Biomedical subjects

Andrew R Collins

Publications and source records attributed to Andrew R Collins.

At least 19 recordsLinked to original sources

PubMed2007

Quantitated transcript haplotypes (QTH) of AGTR1, reduced abundance of mRNA haplotypes containing 1166C (rs5186:A>C), and relevance to metabolic syndrome traits.

The angiotensin II type 1 receptor (AGTR1) is the main target through which angiotensin II influences cardiovascular tone, cell growth, and fluid and electrolyte balance. AGTR1 polymorphism has been reported to associate with hypertension, myocardial infarction (MI), and metabolic traits. Here we describe a novel approach to quantitation of transcript haplotypes (QTH) of AGTR1. To determine relative allelic expression from haplotypes, within-individual-between-allele ratiometric analyses in placental cDNA were developed for the transcribed SNPs rs5182:C>T (encoding p.L191) and rs5186:A>C (3'-noncoding "A1166C"). Additionally, between-individual comparisons were made using TaqMan assays applied to both homozygous and heterozygous genotypes and haplotypes. In conjunction, linkage disequilibrium (LD) and genomic haplotype associations with metabolic syndrome were examined. There was no significant difference of mRNA level for alleles of rs5182:C>T, but allele and mRNA haplotypes carrying 1166C exhibited reduced abundance. The effect was much greater in CC homozygotes than in heterozygotes. The promoter region was confirmed to be in a separate haplotype block from the AGTR1 3' region containing rs5182:C>T and rs5186:A>C. Metabolic syndrome trait associations were strongest for the 3' block generally and for the C allele of rs5186:A>C specifically. All effects were much more prominent in homozygotes, possibly reflecting interallelic interaction through feedback loops of mRNA regulation. Differential abundance of AGTR1 mRNA haplotypes may mediate clinical phenotypic observations of the AGTR1 genotype.

Aged↗

PubMed2006

A map of nuclear matrix attachment regions within the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1.

There is abundant evidence that the DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. To explore the DNA domain organization of the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1, we have identified a significant portion of the scaffold/matrix attachment regions (S/MARs) within this region. Forty independent putative S/MAR elements were assigned within the 16q22.1 locus. More than 90% of these S/MARs are AT rich, with GC contents as low as 27% in 2 cases. Thirty-nine (98%) of the S/MARs are located within genes and 36 (90%) in gene introns, of which 15 are in first introns of different genes. The clear tendency of S/MARs from this region to be located within the introns suggests their regulatory role. The S/MAR resource constructed may contribute to an understanding of how the genes in the region are regulated and of how the structural architecture and functional organization of the DNA are related.

Base Composition↗

PubMed2006

Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup.

As part of the Fourth International Workshop on Genotoxicity Testing (IWGT), held 9-10 September 2005 in San Francisco, California, an expert working group on the Comet assay was convened to review and discuss some of the procedures and methods recommended in previous documents. Particular attention was directed at the in vivo rodent, alkaline (pH >13) version of the assay. The aim was to review those protocol areas which were unclear or which required more detail in order to produce a standardized protocol with maximum acceptability by international regulatory agencies. The areas covered were: number of dose levels required, cell isolation techniques, measures of cytotoxicity, scoring of comets (i.e., manually or by image analysis), and the need for historical negative/positive control data. It was decided that a single limit dose was not sufficient although the required number of dose levels was not stipulated. The method of isolating cells was thought not to have a qualitative effect on the assay but more data were needed before a conclusion could be drawn. Concurrent measures of cytotoxicity were required with histopathological examination of tissues for necrosis or apoptosis as the "Gold Standard". As for analysing the comets, the consensus was that image analysis was preferred but not required. Finally, the minimal number of studies required to generate a historical positive or negative control database was not defined; rather the emphasis was placed on demonstrating the stability of the negative/positive control data. It was also agreed that a minimum reporting standard would be developed which would be consistent with OECD in vivo genotoxicity test method guidelines.

Animals↗

PubMed2006

Life-long vitamin C supplementation in combination with cold exposure does not affect oxidative damage or lifespan in mice, but decreases expression of antioxidant protection genes.

