Karol Bialkowski

Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids.

Abstract A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.

Inter-laboratory validation of procedures for measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2 '-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CV of less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10 6 Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.

Recommendations for standardized description of and nomenclature concerning oxidatively damaged nucleobases in DNA.

We are very grateful to Dr. Gerry Moss, President, IUPAC Division VIII, for his useful comments in the drafting of this document. M.S.C., S.L., P.M., R.O., K.B., and M.D.E. are partners of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (Contract No 513943). J.C. is a member of EU Network COST Action CM0603 “Free Radical in Chemical Biology (CHEMBIO-RADICAL).

STUDIES ON OXIDATIVE MECHANISMS OF METAL-INDUCED CARCINOGENESIS

Two ways by which carcinogenic metals, such as Ni(II), Co(II), Cu(II), or Cd(II), may promote oxidative DNA damage, including direct effects consisting of activation of oxygen species and mediation of their attack on DNA, and indirect effects through suppression of cellular antimutagenic defenses, are discussed. The mechanisms of the direct attack may involve chelation of a metal by nuclear proteins, especially the histones and protamines, and activation of metabolic oxygen species by the resulting metal complexes at close proximity to DNA. We found that human protamine HP2 has a typical binding motif for Ni(II) and Cu(II), Arg-Thr-His-, at its N-terminus. A synthetic pentadecapeptide modeling this terminus formed strong chelates with these metals and, in addition, enhanced oxidative DNA damage by Ni(II) plus H2O2, but suppressed, though not completely, the damage by Cu(II) plus H2O2. Since protamines carry DNA in the sperm, the observed DNA damage may have spermicidal or transgenerational carcinogenic effects in man exposed to metals, as observed epidemiologically. The indirect effects of metals on DNA may involve inhibition of 8-oxo-dGTPases, a class of enzymes preventing incorporation of the 8-oxoguanine lesion from oxidatively-damaged deoxynucleotide pool into DNA. Cd(II) and Cu(II), and to a limited extent also Ni(II) and Co(II), were found to in vitro inhibit the enzymatic activity of a bacterial (MutT) and human (MTH1) 8-oxo dGTPases. This may allow redox-inactive metals, such as Cd(II), to introduce the promutagenic 8-oxoguanine lesion from endogenously damaged 8-oxo-dGTP into DNA.

Up-regulation of 8-oxo-dGTPase Activity of MTH1 Protein in the Brain, Testes and Kidneys of Mice Exposed to 137Cs γ Radiation

Abstract Mammalian MTH1 protein is an antimutagenic (2′-deoxy)ribonucleoside 5′-triphosphate pyrophosphohydrolase that prevents the incorporation of oxidatively modified nucleotides into nucleic acids. It decomposes most specifically the miscoding products of oxidative damage to purine nucleic acid precursors (e.g. 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP, 8-oxo-GTP) that may cause point mutations or transcription errors when incorporated into DNA and RNA, respectively. The increased expression of MTH1 mRNA and MTH1 protein was previously proposed as a molecular marker of oxidative stress. Therefore, we hypothesized that increased 8-oxo-dGTPase activity of MTH1 protein in mouse organs could serve as a dose-dependent marker of exposure to ionizing radiation, which is known to induce oxidative stress. To test our hypothesis, we measured 8-oxo-dGTPase activity in six organs of male BL6 mice after exposure to 0, 10, 25 and 50 cGy and 1 Gy of 137Cs γ radiation given as a single whole-body dose (1 Gy/min). The mice were killed 4, 8 and 24 h after irradiation. A statistically significant induction of 8-oxo-dGTPase was found in brains, testes and kidneys but not in lungs, hearts or livers. Brains, which demonstrated the highest (4.3-fold) increase of 8-oxo-dGTPase activity, were shown to express ∼50% higher levels of MTH1 protein. However, due to the lack of a simple positive correlation between the dose and the observed 8-oxo-dGTPase activity in brain, testes and kidneys, we conclude that measurements of 8-oxo-dGTPase activity in these organs may serve as a rough indicator rather than a quantifiable marker of radiation-induced oxidative stress.

Measurement of DNA oxidation in human cells by chromatographic and enzymic methods

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve problems in the measurement of DNA oxidation that have resulted in varying estimates of the extent of this damage in humans. HeLa cells, sent to members for analysis, were either untreated, or treated with light in the presence of a photosensitizer to induce different amounts of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA. Laboratories employing HPLC with electrochemical detection were able to measure the induced damage with similar efficiency; dose response gradients for seven of the eight sets of results were almost identical. GC-MS and HPLC-MS/MS, employed in three laboratories, did not convincingly detect the dose response. An alternative approach to measuring base oxidation employs the enzyme formamidopyrimidine DNA N-glycosylase (FPG) to convert 8-oxoGua to strand breaks, which are then measured by alkaline unwinding, alkaline elution, or the comet assay. Ten laboratories used this approach; five were able to detect the dose response in cells treated with photosensitizer plus light (at lower doses than for chromatographic methods, because the enzymic methods are more sensitive and less prone to spurious oxidation). Median values for 8-oxoGua (or FPG-sensitive sites) in untreated cells were 4.01 per 106 guanines for chromatographic methods, and 0.53 per 106 guanines for techniques based on FPG.The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve problems in the measurement of DNA oxidation that have resulted in varying estimates of the extent of this damage in humans. HeLa cells, sent to members for analysis, were either untreated, or treated with light in the presence of a photosensitizer to induce different amounts of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA. Laboratories employing HPLC with electrochemical detection were able to measure the induced damage with similar efficiency; dose response gradients for seven of the eight sets of results were almost identical. GC-MS and HPLC-MS/MS, employed in three laboratories, did not convincingly detect the dose response. An alternative approach to measuring base oxidation employs the enzyme formamidopyrimidine DNA N-glycosylase (FPG) to convert 8-oxoGua to strand breaks, which are then measured by alkaline unwinding, alkaline elution, or the comet assay. Ten laboratories used this approach; five were able to detect the dose response in cells treated with photosensitizer plus light (at lower doses than for chromatographic methods, because the enzymic methods are more sensitive and less prone to spurious oxidation). Median values for 8-oxoGua (or FPG-sensitive sites) in untreated cells were 4.01 per 10(6) guanines for chromatographic methods, and 0.53 per 10(6) guanines for techniques based on FPG.