DNA damage and unrepaired or insufficiently repaired DNA double-strand breaks as well as telomere shortening contribute to the formation of structural chromosomal aberrations (CAs). Non-specific CAs ...have been used in the monitoring of individuals exposed to potential carcinogenic chemicals and radiation. The frequency of CAs in peripheral blood lymphocytes (PBLs) has been associated with cancer risk and the association has also been found in incident cancer patients. CAs include chromosome-type aberrations (CSAs) and chromatid-type aberrations (CTAs) and their sum CAtot. In the present study, we used data from our published genome-wide association studies (GWASs) and extracted the results for 153 DNA repair genes for 607 persons who had occupational exposure to diverse harmful substances/radiation and/or personal exposure to tobacco smoking. The analyses were conducted using linear and logistic regression models to study the association of DNA repair gene polymorphisms with CAs. Considering an arbitrary cutoff level of 5 × 10
–3
, 14 loci passed the threshold, and included 7 repair pathways for CTA, 4 for CSA, and 3 for CAtot; 10 SNPs were eQTLs influencing the expression of the target repair gene. For the base excision repair pathway, the implicated genes
PARP1
and
PARP2
encode poly(ADP-ribosyl) transferases with multiple regulatory functions.
PARP1
and
PARP2
have an important role in maintaining genome stability through diverse mechanisms. Other candidate genes with known roles for CSAs included
GTF2H
(general transcription factor IIH subunits 4 and 5), Fanconi anemia pathway genes, and
PMS2
, a mismatch repair gene. The present results suggest pathways with mechanistic rationale for the formation of CAs and emphasize the need to further develop techniques for measuring individual sensitivity to genotoxic exposure.
The genotoxicity of anatase/rutile TiO
nanoparticles (TiO
NPs, NM105 at 3, 15 and 75 µg/cm
) was assessed with the mammalian in-vitro Hypoxanthine guanine phosphoribosyl transferase (
) gene mutation ...test in Chinese hamster lung (V79) fibroblasts after 24 h exposure. Two dispersion procedures giving different size distribution and dispersion stability were used to investigate whether the effects of TiO
NPs depend on the state of agglomeration. TiO
NPs were fully characterised in the previous European FP7 projects NanoTEST and NanoREG2. Uptake of TiO
NPs was measured by transmission electron microscopy (TEM). TiO
NPs were found in cytoplasmic vesicles, as well as close to the nucleus. The internalisation of TiO
NPs did not depend on the state of agglomeration and dispersion used. The cytotoxicity of TiO
NPs was measured by determining both the relative growth activity (RGA) and the plating efficiency (PE). There were no substantial effects of exposure time (24, 48 and 72 h), although a tendency to lower RGA at longer exposure was observed. No significant difference in PE values and no increases in the
gene mutant frequency were found in exposed relative to unexposed cultures in spite of evidence of uptake of NPs by cells.
•The alkaline comet assay is a popular methods for assessing DNA damage in humans.•A database of 19,320 subjects from 44 laboratories in 26 countries was analysed.•A range of measures estimating ...baseline DNA damage is provided for %T, TL, and TM.•None or limited effect was found for sex, age, and major confounding factors.•The comet assay efficiently detected specific genotoxic exposures.
The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations.
The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall ...mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan-Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06-1.90) for overall mortality, and 1.94 (1.04-3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.
As part of a large human biomonitoring study, we conducted occupational monitoring in a glass fibre factory in Slovakia. Shopfloor workers (n = 80), with a matched group of administrators in the same ...factory (n = 36), were monitored for exposure to glass fibres and to polycyclic aromatic hydrocarbons (PAHs). The impact of occupational exposure on chromosomal aberrations, DNA damage and DNA repair, immunomodulatory markers, and the role of nutritional and lifestyle factors, as well as the effect of polymorphisms in metabolic and DNA repair genes on genetic stability, were investigated.
