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The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: −0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%–2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern. [ABSTRACT FROM AUTHOR]

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%–25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I–III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I–III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%–9.6%) as compared to 1 week (CV = 1.3%–6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%–20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets. [ABSTRACT FROM AUTHOR]

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern. [ABSTRACT FROM AUTHOR]

Biofuel is an attractive substitute for petrodiesel because of its lower environmental footprint. For instance, the polycyclic aromatic hydrocarbons (PAH) emission per fuel energy content is lower for rapeseed methyl ester (RME) than for petrodiesel. This study assesses genotoxicity by extractable organic matter (EOM) of exhaust particles from the combustion of petrodiesel, RME, and hydrogenated vegetable oil (HVO) in lung epithelial (A549) cells. Genotoxicity was assessed as DNA strand breaks by the alkaline comet assay. EOM from the combustion of petrodiesel and RME generated the same level of DNA strand breaks based on the equal concentration of total PAH (i.e. net increases of 0.13 [95% confidence interval (CI): 0.002, 0.25, and 0.12 [95% CI: 0.01, 0.24] lesions per million base pairs, respectively). In comparison, the positive control (etoposide) generated a much higher level of DNA strand breaks (i.e. 0.84, 95% CI: 0.72, 0.97) lesions per million base pairs. Relatively low concentrations of EOM from RME and HVO combustion particles (<116 ng/ml total PAH) did not cause DNA strand breaks in A549 cells, whereas benzo[a]pyrene and PAH-rich EOM from petrodiesel combusted using low oxygen inlet concentration were genotoxic. The genotoxicity was attributed to high molecular weight PAH isomers with 5–6 rings. In summary, the results show that EOM from the combustion of petrodiesel and RME generate the same level of DNA strand breaks on an equal total PAH basis. However, the genotoxic hazard of engine exhaust from on-road vehicles is lower for RME than petrodiesel because of lower PAH emission per fuel energy content. [ABSTRACT FROM AUTHOR]

Humans ingest particles and fibers on daily basis. Non-digestible carbohydrates are beneficial to health and food additives are considered safe. However, titanium dioxide (E171) has been banned in the European Union because the European Food Safety Authority no longer considers it non-genotoxic. Ingestion of microplastics and nanoplastics are novel exposures; their potential hazardous effects to humans have been under the radar for many years. In this review, we have assessed the association between oral exposure to man-made particles/fibers and genotoxicity in gastrointestinal tract cells and secondary tissues. We identified a total of 137 studies on oral exposure to particles and fibers. This was reduced to 49 papers with sufficient quality and relevance, including exposures to asbestos, diesel exhaust particles, titanium dioxide, silver nanoparticles, zinc oxide, synthetic amorphous silica and certain other nanomaterials. Nineteen studies show positive results, 25 studies show null results, and 5 papers show equivocal results on genotoxicity. Recent studies seem to show null effects, whereas there is a higher proportion of positive genotoxicity results in early studies. Genotoxic effects seem to cluster in studies on diesel exhaust particles and titanium dioxide, whereas studies on silver nanoparticles, zinc oxide and synthetic amorphous silica seem to show mainly null effects. The most widely used genotoxic tests are the alkaline comet assay and micronucleus assay. There are relatively few results on genotoxicity using reliable measurements of oxidatively damaged DNA, DNA double strand breaks (γH2AX assay) and mutations. In general, evidence suggest that oral exposure to particles and fibers is associated with genotoxicity in animals. [ABSTRACT FROM AUTHOR]

DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1–2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer's protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer's protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO 3 , 0–4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer's protocol), using TK6 cells with MMS (100–800 µM, 1 h) and hydrogen peroxide (H 2 O 2, 7.7–122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required. [ABSTRACT FROM AUTHOR]

The comet assay is widely used in studies on genotoxicity testing, human biomonitoring and clinical studies. The simple version of the assay detects a mixture of DNA strand breaks and alkali-labile sites; these lesions are typically described as DNA strand breaks to distinguish them from oxidatively damaged DNA that are measured with the enzyme-modified comet assay. This review assesses the association between high-prevalence diseases in high-income countries and DNA damage measured with the comet assay in humans. The majority of case–control studies have assessed genotoxicity in white blood cells. Patients with coronary artery disease, diabetes, kidney disease, chronic obstructive pulmonary disease and Alzheimer's disease have on average 2-fold higher levels of DNA strand breaks compared with healthy controls. Patients with coronary artery disease, diabetes, kidney disease and chronic obstructive pulmonary disease also have 2- to 3-fold higher levels of oxidatively damaged DNA in white blood cells than controls, although there is not a clear difference in DNA damage levels between the different diseases. Case–control studies have shown elevated levels of DNA strand breaks in patients with breast cancer, whereas there are only few studies on colorectal and lung cancers. At present, it is not possible to assess if these neoplastic diseases are associated with a different level of DNA damage compared with non-neoplastic diseases. [ABSTRACT FROM AUTHOR]

