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A R Collins

Publications and source records attributed to A R Collins.

At least 73 records · Page 4Linked to original sources
PubMed1998

Oxidative DNA damage measured in human lymphocytes: large differences between sexes and between countries, and correlations with heart disease mortality rates.

The 'antioxidant hypothesis' proposes that vitamin C, vitamin E, carotenoids, and other antioxidants occurring in fruit and vegetables afford protection against heart disease and cancer by preventing oxidative damage to lipids and to DNA, respectively. To test elements of this hypothesis, we have measured blood levels of dietary antioxidants, and 8-oxodeoxyguanosine (8-oxo-dG) concentrations in lymphocyte DNA, in healthy men and women from five European countries: France, Ireland, The Netherlands, Spain, and the U.K. Volunteers, aged 25 45, all nonsmokers, gave blood samples before and after a 12-wk carotenoid supplementation regime. Vitamin C was measured in plasma and vitamin E and carotenoids were measured in serum by high-performance liquid chromatography (HPLC). 8-oxo-dG was assayed by HPLC (with coulometric detection) in DNA isolated from lymphocytes from the same blood samples. Mean values were calculated for groups of volunteers at each sampling time according to country, sex, and supplementation (between 9 and 24 individual samples contributing to each mean). We found that 8-oxo-dG levels in lymphocyte DNA vary significantly according to sex and country. A low mean 8-oxo-dG concentration is seen in DNA of women from all five countries, and of men from France and Spain. 8-oxo-dG is significantly higher (up to about threefold) in lymphocyte DNA from men in Ireland and the U.K. Oxidative DNA damage is not significantly affected by carotenoid supplementation; nor is there any association with mean baseline levels of antioxidants, which are generally similar in the five countries. The five countries sampled lie on an axis from northern to southern Europe with a steep gradient in terms of premature heart disease. There is a strong association between premature coronary heart disease mortality in men and the mean levels of 8-oxo-dG for the five countries (r = 0.95, P < 0.01). Women have low coronary heart disease mortality rates, which do not correlate with 8-oxo-dG. In terms of cancer deaths, only colorectal cancer in men shows a significant positive correlation (r = 0.91, P < 0.05), and stomach cancer in women is negatively correlated with DNA oxidation (r = -0.92, P = 0.01).

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes.

The effects of the flavonoids quercetin and myricetin, and the antihepatotoxic agent silymarin, on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide increased the levels of DNA strand breaks and oxidised pyrimidine bases in these cells. Quercetin was protective at concentrations above 10 microM and myricetin decreased oxidant-induced DNA strand breakage at concentrations of 100 microM. Cellular metabolism may alter the antioxidant efficacy of the flavonoids. Silymarin had no protective effect at any of the concentrations tested. None of these flavonoids was itself genotoxic. Neither alpha-tocopherol nor beta-carotene decreased hydrogen peroxide-induced DNA breakage. The differences in effectiveness of these dietary compounds against oxidative DNA damage may be explained by differences in their chemical structure or location within the cell.

Cell Count↗
PubMed1997

The comet assay: what can it really tell us?

A range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behaviour of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations. The underlying principles of this assay are discussed, and new evidence presented which supports the concept of relaxation of supercoiled loops, rather than alkaline unwinding, as the primary reason for comet tail formation. DNA-damaging agents that do not induce strand breaks directly can be detected when cellular repair processes convert lesions to transient strand breaks; an approach is outlined here which maximises this effect and thus widens the scope of the assay. Purified repair enzymes, applied to DNA during the course of the comet assay procedure, greatly increase the sensitivity and specificity of the assay; recent developments with formamidopyrimidine glycosylase (recognising 8-OH-gua and other damaged purines) and uvrABC (for bulky lesions) are presented. The kinetics of cellular repair after low doses of oxidative damage have been followed with this modified comet assay. Finally, the successful measurement of biomarkers of oxidative damage in human populations establishes the comet assay as a valuable tool in molecular epidemiology.