The (enzyme-modified) comet assay was employed to measure DNA strand breaks (SBs) and apurinic sites, oxidised and alkylated bases. Antioxidant status was estimated by resistance to H2O2-induced DNA damage. Base excision repair capacity was measured with an in vitro assay (based on the comet assay).
Exposure of workers to fibres was low, but still was associated with higher levels of SBs, and SBs plus oxidised bases, and higher sensitivity to H2O2. Multivariate analysis showed that exposure increased the risk of high levels of SBs by 20%. DNA damage was influenced by antioxidant enzymes catalase and glutathione S-transferase (measured in blood). DNA repair capacity was inversely correlated with DNA damage and positively with antioxidant status. An inverse correlation was found between DNA base oxidation and the percentage of eosinophils (involved in the inflammatory response) in peripheral blood of both exposed and reference groups. Genotypes of XRCC1 variants rs3213245 and rs25487 significantly decreased the risk of high levels of base oxidation, to 0.50 (p = 0.001) and 0.59 (p = 0.001), respectively.
Increases in DNA damage owing to glass fibre exposure were significant but modest, and no increases were seen in chromosome aberrations or micronuclei. However, it is of concern that even low levels of exposure to these fibres can cause significant genetic damage.
•Exposure of workers to glass fibres was associated with increased levels of DNA damage and higher sensitivity to H2O2.•DNA damage was influenced by catalase activity and glutathione S-transferase levels measured in peripheral blood.•XRCC1 variants rs3213245 and rs25487 were associated with a decrease in the risk of high DNA oxidation damage.•Glass fibre exposure did not affect the levels of chromosome aberrations or micronuclei in exposed workers.
The in vitro genotoxicity of PLGA–PEO (poly-lactic-co-glycolic acid–polyethylene oxide copolymer) nanoparticles was assessed in TK6 cells using the comet assay as well as cytokinesis-block ...micronucleus (CBMN) assay. The cells were exposed to 0.12–75μg/cm2 of PLGA–PEO nanoparticles during 2 and 24h for analysis in the comet assay, and to 3–75μg/cm2 of these nanoparticles during 4, 24, 48 and 72h, respectively, for analysis in the CBMN assay. Two different protocols for treatment with cytochalasin B were used. We found that PLGA–PEO was neither cytotoxic (measured by relative cell growth activity and cytokinesis-block proliferation index (CBPI)), nor did it induce DNA strand-breaks (detected by the comet assay) or oxidative DNA lesions (measured by the comet assay modified with lesion-specific enzyme formamidopyrimidine-DNA-glycosylase). There were no statistically significant differences in the frequencies of micronucleated binucleated cells (MNBNCs) between untreated and treated cells in either of the conditions used. This suggests that PLGA–PEO did not have potential genotoxicity. However, using two experimental protocols of the micronucleus assay, PLGA–PEO nanoparticles showed a weak but significant increase in the level of MN in mononucleated cells, in cells treated for 48h with PLGA–PEO nanoparticles when cytochalasin B was added for the last 24h (1st protocol), and in cells treated for 24h with PLGA–PEO nanoparticles followed by washing of NPs and addition of cytochalasin B for another 24h (2nd protocol). It remains unclear whether the increase of MNMNC after treatment with PLGA–PEO nanoparticles is the effect of a possible, weak aneugenic potential or early effect of these particles, or due to another reason. These results suggest that aneugenicity in addition to clastogenicity may be considered as an important biomarker when assessing the genotoxic potential of polymeric nanoparticles.
•Age influences CA formation in healthy individuals.•A wide variety of DNA repair pathways are associated with CA frequency.•Implicated BER, NER pathways’ genes point to CA association with ...telomerase function.
