Comparison of the Formation of 8-Hydroxy-2‘-deoxyguanosine and Single- and Double-Strand Breaks in DNA Mediated by Fenton Reactions

Abstract

The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single- and double-strand breaks in DNA by Fenton-type reactions has been investigated. Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and one of nine different transition-metal ions (25 microM-1 mM). Modified DNA was isolated and subjected to analysis by liquid chromatography coupled to an electrochemical detection system (LC-ECD), to evaluate the formation of 8-OHdG. The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III) (5800), and copper(II) (5200). The chromium(VI) Fenton reaction generated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the yield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in control incubations of DNA with hydrogen peroxide alone. Similar treatment of the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transition-metal ion (1-100 microM) followed by quantitative agarose gel electrophoresis demonstrated that open-circle DNA, resulting from single-strand breaks, was generated in Fenton reactions involving all nine metal ions. In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and vanadium(III) ions. Formation of linear DNA, under conditions that generated relatively few single-strand breaks, suggests that these four transition-metal ions partake in Fenton reactions to generate true double-strand breaks. Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting that they arise by a similar mechanism.

Oxidised guanidinohydantoin (Ghox) and spiroiminodihydantoin (Sp) are major products of iron- and copper-mediated 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine oxidation

“…2 Steady state levels of oxidatively generated damage are one or more orders of magnitude higher than those of non-oxidatively generated nucleobase adducts. 3 This result of so called "oxidatively generated stress" has been implicated in mutagenesis, disease 35 and aging. [4][5][6] One of the primary oxidatively generated stress biomarkers to have emerged in recent years is 8-oxoGua, a primary product of Guanine oxidation.…”

DNA-bound metal ions: recent developments

“…A prime example is Hg(II) ion and Hg(II) compounds, which disrupt the hydrogen bonds between naturally occurring A-T base pairs and form T-Hg 2+ -T pairs (8,9). Redox-active metal ions are receiving much attention because of their ubiquitous presence in biological systems and their abilities to react with H 2 O 2 and other endogenous oxidizing agents to produce damaging reactive oxygen species (ROS), and an important topic in metal toxicity and carcinogenesis is metal-mediated oxidative DNA damage (10)(11)(12)(13) (15)(16)(17)(18). The suggested mechanism of oxidative DNA damage arising from Cu(II) involves a process in which Cu(II) is bound to a G-containing moiety in DNA and reduced to Cu(I) (16,(19)(20)(21).…”

“…The structures of the mononucleoside 2′-deoxyguanosine (dG) and the accepted oxidative damage marker 8-OH-dG and its keto form 8-oxo-2′-deoxyguanosine (8-oxo-dG) are presented in Figure 2. The transition metal ions Fe(II), Cu(II), Cr(III), and V(III) produce significant levels of 8-OH-dG in the presence of H 2 O 2 (15), and the Cu(I)/H 2 O 2 system also yields significant 8-OH-dG levels (17). The reactive nature of radical species suggests that they are generated close to the site at which they react.…”

“…The reactive nature of radical species suggests that they are generated close to the site at which they react. In general, single-strand breaks are a form of generalized damage associated with ROS generated free in solution, and double-strand breaks are closely correlated with 8-OH-dG levels, suggesting that they arise from ROS generated in close proximity to the G base (15,18,39). Figure 3 illustrates ROS production and the resulting types of damage from metal ions free in solution and those bound to the phosphate backbone and/or individual bases of DNA.…”

Engaging Practicing Gynecologists in the Management of Infertile Men

“…Improvements in sperm motility, sperm DNA fragmentation, fertilization capacity, and odds of normal sperm count were observed in most studies [120][121][122][123], albeit, in a few of them, no advantage was documented [124,130]. A Cochrane metaanalysis on the use of oral antioxidants in male infertility found that these agents significantly improved pregnancy rates and live births and decreased sperm DNA damage [131].…”