Cell Nucleus↗
PubMed1997

The influence of cell growth, detoxifying enzymes and DNA repair on hydrogen peroxide-mediated DNA damage (measured using the comet assay) in human cells.

Single-cell gel electrophoresis (the comet assay) is a sensitive method for detecting strand breaks at the level of individual cells. Cells embedded in agarose are lysed, electrophoresed, and fluorescently stained. Breaks in the DNA release its supercoiling and allow DNA to extend toward the anode, resembling a comet. We have used the comet assay to investigate the influence of growth state, xenobiotic detoxifying enzymes, and DNA repair processes on the response of cultured human cells to oxidative damage. HepG2 and Caco-2 cells are differentiated liver and colon cell lines, respectively. HeLa and GM1899A cells are relatively unspecialized epithelial and lymphoblastoid cells. Substrate-dependent cells showed a cyclical fluctuation of glutathione (GSH) with respect to growth. Enzyme activities (glutathione reductase, glutathione peroxidase, and catalase) varied considerably between cell types and changed with cell growth state. Hydrogen peroxide induced more DNA damage in actively dividing cells than in confluent cultures. Sensitivity to oxidative injury did not correlate with detoxifying enzyme activity. Rather, differences in susceptibility between cells could be correlated with differences in DNA repair capacity. This study highlights the need to standardize experimental conditions if the comet assay is to be employed in the study of genotoxicity.

Catalase↗
PubMed1997

Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-alpha concentration.

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-alpha (TNF-alpha) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34+ compartment from MDS patients (P=0.039), which are absent in both CD34- MDS cells (P=0.53) and also CD34+ cells from normal subjects (P=0.55). MDS CD34+ blood cells also showed oxidized pyrimidine nucleotides compared with CD34- cells (P=0.029). Within normal subjects no differences were seen between CD34+ and CD34- bone marrow cell compartments. CD34+ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-alpha and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-alpha, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.

Aged↗
PubMed1997

Ex vivo assessment of lymphocyte antioxidant status using the comet assay.

Lymphocytes were isolated from volunteers before and after receiving a single supplement of vitamin C, vitamin E or beta-carotene. The lymphocytes were treated with H2O2, and DNA strand breaks were measured by single cell gel electrophoresis (the comet assay). Significant protection against oxidative DNA damage was evident 2-4 h after vitamin C intake, and 18-24 h after consumption of the other antioxidants. Lymphocytes from smokers were more sensitive to DNA damage than those from non-smokers, and they showed at least as great a protective effect with antioxidants.

Administration, Oral↗
PubMed1997

UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts.

Rodent UV-sensitive mutant cell lines of complementation groups 6 and 8 are the genetic counterparts of human Cockayne syndrome CS-B and CS-A, respectively. The original mutant in this group, UV61, was described as defective in cyclobutane pyrimidine dimer removal after high doses of UV. We have examined the responses of several cell lines from group 6 to low doses of UV irradiation, and find that these mutants have wild-type capacity for DNA repair as indicated by incision, cyclobutane pyrimidine dimer, and (6-4) photoproduct removal. ERCC6, the product of the gene defective in CS-B and group 6 mutants, is implicated in the regulation of repair of actively transcribed genes in Cockayne syndrome; however, this protein clearly is not required for the processing of low levels of damage in CHO cells, which occurs remarkably efficiently, 40-50% of dimers being removed in both wild-type and group 6 mutants in 5 hours following 0.1 Jm(-2) of UV. The group 8 mutant cell line US31, on the other hand, is very deficient in repair of UV damage, showing a more extreme phenotype than is seen in the corresponding human syndrome CS-A. In both complementation groups, expression of mutations in a gene involved in regulation of DNA repair takes very different forms in human and rodent cells.

Animals↗
PubMed1996

Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes.

