Ryszard Olinski

Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome

Free radical attack upon DNA generates a multiplicity of DNA damage, including modified bases. Some of these modifications have considerable potential to damage the integrity of the genome. This article reviews recent data that suggest the involvement of oxidative DNA damage in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome (AIDS). There is evidence that oxidative DNA damage may play a causative role in atherosclerosis. Oxidative DNA damage may lead to apoptotic cell death of patients infected with human immunodeficiency virus (HIV) and may influence the progression of AIDS. While many details regarding the role of reactive oxygen species-induced DNA damage in the etiology of complex multifactorial diseases like cancer are yet to be discovered, evidence suggests that oxidants act at several stages in the malignant transformation of cells. However, the quantitative relationship between the measured DNA damage and the development of cancer is still lacking.

Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer

We have investigated levels of antioxidant enzymes and free radical‐induced DNA base modifications in human cancerous lung tissues and in their cancer‐free surrounding tissues. Various DNA base lesions in chromatin of lung tissues were measured by gas chromatography‐mass spectrometry. Activities of superoxide dismutase, catalase and glutathione peroxidase were also measured in lung tissues. Higher levels of DNA lesions were observed in cancerous tissues than in cancer‐free surrounding tissues. Antioxidant enzyme levels were lower in cancerous tissues. The results indicate an association between decreased activities of antioxidant enzymes and increased levels of DNA lesions in cancerous tissues. Higher levels of DNA lesions suggest that free radical reactions may be increased in malignant tumor cells.

Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine

Background: Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. The noninvasive assessment of such damage, i.e., in urine, and application to large-scale human studies are vital to understanding this role and devising intervention strategies. Methods: We have reviewed the literature to establish the status quo with regard to the methods and meaning of measuring DNA oxidation products in urine. Results: Most of the literature focus upon 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), and whereas a large number of these reports concern clinical conditions, there remains (a) lack of consensus between methods, (b) possible contribution from diet and/or cell death, (c) no definitive DNA repair source of urinary 2′-deoxyribonucleoside lesions, and (d) no reference ranges for healthy or diseased individuals. Conclusions: The origin of 8-oxodG is not identified; however, recent cell culture studies suggest that the action of Nudix hydrolase(s) on oxidative modification of the nucleotide pool is a likely candidate for the 8-oxodG found in urine and, potentially, of other oxidized 2′-deoxyribonucleoside lesions. Literature reports suggest that diet and cell death have minimal, if any, influence upon urinary levels of 8-oxodG and 8-oxo-7,8-dihydroguanine, although this should be assessed on a lesion-by-lesion basis. Broadly speaking, there is consensus between chromatographic techniques; however, ELISA approaches continue to overestimate 8-oxodG levels and is not sufficiently specific for accurate quantification. With increasing numbers of lesions being studied, it is vital that these fundamental issues are addressed. We report the formation of the European Standards Committee on Urinary (DNA) Lesion Analysis whose primary goal is to achieve consensus between methods and establish reference ranges in health and disease. (Cancer Epidemiol Biomarkers Prev 2008;17(1):3–14)

Toward consensus in the analysis of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine as a noninvasive biomarker of oxidative stress

Of the DNA‐derived biomarkers of oxidative stress, urinary 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography‐based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8‐oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8‐oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme‐linked immunosorbant assay (ELISA). Overall, there was good within‐technique agreement, with the majority of laboratories’ results lying within 1 SD of their consensus mean. However, ELISA showed more within‐technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland‐Altman plots revealed good agreement between MS and EC methods but concentration‐dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 SD of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA.—ESCULA [European Standards Committee on Urinary (DNA) Lesion Analysis], Evans, M. D., Olinski, R., Loft, S., Cooke, M. S. Toward consensus in the analysis of urinary 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine as a noninvasive biomarker of oxidative stress. FASEB J. 24, 1249–1260 (2010). www.fasebj.org

Persistent oxidative stress in colorectal carcinoma patients

We examine whether the level of 8‐oxo‐2′‐deoxyguanosine (8‐oxodGuo) in lymphocytes DNA is higher in colon cancer when compared to the control group. Factors that may influence oxidative stress such as antioxidant vitamins and uric acid were also determined. Blood samples were obtained from a control group of 55 healthy persons and 43 colon cancers. 8‐OxodGuo level and the vitamins concentration were measured by high‐performance liquid chromatography. The levels of 8‐oxodGuo were significantly higher whereas the concentrations of the vitamins and uric acid were significantly lower in colon cancer patients than in control group. Therefore, the decreased concentration of antioxidant vitamins together with lower amount of uric acid may be responsible for the formation of pro‐oxidative environment in blood of colorectal carcinoma patients.

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet.

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (±0.90) and 0.83 nmol/kg 24h (±0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).

SUPPLEMENTATION WITH ANTIOXIDANT VITAMINS PREVENTS OXIDATIVE MODIFICATION OF DNA IN LYMPHOCYTES OF HIV-INFECTED PATIENTS

There is evidence suggesting that patients infected with human immunodeficiency virus (HIV) are under chronic oxidative stress. In the present study, the level of oxidatively modified bases in lymphocyte DNA and some other parameters of oxidative stress were measured in HIV-infected patients (n = 30), as well as in control groups (10 healthy volunteers and 15 HIV-seronegative injected drug users). Additional experiments were conducted using lymphocyte DNA samples from asymptomatic seropositive, HIV-infected patients who were supplemented with antioxidant vitamins A, C, and E or received placebo. Significant increases in the amount of the modified DNA bases were observed in HIV-infected patients when compared with the control group. The concentration of thiobarbituric acid reactive substances (TBARS) was higher and activities of antioxidant enzymes (superoxide dismutase and catalase) were lower in the group of HIV-infected patients in comparison to the control group. Vitamin supplementation resulted in the significant decrease in the levels of all modified DNA bases when compared to the patients who received placebo. The reduction of TBARS and the restoration of the activity of the enzymes were also observed. Our data suggest that people infected with HIV can benefit from treatment with antioxidant vitamins.

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.

COMPARISON OF OXIDATIVE BASE DAMAGE IN MITOCHONDRIAL AND NUCLEAR DNA

The levels of endogenous pig liver cells mitochondrial DNA oxidative base damage have been investigated using isotope dilution gas chromatography mass spectrometry (GC/MS). Higher levels of five measured bases were found in mtDNA in relation to nuclear DNA. We have also detected large differences in the modified base ratios of mitochondrial versus nuclear DNA. These ratios for the bases with promutagenic properties as 8OHGua and 5OHCyt are much lower than for other bases (5OHHyd, 5OHMeHyd, 5OHMeUra).