Metal Ions Modify In Vitro DNA Damage Yields with High-LET Radiation

Abstract

Cu²⁺ and Co²⁺ are metals known to increase DNA damage in the presence of hydrogen peroxide through a Fenton-type reaction. We hypothesized that these metals could increase DNA damage following irradiations of increasing LET values as hydrogen peroxide is a product of the radiolysis of water. The reaction mixtures contain either double- or single-stranded DNA in solution with Cu²⁺ or Co²⁺ and were irradiated either with X-ray, carbon-ion or iron-ion beams, or they were treated with hydrogen peroxide or bleomycin at increasing radiation dosages or chemical concentrations. DNA damage was then assessed via gel electrophoresis followed with a band intensity analysis. DNA damage was the greatest when DNA in the solution with either metal was treated with only hydrogen peroxide followed by the DNA damage of DNA in the solution with either metal post irradiation of low-LET (X-Ray) or high-LET (carbon-ion and iron-ion), respectively, and demonstrated the least damage after treatment with bleomycin. Cu²⁺ portrayed greater DNA damage than Co²⁺ following all experimental conditions. The metals' effect caused more DNA damage and was observed to be LET-dependent for single-strand break formation but inversely dependent for double-strand break formation. These results suggest that Cu²⁺ is more efficient than Co²⁺ at inducing both DNA single-strand and double-strand breaks following all irradiations and chemical treatments.

Keywords: DNA breaks; DNA damage; Fenton reaction; carbon-ion radiation; high-LET radiation.

Figure 1

Cu²⁺ and Co²⁺ effect on DNA DSBs and SSBs at increasing radiation dosage (Gy) with radiation sources of increasing LET values. (a) Low-LET X-ray for DSBs; (b) high-LET carbon-ion for DSBs; (c) high-LET iron-ion for DSBs; (d) low-LET X-ray for SSBs; (e) high-LET carbon-ion for SSBs and (f) high-LET iron-ion for SSBs. Error bars indicate standard error of the means from at least three independent experiments. * indicates statistical differences (p < 0.05).

Figure 2

Cu²⁺ and Co²⁺ effect on DNA DSBs and SSBs at increasing drug concentrations of either Bleomycin or hydrogen peroxide. (a) Bleomycin for DSBs; (b) hydrogen peroxide for DSBs; (c) Bleomycin for SSBs and (d) hydrogen peroxide for SSBs. Error bars indicate standard error of the means from at least three independent experiments. * indicates statistical differences (p < 0.05).

Figure 3

Metal enhancement ratio (MER) for DNA break formation comparison between ionizing radiation and chemical treatment with metals in solution with DNA (a) dsDNA; (b) ssDNA.

Figure 4

Proposed mechanisms of metal enhancement of ionizing radiation, bleomycin and H₂O₂. Thickness of arrows associated with the degree of effects.