Nonspecific structural chromosomal aberrations (CAs) can be found at around 1% of circulating lymphocytes from healthy individuals but the frequency may be higher after exposure to carcinogenic chemicals or radiation. The frequency of CAs has been measured in occupational monitoring and an increased frequency of CAs has also been associated with cancer risk. Alterations in DNA damage repair and telomere maintenance are thought to contribute to the formation of CAs, which include chromosome type of aberrations and chromatid type of aberrations. In the present study, we used the result of our published genome-wide association studies to extract data on 153 DNA repair genes from 866 nonsmoking persons who had no known occupational exposure to genotoxic substances. Considering an arbitrary cut-off level of P< 5 × 10−3, single nucleotide polymorphisms (SNPs) tagging 22 DNA repair genes were significantly associated with CAs and they remained significant at P < 0.05 when adjustment for multiple comparisons was done by the Binomial Sequential Goodness of Fit test. Nucleotide excision repair pathway genes showed most associations with 6 genes. Among the associated genes were several in which mutations manifest CA phenotype, including Fanconi anemia, WRN, BLM and genes that are important in maintaining genome stability, as well as PARP2 and mismatch repair genes. RPA2 and RPA3 may participate in telomere maintenance through the synthesis of the C strand of telomeres. Errors in NHEJ1 function may lead to translocations. The present results show associations with some genes with known CA phenotype and suggest other pathways with mechanistic rationale for the formation of CAs in healthy nonsmoking population.
Human cancers are often associated with numerical and structural chromosomal instability. Structural chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBL) arise as consequences of ...direct DNA damage or due to replication on a damaged DNA template. In both cases, DNA repair is critical and inter-individual differences in its capacity are probably due to corresponding genetic variations. We investigated functional variants in DNA repair genes (base and nucleotide excision repair, double-strand break repair) in relation to CAs, chromatid-type aberrations (CTAs) and chromosome-type aberrations (CSAs) in healthy individuals. Chromosomal damage was determined by conventional cytogenetic analysis. The genotyping was performed by both restriction fragment length polymorphism and TaqMan allelic discrimination assays. Multivariate logistic regression was applied for testing individual factors on CAs, CTAs and CSAs. Pair-wise genotype interactions of 11 genes were constructed for all possible pairs of single-nucleotide polymorphisms. Analysed individually, we observed significantly lower CTA frequencies in association with XPD Lys751Gln homozygous variant genotype odds ratio (OR) 0.64, 95% confidence interval (CI) 0.48-0.85, P = 0.004; n = 1777. A significant association of heterozygous variant genotype in RAD54L with increased CSA frequency (OR 1.96, 95% CI 1.01-4.02, P = 0.03) was determined in 282 subjects with available genotype. By addressing gene-gene interactions, we discovered 14 interactions significantly modulating CAs, 9 CTAs and 12 CSAs frequencies. Highly significant interactions included always pairs from two different pathways. Although individual variants in genes encoding DNA repair proteins modulate CAs only modestly, several gene-gene interactions in DNA repair genes evinced either enhanced or decreased CA frequencies suggesting that CAs accumulation requires complex interplay between different DNA repair pathways.
Purpose
To determine the DNA protective effects of a standard coffee beverage in comparison to water consumption.
Methods
The single-blind, randomised controlled study with parallel design included ...healthy women (
n
= 50) and men (
n
= 50) recruited from the general Central European population. The subjects were randomised in a coffee and a control group, with stratification for sex and body mass index. The study comprised two periods of 4 weeks: a preconditioning period, with daily consumption of at least 500 ml water but no coffee, nor tea, nor any other caffeine-containing product. During the subsequent intervention period the coffee group consumed 500 ml of freshly brewed dark roast coffee blend per day, the control group consumed water instead. On the last day of each period, blood was drawn and analysed by comet assay (single-cell gel electrophoresis) to assess the level of DNA damage (strand breakage).
Results
At the end of the intervention period the mean level of DNA strand breaks in the coffee group has decreased in comparison to the control group difference in means 0.23% TI (tail intensity),
p
= 0.028. The mean change from baseline (delta value) was − 23% in the coffee group (
p
= 0.0012). Effects of coffee intake were similar for men and women. During intervention, neither group showed any significant change in body weight or calorie intake.
Conclusions
Our results indicate that regular consumption of a dark roast coffee blend has a beneficial protective effect on human DNA integrity in both, men and women.
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