DNA plasmid relaxation (Form II) after exposure to uranyl acetate and ascorbate. (A) Representative gel illustrating reactions of UA (0.50 mM) and ascorbate (0.50 mM) with pBR322 DNA (0.2 mM DNA-P, 25.0 mM ACES, pH 7.4, 37 °C, 30 min) followed by post-treatment exposure to either water (30 min, RT) (left); water (30 min, 60 °C) (center); or 30 μM piperidine (30 min, 60 °C) (right). (B) Quantification of DNA degradation as % DNA migrating as Form II for post-treatment exposure to either water (30 min, RT) (open bars); water (30 min, 60 °C) (grey bars); or 30 μM piperidine (30 min, 60 °C) (black bars). Data represent mean ± SEM for n = 4–14 independent experiments. Statistical significance of the effect of ± heat or ± piperidine was determined by ANOVA (NS not significant, *p<0.05, **p<0.01, ****p<0.0001).

Figure 5

Effect of mannitol and catalase on % DNA plasmid relaxation (Form II) in the absence and presence of piperidine. pBR322 DNA was reacted with UA and ascorbate as described in Figure 4. (A) For comparison purposes, the reactions from Figure 4B are shown as difference in % Form II DNA for [piperidine vs. no piperidine]. Reactions were also carried out in the added presence of: (B) 500 μM mannitol, followed by addition of water or 30 μM piperidine and incubation for 30 min at 60 °C. (C) 70 U/mL catalase, followed by addition of water and incubation for 30 min at 60 °C. (D) 500 μM mannitol, followed by addition of 30 μM piperidine and incubation for 30 min at 60 °C. (E) 70 U/mL catalase, followed by addition of 30 μM piperidine and incubation for 30 min at 60 °C. In all cases data represent mean ± SEM for differences in % Form II for [treatment vs. no treatment], for n = 5 independent experiments. Differences were significantly different than 0 by Student’s t-test (*p<0.05, **p<0.01, ****p<0.0001).

Scheme I

Proposed mechanism for conversion of a uranyl-DNA adduct to an AP site