Oxidative stress is suggested to be central to the ageing process, with endogenous antioxidant defence and repair mechanisms in place to minimize damage. Theoretically, supplementation with exogenous antioxidants might support the endogenous antioxidant system, thereby reducing oxidative damage, ageing-related functional decline and prolonging life- and health-span. Yet supplementation trials with antioxidants in animal models have had minimal success. Human epidemiological data are similarly unimpressive, leading some to question whether vitamin C, for example, might have pro-oxidant properties in vivo. We supplemented cold exposed (7+/-2 degrees C) female C57BL/6 mice over their lifespan with vitamin C (ascorbyl-2-polyphosphate), widely advocated and self administered to reduce oxidative stress, retard ageing and increase healthy lifespan. No effect on mean or maximum lifespan following vitamin C treatment or any significant impact on body mass, or on parameters of energy metabolism was observed. Moreover, no differences in hepatocyte and lymphocyte DNA oxidative damage or hepatic lipid peroxidation was seen between supplemented and control mice. Using a DNA macroarray specific for oxidative stress-related genes, we found that after 18 months of supplementation, mice exhibited a significantly reduced expression of several genes in the liver linked to free-radical scavenging, including Mn-superoxide dismutase. We confirmed these effects by Northern blotting and found additional down-regulation of glutathione peroxidase (not present on macroarray) in the vitamin C treated group. We suggest that high dietary doses of vitamin C are ineffective at prolonging lifespan in mice because any positive benefits derived as an antioxidant are offset by compensatory reductions in endogenous protection mechanisms, leading to no net reduction in accumulated oxidative damage.

Animals↗

PubMed2006

Possible involvement of XPA in repair of oxidative DNA damage deduced from analysis of damage, repair and genotype in a human population study.

Participants in a study of occupational exposure to mineral fibres in Slovakia were analysed for the polymorphism 23A-->G in the DNA repair gene XPA. Of the 388 subjects, 239 were exposed to asbestos, stonewool or glass fibre; the rest were unexposed controls. Levels of DNA base alterations (oxidation and alkylation) in lymphocytes were measured using the comet assay with lesion-specific endonucleases. 8-oxoguanine DNA glycosylase (OGG1) DNA repair activity was measured, as incision activity of a lymphocyte extract on DNA containing the OGG1 substrate 8-oxoguanine. Presence of the A allele was associated with higher levels of DNA damage (sites sensitive to formamidopyrimidine DNA glycosylase, endonuclease III or 3-methyladenine DNA glycosylase II) as well as with higher activity of OGG1 repair enzyme. DNA base damage increased with age, showing highly significant correlations when the whole population or subgroups of the population were analysed. OGG1 repair activity also increased with age, but when analysed according to XPA genotype, the increase was observed only in those individuals with an A allele. Although XPA is known as a protein involved in nucleotide excision repair of UV-induced damage and bulky DNA adducts, it may also have a role in the repair of oxidized bases.

Age Factors↗

PubMed2005

Antioxidant intervention as a route to cancer prevention.

The notion of cancer prevention through antioxidant intervention arises from the fact that fruits and vegetables contain antioxidants and are linked to low cancer rates in those who consume them. Protection against DNA damage by plant food products can be demonstrated in vitro. However, particular care is needed when measuring the damage, since oxidation readily occurs during sample preparation, creating a serious artefact. In the case of DNA oxidation, estimates of background levels in human cells range over 3 orders of magnitude, depending on the method used. Using validated, reliable biomarker assays for DNA oxidation, it is possible to demonstrate a decrease in oxidative damage after supplementation with isolated antioxidants or whole plant foods in humans. In contrast, in several large-scale interventions with disease or death as the endpoint, supplementation with beta-carotene resulted in no effect or an increase in cancer incidence. It is certainly true that we do not yet fully understand the role of phytochemicals as antioxidants, or as modulators of other processes related to carcinogenesis and its prevention.

Antioxidants↗

PubMed2005

Assays for oxidative stress and antioxidant status: applications to research into the biological effectiveness of polyphenols.

