Valerie J. McKelvey-Martin

Bio: Valerie J. McKelvey-Martin is an academic researcher from Ulster University. The author has contributed to research in topics: Comet assay & DNA damage. The author has an hindex of 22, co-authored 43 publications receiving 2763 citations.

The single cell gel electrophoresis assay (comet assay): A European review

Abstract: The single cell gel electrophoresis (SCGE) assay is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells. Here we review the development of the SCGE assay (with particular reference to the alkaline version), existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA, and the potential applications of the technique.

A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay.

Abstract: Baseline DNA damage in spermatozoa from fertile and infertile men was compared using a modified alkali single cell gel electrophoresis (comet) assay. Semen from normozoospermic fertile, normozoospermic infertile and asthenozoospermic infertile (World Health Organization criteria, 1992) samples were studied. No significant difference was observed in levels of baseline damage between the three groups. A median value for baseline damage of approximately 20% (80% head DNA) was obtained in all samples. Irradiation with X-rays (5-30 Gy) produced no additional damage in fertile samples when median values were examined. However, irradiation with 30 Gy X-rays produced significant damage in both infertile groups. Hydrogen peroxide (40 microM) treatment induced significant damage in the asthenozoospermic group, whereas 100 microM H2O2 was required to cause significant damage in the normozoospermic fertile and infertile samples. Within the fertile population a subgroup in which percentage head DNA was greater than 80% was observed in both treated and untreated specimens. This subgroup significantly decreased with treatment in both infertile groups. We conclude that the asthenozoospermic infertile group is more susceptible to damage than the normozoospermic infertile group, which in turn is more susceptible than the fertile group. The fertile group contains a resistant subpopulation of spermatozoa with relatively intact DNA.

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity.

Abstract: The integrity of sperm DNA is crucial for the maintenance of genetic health. A major source of damage is reactive oxygen species (ROS) generation; therefore, antioxidants may afford protection to sperm DNA. The objectives of the study were, first, to measure the effects of antioxidant supplementation in vitro on endogenous DNA damage in spermatozoa using the single cell gel electrophoresis (comet) assay and, second, to assess the effect of antioxidant supplementation given prior to X-ray irradiation on induced DNA damage. Spermatozoa from 150 patients were prepared by Percoll centrifugation in the presence of ascorbic acid (300, 600 microM), alpha tocopherol (30, 60 microM), urate (200, 400 microM), or acetyl cysteine (5, 10 microM). DNA damage was induced by 30 Gy X-irradiation. DNA strand breakage was measured using the comet assay. Sperm DNA was protected from DNA damage by ascorbic acid (600 microM), alpha tocopherol (30 and 60 microM) and urate (400 microM). These antioxidants provided protection from subsequent DNA damage by X-ray irradiation. In contrast, acetyl cysteine or ascorbate and alpha tocopherol together induced further DNA damage. Supplementation in vitro with the antioxidants ascorbate, urate and alpha tocopherol separately has beneficial effects for sperm DNA integrity.

Global DNA and p53 Region-Specific Hypomethylation in Human Colonic Cells Is Induced by Folate Depletion and Reversed by Folate Supplementation

Abstract: There is increasing evidence to suggest that reduced folate status may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Folate is essential for the synthesis of S-adenosylmethionine, the methyl donor required for all methylation reactions in the cell, including the methylation of DNA. Global DNA hypomethylation appears to be an early, and consistent, molecular event in carcinogenesis. We have examined the effects of folate depletion on human-derived cultured colon carcinoma cells using 2 novel modifications to the Comet (single cell gel electrophoresis) assay to detect global DNA hypomethylation and gene region-specific DNA hypomethylation. Colon cells cultured in folate-free medium for 14 d showed a significant increase in global DNA hypomethylation compared with cells grown in medium containing 3 micromol/L folic acid. This was also true at a gene level, with folate-deprived cells showing significantly more DNA hypomethylation in the region of the p53 gene. In both cases, the effects of folate depletion were completely reversed by the reintroduction of folic acid to the cells. These results confirm that decreased folate levels are capable of inducing DNA hypomethylation in colon cells and particularly in the region of the p53 gene, suggesting that a more optimal folate status in vivo may normalize any DNA hypomethylation, offering potential protective effects against carcinogenesis. This study also introduces 2 novel functional biomarkers of DNA hypomethylation and demonstrates their suitability to detect folate depletion-induced molecular changes.

Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay.

Abstract: The single cell gel electrophoresis (SCGE) assay is a simple visual technique used to assess DNA integrity in individual cells by measuring damage reflected as strand breaks under alkaline conditions. Cells are embedded in agarose on glass slides followed by lysis of the cell membranes after which damaged DNA strands are electrophoresed away from the nucleus towards the anode and deposited to one side giving the appearance of a tail. DNA damage may be measured by assessing the relative amounts of DNA remaining in the head as opposed to those strands which have formed the tail. The assay has been used to determine DNA quality in human sperm (Hughes, C.M., S.E.M. Lewis, V.J. McKelvey-Martin, W. Thompson, A comparison of baseline and induced DNA damage in human sperm from fertile and infertile man, using a modified comet assay. Mol. Human Reprod., in press) by measuring fifty cells on one slide for each individual. Coefficients of variation between three control slides prepared for ten individuals were less than 4% and less than 9% for three slides prepared using irradiated sperm. Ten readings of fifty sperm each from a single slide showed a coefficient of variation of less than 6% for ten individuals studied. These results indicate that the measurement of fifty sperm from a single slide is sufficient to assess the DNA damage within a sperm population. Coefficients of variation of less than 5.4% for repeated analysis of individual cells were obtained which demonstrates the reproducibility of the image analysis software.

Democracy and education.

Abstract: Course Description In this course, we will explore the question of the actual and potential connections between democracy and education. Our focus of attention will be placed on a critical examination of democratic theory and its implications for the civic education roles and contributions of teachers, adult educators, community development practitioners, and community organizers. We will survey and deal critically with a range of competing conceptions of democracy, variously described as classical, republican, liberal, radical, marxist, neomarxist, pragmatist, feminist, populist, pluralist, postmodern, and/or participatory. Using narrative inquiry as a means for illuminating and interpreting contemporary practice, we will analyze the implications of different conceptions of democracy for the practical work of civic education.

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Abstract: Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

The Comet Assay for DNA Damage and Repair: Principles, Applications, and Limitations

Abstract: 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.

Health Belief Model

Abstract: This article presents an introduction to the Health Belief Model (HBM). The HBM states that the perception of a personal health behavior threat is influenced by at least three factors: general health values, interest and concern about health; specific beliefs about vulnerability to a particular health threat; and beliefs about the consequences of the health problem. Once an individual perceives a threat to his health and is simultaneously cued to action, if his perceived benefits outweighs his perceived costs, then the individual is most likely to undertake the recommended preventive health action. Key words: health promotion, health belief model, perceived susceptibility, perceived severity, perceived benefits, perceived barriers, cues to action, self-efficacy. Content available only in Romanian.