•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.
Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been ...conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12µg/cm2 At these range we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure.
•MWCNT were tested in V79 cells.•Cellular uptake of MWCNT was detected using TEM.•Intracellular ROS induction was observed after MWCNT exposure.•MWCNT induced a concentration-dependent increase of HPRT mutations.
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 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.
•TiO2 NPs dispersed according to DP1 induced DNA strand breaks in comet assay.•There were no significant increases in MNBNC neither in PBL nor TK6 cells.•DNA strand breaks in rat PBMCs were elevated ...after one day of exposure to TiO2 NPs.•No cytotoxicity or micronucleus formation was seen in bone marrow PCEs.
The genotoxicity of TiO2 nanoparticles (NPs) was assessed with the cytokinesis-block micronucleus (CBMN) assay in TK6 lymphoblastoid cells, lymphocytes from human volunteers, and bone marrow erythrocytes from rats exposed in vivo; and with the comet assay (detecting both strand breaks and oxidised purines) in human and rat peripheral blood mononuclear cells (PBMCs). NPs were dispersed using three different methods giving different size distribution and stability.
On average, TiO2 NPs caused no increase in micronuclei in TK6 cells, rat bone marrow erythrocytes or human lymphocytes (though lymphocytes from 3 out of 13 human subjects showed significant increases).
PBMCs from rats treated in vivo with a single dose of NPs dispersed by a method with low agglomeration showed an increase in strand breaks after 1 day.
TiO2 NPs dispersed in a stable, non-agglomerated state induced DNA strand breaks at 75 μg/cm2 after 4 h exposure of human PBMCs and at 15 μg/cm2 and 75 μg/cm2 after 24 h exposure, but no increase in DNA oxidation was seen. Overall, NPs in an agglomerated state did not cause DNA damage. However, at the individual level, significant increases in strand breaks were seen in PBMCs from most of the volunteers. Cells from one volunteer showed positive effects in all conditions and both tests, while cells from another volunteer appeared to be completely resitant to TiO2 NPs. The implication is that some individuals may be more sensitive than others to effects of this nanomaterial.
Differences seen in results obtained with the micronucleus and the comet assay may be due to the mechanisms underlying the genotoxic effects of TiO2 NPs and the different endpoints represented by the two assays.
Life expectancy in central-Eastern European countries is more than 10 years lower compared with Northern or Western countries which could be the result of complex factors including genetics, ...nutrition and life style. We conducted a molecular epidemiological study with the aim of investigating links between DNA instability, genetic polymorphisms in nucleotide excision repair genes and ageing. Two groups-151 young people (78 women and 73 men) aged 20-25, and 140 elderly subjects (101 women and 39 men), aged 65-70 have been investigated. Results show elevated levels of micronuclei and chromosome aberrations in elderly compared with young groups (P<0.001); women had more micronuclei than men (P<0.001). Micronucleus frequencies were influenced by age (P<0.001). In the group of elderly people those who were homozygous with C/C or A/A in XPC IVS11 had more aberrant cells compared with C/A heterozygotes (P=0.04). When the dependent variable was break per cell, elderly people A/A homozygous in XPC IVS11 had more breaks per cell compared with C/A heterozygous or C/C homozygous subjects (P=0.03). Significantly the most chromatid breaks were found in elderly people both Lys/Lys homozygous in the XPD Lys751Gln genotype and C/C or A/A homozygous in the XPC IVS11 genotype (P<0.05). A General Linear Model analysis shows a statistically significant effect of interactions between age, sex and genotype XPC IVS11 (P=0.001) and age, sex and genotype XPCin9 (P=0.007) on number of chromatid breaks. When we divided people into two subgroups (without mutant allele and with one or two mutant alleles) we found a significantly higher number of chromosome exchanges in people with one or two variant polymorphism XPCin9 (P=0.04), XPC IVS11 (P=0.004) or XPCex15 (P=0.001). Level of cells with micronuclei was influenced by polymorphisms XPD Lys751Gln (P=0.03). However, we did not find any relationship between XPA polymorphism and studied cytogenetic biomarkers.
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.
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