Oxidative stress is a factor in many human diseases, as either cause or effect. A convenient biomarker of oxidative stress is the extent of oxidation of bases in DNA (although measures of lipid or protein oxidation may be equally informative). 8-Oxo-7,8-dihydroguanine or the corresponding nucleoside is most often measured, either chromatographically (gas chromatography-mass spectrometry, HPLC with electrochemical detection, or HPLC-tandem mass spectrometry) or enzymically, with the use of the enzyme formamidopyrimidine DNA glycosylase to convert 8-oxo-7,8-dihydroguanine to DNA breaks, which are detected with alkaline elution, alkaline unwinding, or the comet assay. Estimates of background levels of 8-oxo-7,8-dihydroguanine in normal human cells vary 1000-fold, depending on the technique used. Gas chromatography-mass spectrometry is particularly prone to oxidation of samples during derivatization, whereas HPLC suffers from this artifact to a lesser degree. In a recent interlaboratory study that measured the same samples of human cells, median values obtained with HPLC with electrochemical detection and with formamidopyrimidine DNA glycosylase differed by approximately 10-fold. There are still questions regarding the actual level of damage, but it is probably approximately one 8-oxo-7,8-dihydroguanine residue per 10(6) guanines. Assays for antioxidant protection against oxidative damage generally depend on measurements of decreases in a marker of oxidation. Potential dietary antioxidants can be screened with in vitro antioxidant assays or tested in cell culture systems. The best test, however, is in humans. The total antioxidant capacity of plasma is generally insensitive to dietary supplementation with antioxidants or antioxidant-rich foods. An increase in the resistance of lymphocyte DNA to oxidation in vitro is commonly seen, however, and a decrease in endogenous oxidation of DNA may be detected, especially after prolonged supplementation.

Antioxidants↗

PubMed2004

Are environmental electromagnetic fields genotoxic?

Long-term exposure to extremely-low-frequency electromagnetic fields (ELF EMFs) greater than 0.4 microT has been linked, by epidemiological studies, to a small elevated risk of childhood leukaemia. Laboratory-based experiments have been claimed to show that ELF EMFs induce a variety of biological responses, although these claims are controversial. Recent experiments by Ivancsits et al. [Mutat. Res. 519 (2002) 1; Int. Arch. Occup. Environ. Health 76 (2003) 431; Mech. Age. Dev. 124 (2003) 847; H.W. Rüdiger, S. Ivancsits, E. Diem, O. Jahn, Genotoxic effects of ELF-EMF on human cells in vitro, Bioelectromagnetics Society 25th Annual Meeting, Maui, USA, 2003] suggest that ELF EMFs are genotoxic, on the basis of observations that intermittent exposures induce single-strand breaks (SSB) and double-strand DNA breaks (DSB) in the DNA of cultured human fibroblasts. The implications of these findings are discussed.

Animals↗

PubMed2004

Nutrition and carcinogenesis.

Traditional views of nutritional carcinogenesis depend on the identification of exogenous carcinogens as major risk factors. As our understanding evolves, it is clear that the pattern of events involves not only exogenous carcinogens, but also metabolic processes and endogenous and exogenous anticarcinogens. The process is modulated by the immune system, and genetics plays a significant role. New monitoring methods provide much-needed tools for providing proof of involvement of various factors at the level of human populations.

Animals↗

PubMed2004

Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells?

The most commonly measured marker of oxidative DNA damage is 8-oxo-7,8-dihydroguanine (8-oxoGua) or its deoxyribonucleoside (8-oxodGuo). Published estimates of the concentration of 8-oxoGua/8-oxodGuo in DNA of normal human cells vary over a range of three orders of magnitude. Analysis by chromatographic methods (GC-MS, HPLC with electrochemical detection (ECD) or HPLC-MS/MS) is beset by the problem of adventitious oxidation of guanine during sample preparation. An alternative approach, based on the use of the DNA repair enzyme formamidopyrimidine DNA N-glycosylase (FPG) to make breaks in the DNA at sites of the oxidised base, gives much lower values. ESCODD, the European Standards Committee on Oxidative DNA Damage, has been testing the ability of different laboratories using a variety of methods to measure 8-oxoGua in standard samples of 8-oxodGuo, calf thymus DNA, pig liver, oligonucleotides, and HeLa cells, and in lymphocytes isolated from blood of volunteers. HPLC-ECD is capable of measuring 8-oxodGuo induced experimentally in calf thymus DNA or HeLa cells with high accuracy. However, there is no sign of consensus over the background level of this damage, suggesting that, even though standard extraction procedures were used, variable oxidation of Gua is still occurring. GC-MS failed to detect a dose response of induced 8-oxoGua and cannot be regarded as a reliable method for measuring low levels of damage. HPLC-MS/MS as yet has not proved capable of measuring low levels of oxidative DNA damage. FPG-based methods seem to be less prone to the artefact of additional oxidation. Although they can be used quantitatively, they require careful calibration and standardisation if they are to be used in human biomonitoring. The background level of DNA oxidation in normal human cells is likely to be around 0.3-4.2 8-oxoGua per 10(6) Gua. An effort should be made to develop alternative, validated methods for estimating oxidative DNA damage.