The association between high intake of fruit and vegetables and low incidence of certain cancers is well established. Dietary antioxidants present in these foods are thought to decrease free radical attack on DNA and hence to protect against mutations that cause cancer, but this causal mechanism remains conjectural. We have adopted a molecular epidemiological approach to this question, based on a modified alkaline single-cell gel electrophoresis assay ("comet assay") which specifically detects oxidation of pyrimidines in the DNA of human lymphocytes. In a survey of men 50-59 years of age living in the northeast of Scotland, smokers initially showed significantly more base damage than nonsmokers. Correlations between oxidative base damage and plasma concentrations of various antioxidants were generally negative but not statistically significant. Supplementation of the diet for 20 weeks with vitamin C (100 mg/day), vitamin E (280 mg/day), and beta-carotene (25 mg/day) resulted in a highly significant (P < 0.002) decrease in endogenous oxidative base damage in the lymphocyte DNA of both smokers and nonsmokers. In addition, lymphocytes of antioxidant-supplemented subjects showed an increased resistance to oxidative damage when challenged in vitro with H2O2. These findings strongly support the hypothesis that fruit and vegetables exert a cancer-protective effect via a decrease in oxidative damage to DNA.

Antioxidants↗
PubMed1996

Oxidative damage to DNA: do we have a reliable biomarker?

Oxidized bases in DNA can be measured directly by high-performance liquid chromatography (HPLC). 7,8-Dihydro-8-oxo-guanine (8-OHgua), as the most abundant oxidation product, is often regarded as an indicator of oxidative stress. Estimates of endogenous 8-OHgua levels in human lymphocyte DNA are between 2 and 8 for every 10(5) unaltered bases--a high frequency in view of the potential mutagenicity of this base alteration and of the presence of an effective base excision repair pathway in eukaryotic cells. An alternative approach to the measurement of oxidized bases makes use of repair endonucleases with appropriate lesion specificities--endonuclease III, for oxidized pyrimidines and formamidopyrimidine glycosylase for 8-OHgua. These enzymes introduce breaks at sites of damage in DNA. The comet assay (single cell gel electrophoresis) can then be used to detect the DNA breaks. This modified comet assay, like other enzyme-linked DNA breakage assays, gives a value for endogenous oxidative base damage that is more than 10-fold lower than most estimated from HPLC. It is possible that HPLC-based estimates are artificially high because oxidation of guanine occurs during isolation, storage, or hydrolysis of DNA. Using a revised DNA isolation procedure designed to decrease in vitro oxidation, we have obtained results for 8-OHgua concentrations in human lymphocytes that are closure to the figures obtained by the comet assay. It is still an open question whether 8-OHgua, measured by HPLC or by the comet assay, is a valid marker for oxidative damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1995

Identification of 120 kD and 30 kD receptors for human coronavirus OC43 in cell membrane preparations from newborn mouse brain.

A biotinylated virus overlay was used to identify a 120kD virus-binding molecule in dissociated newborn mouse brain (nmb) cell suspensions after separation of the proteins by polyacrylamide gel electrophoresis, electroblotting, and blockage of non-specific binding sites. The virus-binding molecule was not detected in adult mouse brain cell suspensions. Mannose- and glucose-rich glycoproteins from nmb cell membranes were selected by ConA-Sepharose (Pharmacia) chromatography. A 30kD virus-binding molecule was eluted by 0.2 M alpha-methyl-D-mannoside. O-linked sialic acid, a receptor component, was identified in the eluate.

Aging↗
PubMed1995

The influence of smoking and diet on the hypoxanthine phosphoribosyltransferase (hprt) mutant frequency in circulating T lymphocytes from a normal human population.