Animals↗

PubMed2004

Does a vegetarian diet influence genomic stability?

BACKGROUND: The vegetarian lifestyle is supposedly healthy, and differences between vegetarians and non-vegetarians in biomarkers related to diseases such as cancer might be expected. AIM OF THE STUDY: To investigate the possible role of different diets in maintaining genomic stability. METHODS: The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among vegetarians there were 13 lacto-ovo-vegetarians (8 women, 5 men) with average length of vegetarian diet 10.8 years (ranging from 5 to 26) and 11 lacto-vegetarians (5 women, 6 men) with average length of vegetarian diet 8.2 years (ranging from 3 to15). All volunteers were nonsmokers, non-consumers of alcohol and had similar education and patterns of physical activity. Chromosome aberrations, micronuclei and DNA damage (strand breaks, oxidised bases and H(2)O(2)-sensitivity) were examined in peripheral blood lymphocytes of vegetarians and non-vegetarians. Plasma antioxidant status was assessed with the FRAP assay. RESULTS: We did not find any differences in percentage of cells with chromosome aberrations or in the frequency of micronuclei between vegetarians and non-vegetarians or between lacto-ovo and lacto-vegetarians. There was no statistically significant difference in total antioxidant capacity between the groups. The group with traditional dietary habits had significantly higher levels of oxidative DNA damage (strand breaks and oxidised purines, P = 0.005) compared with vegetarians. A significant positive correlation between age and oxidative DNA damage (net FPG-sensitive sites) was found in non-vegetarians, while there was an opposite trend towards a negative association in vegetarians. On the other hand chromosome aberrations correlated with age in vegetarians (r = 0.48, P = 0.017) but not in non-vegetarians. CONCLUSIONS: Our results indicate that a vegetarian diet can lead to a slight decrease in oxidative DNA damage in lymphocytes, but other markers of genetic stability are not affected. The lowest level of DNA damage was found in lymphocytes of lactovegetarians, (especially oxidised pyrimidines, P = 0.0017), suggesting that this diet provides some protection against oxidative stress.

Adult↗

PubMed2004

DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization.

The comet assay is a sensitive method for measuring DNA strand breaks in eukaryotic cells. After embedding in agarose, cells are lysed and electrophoresed at high pH. DNA loops containing breaks (in which supercoiling is relaxed) escape from the nucleoid comet head to form a tail. Oligonucleotide probes were designed for 5' and 3' regions of the genes for dihydrofolate reductase (DHFR) and O6-methylguanine DNA methyltransferase (MGMT), both from the Chinese hamster, and the human tumour suppressor p53 gene. Alternate ends were labelled with either biotin or fluorescein. These probes were hybridized to the DNA of comets from Chinese hamster ovary (CHO) cells or human lymphocytes treated with H2O2 or photosensitizer plus light to induce oxidative damage. Amplification with Texas red- and fluorescein-tagged antibodies led, in the case of p53 in human cells, to red and green signals located in the comet tail (as well as in the head), indicating the presence of breaks in the vicinity of the gene. However, only one end of the MGMT gene appeared in the tail and almost no signals from the DHFR gene, either red or green, were in the tail of comets from CHO cells. Restriction on movement from the head to tail may result from the presence of a 'matrix-associated region' in the gene. The kinetics of repair of oxidative damage were followed; strand breaks in the p53 gene were repaired more rapidly than total DNA. Thus, fluorescent in situ hybridization in combination with the comet assay provides a powerful method for studying repair of specific genes in relation to chromatin structure.

Animals↗

PubMed2004

The comet assay for DNA damage and repair: principles, applications, and limitations.

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

Animals↗

PubMed2003

Independent effects of the -219 G>T and epsilon 2/ epsilon 3/ epsilon 4 polymorphisms in the apolipoprotein E gene on coronary artery disease: the Southampton Atherosclerosis Study.

A number of studies have shown that coronary artery disease severity is associated with the epsilon 2/ epsilon 3/ epsilon 4 polymorphism in the coding region of the apolipoprotein E gene. In this study, we investigated whether the severity of the disease was also influenced by a functional polymorphism (-219 G>T) in the promoter of the gene, and if so, whether the effects of the two polymorphisms were independent. A cohort of 1170 patients with angiographically documented coronary artery disease were genotyped for the two polymorphisms. The frequency of the epsilon 4 allele of the epsilon 2/ epsilon 3/ epsilon 4 polymorphism increased linearly with increasing number of diseased vessels, so did the -219T allele of the -219 G>T polymorphism. In the sample as a whole, logistic regression analyses indicated that compared with the G/G genotype, the T/T genotype conferred an odds ratio of 1.598 (95% CI=1.161-2.201, P=0.004) in favor of increased disease severity, and the relationship remained significant after adjustment for epsilon 2/ epsilon 3/ epsilon 4 polymorphism genotypes, plasma cholesterol and triglyceride levels, and other risk factors. The effect of the T/T genotype on disease severity was more significant in patients who did not carry the epsilon 4 allele (OR=1.510, 95% CI=1.028-2.221) than in epsilon 4 allele carriers (OR=1.303, 95% CI=0.619-2.742). There was considerable linkage disequilibrium between the two polymorphisms (rho=0.9, P<0.001). Logistic regression analysis showed that the -219T- epsilon 4 haplotype conferred an odds ratio of 1.488 (95% CI=1.133-1.954). These findings suggest that the -219 G>T and epsilon 2/ epsilon 3/ epsilon 4 polymorphisms, which may affect respectively the quantity and quality of apoE, have independent and possibly additive effects on coronary artery disease severity.

Aged↗

PubMed2003

Nutritional modulation of DNA repair in a human intervention study.

DNA oxidation is a potential cause of cancer in humans. It is well-known that fruits and vegetables protect against cancer, and this may be in part because they contain antioxidants, which decrease the level of oxidation of DNA. However, there are other possible mechanisms, such as an enhancement of cellular repair of this damage. A randomized cross-over study was carried out on healthy human subjects, who were given kiwifruit as a supplement to their normal diet, for 3-week periods at different 'doses', with 2-week washout periods between doses. Endogenous oxidation of bases in lymphocyte DNA, and the resistance of the DNA to oxidation ex vivo, were assessed using single cell gel electrophoresis (the 'comet assay'). The capacity to repair DNA base oxidation was measured with an in vitro test, and levels of expression of repair-related genes OGG1 and APE1 were assessed by semi-quantitative RT-PCR. Concentrations of dietary antioxidants were measured in plasma. The antioxidant status of plasma and of lymphocytes was increased by consumption of kiwifruit. Levels of endogenous oxidation of pyrimidines and purines in DNA were markedly decreased, and DNA repair measured on a substrate containing 8-oxo-7,8-dihydroguanine was substantially increased (without change in levels of OGG1 or APE1 mRNA). The magnitude of these effects was generally not related to the number of kiwifruits consumed per day. Kiwifruit provides a dual protection against oxidative DNA damage, enhancing antioxidant levels and stimulating DNA repair. It is probable that together these effects would decrease the risk of mutagenic changes leading to cancer.

Adult↗

PubMed2002

Antioxidant enzyme activities, lipid peroxidation, and DNA oxidative damage: the effects of short-term voluntary wheel running.