The influence of the dietary antioxidants vitamin C, alpha- and beta-carotene, lycopene, lutein/zeaxanthin, phytofluene, beta-cryptoxanthin, retinol and alpha- and gamma-tocopherol on the hypoxanthine phosphoribosyltransferase (hprt) mutant frequency in human peripheral T lymphocytes was investigated. Twenty-five male non-smokers and 27 male smokers in the age range 50-59 years were recruited. Smokers showed a significantly higher mutant frequency compared with non-smokers (X1.5, P < 0.01). In addition, there was a significant positive relationship between hprt mutant frequency and the number of cigarettes that individuals reported smoking daily (P < 0.01). Smokers showed significantly lower levels of plasma vitamin C and the carotenoid alpha-carotene than non-smokers (P < 0.01 and P < 0.05 respectively). Both hprt mutant frequency and lymphocyte plating efficiency were weakly inversely associated with plasma vitamin C levels (P < 0.07 and P < 0.06 respectively) suggesting that vitamin C may be protective against mutation at the hprt locus. This relationship was markedly stronger in smokers (P < 0.01).

Adult↗
PubMed1995

The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells.

Single cell gel electrophoresis is a sensitive method for detecting DNA strand breaks. Cells embedded in agarose are converted to nucleoids by treating with detergent and high salt. DNA breaks render the nucleoid DNA susceptible to extension by electrophoresis, forming 'comets'. We find that when DNA breakage resulting from H2O2 treatment is examined, freshly isolated normal human lymphocytes are relatively resistant compared with transformed human cells. When incubated after treatment with H2O2, HeLa cells repair most strand breaks within 1 h, and a substantial fraction of the oxidised pyrimidines (detected by converting them to DNA breaks with endonuclease III) within 4 h. However, lymphocytes are less proficient at repair; during incubation for 4 h after treatment with H2O2, no detectable removal of endonuclease III-sensitive sites is seen. While the addition of deoxyribonucleosides promotes completion of repair of UV damage by lymphocytes, it has no significant effect on repair of oxidative damage.

DNA↗
PubMed1995

DNA damage and repair after low doses of UV-C radiation; comparable rates of repair in rodent and human cells.

The UV endonuclease assay for cyclobutane pyrimidine dimers in the DNA of UV-C-irradiated mammalian cells has been modified by replacing alkaline sucrose sedimentation with alkaline unwinding and hydroxylapatite chromatography to determine the number of DNA breaks introduced by the endonuclease. Dimers induced by doses as low as 0.25 Jm-2 can be detected and the assay has been used to examine the capacity of human, hamster and mouse cells to remove damage inflicted by sublethal doses of UV-C. In addition, incision activity has been measured by incubating cells with DNA synthesis inhibitors after irradiation with UV-C. In rodent and human cells, given a dose of UV-C of 1 Jm-2 about half of the endonuclease-sensitive sites are lost in 5-6 h. The incision capacity of these cells corresponds well with the extent of removal of dimers. Thus, although rodent cells are normally considered to be relatively deficient in nucleotide excision repair, we find that rodent and human cells have comparable capacities to deal with low levels of UV-C-induced damage.

3T3 Cells↗
PubMed1995

Identification of salivary proteins inhibiting herpes simplex virus 1 replication.

Salivary proteins play an important role in the maintenance of the oral ecology. Previous studies have indicated that human submandibular-sublingual and parotid salivas can selectively suppress the in vitro infectivity of herpes simplex virus 1. The purpose of this study was to identify the salivary components in human submandibular-sublingual saliva that modulate in vitro infectivity. Assessment of the interaction of viral particles with salivary components was accomplished using an in vitro solid-phase assay. These experiments revealed that herpes simplex virus particles selectively interact with the members of the salivary proline-rich protein and cystatin families. Subsequent yield reduction assays demonstrated the ability of proline-rich proteins and salivary cystatins to inhibit the viral replication, with basic proline-rich peptides being more effective. Subsequent assays suggest that basic proline-rich peptides reduced the virus titer by interfering with penetration and/or cellular processing of virus within the target cell. Collectively, these results further suggest that salivary proteins have an important role in the host defense mechanism against recurrent herpesvirus infection.

Animals↗