We examined the effect of voluntary exercise on antioxidant enzyme activities (catalase, glutathione peroxidase, superoxide dismutase) in skeletal muscle (hind- and forelimb) and heart of a model small mammal species: short-tailed field vole Microtus agrestis. In addition, DNA oxidation was determined in lymphocytes and hepatocytes using the comet assay and lipid peroxidation estimated in hindlimb muscle by measurement of thiobarbituric-acid-reactive substances. Voles (approximately 6 weeks old), exposed to a 16L:8D photoperiod (lights on 0500 h), ran almost continuously during darkness. We studied the effects of voluntary running over 1 or 7 days duration, with or without an 8-h rest period, on various biomarkers of oxidative stress compared to nonrunning controls. No differences were observed in antioxidant enzyme activities, except in heart total superoxide dismutase activity (P=0.037), with the lowest levels in 1- and 7-day runners at 0500 h. DNA oxidative damage, in lymphocytes or hepatocytes, and lipid peroxidation did not differ between groups. There was no evidence of any significant increase in any oxidative stress parameter in running individuals, despite having significantly elevated energy expenditures compared to sedentary controls.

Animals↗

PubMed2002

Homocysteine, B vitamin status, and cognitive function in the elderly.

BACKGROUND: Old age is associated with reduced cognitive performance. Nutritional factors may contribute to this association. OBJECTIVE: We tested associations between cognitive performance and plasma vitamin B-12, folate, and homocysteine concentrations in the elderly. DESIGN: We studied survivors of the Scottish Mental Surveys of 1932 (Aberdeen 1921 Birth Cohort, or ABC21) and 1947 (Aberdeen 1936 Birth Cohort, or ABC36), which surveyed childhood intelligence quotient. We measured folate, vitamin B-12, and homocysteine concentrations in fasting blood samples and cognitive performance by the Mini Mental State Examination (MMSE), National Adult Reading Test (NART), Raven's Progressive Matrices (RPM), Auditory Verbal Learning Test (AVLT), digit symbol (DS) subtest, and block design (BD) subtest. RESULTS: Homocysteine was higher in the ABC21 than in the ABC36 (P < 0.0001). There were positive correlations between folate and vitamin B-12 and negative correlations between homocysteine and both folate and vitamin B-12. MMSE, RPM, AVLT, DS, and BD scores were higher in the ABC36. In the ABC21, folate, vitamin B-12, and MMSE score were positively correlated and homocysteine was negatively correlated with RPM, DS, and BD scores. Folic acid was positively correlated with AVLT and DS scores. In the ABC36, folate was positively correlated with BD score. After adjustment for childhood intelligence quotient, partial correlations were strengthened between vitamin B-12 and NART score and between homocysteine and RPM score but weakened between red blood cell folate and DS score. CONCLUSIONS: B vitamins and homocysteine are associated with cognitive variation in old age. In the ABC21 but not the ABC36, homocysteine accounted for approximately 7-8% of the variance in cognitive performance. This may prove relevant to the design of neuroprotective studies in late life.

Aged↗

PubMed2002

Oxidative stress in humans: validation of biomarkers of DNA damage.

Two studies have been performed to clarify the relationship between different markers of oxidative DNA damage commonly employed in molecular epidemiological studies. In the first, 8-Oxo-7,8-dihydroguanine (8-oxoGua) was induced in DNA of HeLa cells by treatment with different concentrations of photosensitizer Ro 19-8022 together with visible light. 8-OxoGua was estimated by the comet assay (alkaline single cell gel electrophoresis) with formamidopyrimidine DNA glycosylase and by HPLC with electrochemical detection. The dose-response curves indicate that the comet assay and HPLC are equally efficient at detecting induced damage. Background levels of 8-oxoGua in HeLa cells were 0.92 +/- 0.22 per 10(6) guanines by the comet assay and 2.09 +/- 0.13 per 10(6) guanines by HPLC. The second study was a small human trial, in which lymphocytes were collected for analysis of background levels of 8-oxoGua, as well as overnight and 24 h urine samples for measurement of excreted 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) by ELISA. The mean level of 8-oxoGua in lymphocytes was determined as 1.33 +/- 0.21 per 10(6) guanines by the comet assay and 3.72 +/- 1.06 per 10(6) guanines by HPLC. A strong correlation was seen between overnight and 24 h urinary 8-oxodGuo (r = 0.93, P < 0.01). Overnight urinary 8-oxodGuo concentrations correlated with 8-oxoGua in lymphocytes measured by HPLC (r = 0.85, P < 0.05) or by the comet assay (r = 0.86, P < 0.05), although individual values from HPLC and the comet assay did not correlate with each other. It is reasonable to assess oxidative stress by any of these methods.

8-Hydroxy-2'-Deoxyguanosine↗