• Oxidatively damaged DNA was measured with the comet assay in blood cells. • Intake of fruit, vegetable, whole grains, fish, salad and potatoes were assessed. • Levels of Fpg-sensitive sites were inversely associated with intake of fish. • Levels of Fpg-sensitive sites were inversely associated with intake of salad. • There was no association between dietary items and DNA repair activity. This study investigated associations between levels of oxidatively damaged DNA, measured by the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay and intake of fish, salad, fruits, vegetables, wholegrain items, and potatoes in a cross-sectional study of 382 men and 591 women between 18 and 93 years. Intake of dietary items was obtained from questionnaires, and stratified into less than once per week, weekly or daily consumption. Intake of fish as main course was inversely associated with levels of Fpg-sensitive sites in peripheral blood mononuclear cells (PBMCs) in especially women (P < 0.001 multivariate linear regression). Intake of fish was also inversely associated with lower levels of Fpg-sensitive sites in men (P < 0.05, univariate analysis), although it was not statistically significant in analysis adjusted for lifestyle and other dietary factors. Intake of salad was inversely associated with levels of Fpg-sensitive sites in men (P < 0.001, multivariate linear regression). Statistically significant associations were also observed for intake of vegetables and potatoes in men, although these were weak and not robust in all statistical models. The sum the six individual dietary items was inversely associated with levels of Fpg-sensitive sites in the strata of men (P < 0.001, multivariate linear regression). Finally, levels of DNA repair incision activity were not associated with individual food categories or the total dietary food score. In summary, consumption of health-promoting foods is associated with lower levels of Fpg-sensitive sites in human PBMCs and strongest effects in the present population were ingestions of fish and salad. [ABSTRACT FROM AUTHOR]

Highlights • This review describes studies of the comet assay detected DNA damage in leukocytes. • DNA strand break levels are positively associated with age. • DNA strand break levels are higher in men than women. • Age and sex may affect the level of DNA strand break through lifestyle factors. • There are inconsistent data on the effect of age and sex on oxidatively damaged DNA. Abstract DNA damage measured by the comet assay is a well-established biomarker in studies on environmental and occupational exposures, dietary factors and clinical outcomes. Age and sex are typically regarded as confounding factors that are controlled by either selective inclusion criteria of subjects or adjustment in the statistical analysis. This review assesses the influence of age and sex on levels of DNA damage in leukocytes in study populations of healthy subjects. Analysis of unadjusted results in the studies indicates that the level of DNA strand breaks increases by 1% per year (95% CI: 0.9%–1.1%, linear regression analysis weighted for the number of subjects in the individual studies). The studies also show a slightly higher level of DNA strand breaks in men than women (8%, 95% CI: 0.4%–17%) in unadjusted analyses, which is not robust in studies with adjusted analyses. The attenuation of effect in adjusted analyses of DNA strand breaks in leukocytes indicates that the effect of age and sex may be driven by differences in lifestyle factors or other exposures. There do not appear to be differences related to age and sex on basal levels of oxidatively damaged DNA in leukocytes. In summary, the results indicate influences of both age and sex on DNA damage in the comet assay, which may be mediated by lifestyle factors or external exposures rather than direct effects of age and sex. [ABSTRACT FROM AUTHOR]

During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years. [ABSTRACT FROM AUTHOR]

The formamidopyrimidine DNA glycosylase (Fpg) and human 8-oxoguanine DNA glycosylase (hOGG1)-modified comet assays have been widely used in human biomonitoring studies. The purpose of this article is to assess differences in reported levels of Fpg and hOGG1-sensitive sites in leukocytes and suggest suitable assay controls for the measurement of oxidatively damaged DNA. An assessment of the literature showed a large variation in the reported levels of Fpgsensitive sites (range 0.05-1.31 lesions/106 bp). The levels of Fpg-sensitive sites are lower in studies where Fpg has been obtained from commercial suppliers or unknown sources as compared to Fpg from one particular non-commercial source (x2 = 7.14, P = 0.028). The levels of hOGG1-sensitive sites are lower (range: 0.04-0.18 lesions/106 bp in leukocytes) compared to the Fpg-sensitive sites. Surprisingly, few publications have reported the use of oxidising agents as assay controls, with the exception of hydrogen peroxide. This may be due to a lack of consensus about suitable controls for the Fpg and hOGG1-modified comet assay. A major challenge is to find an oxidising agent that only oxidises nucleobases and does not generate DNA strand breaks because this reduces the dynamic range of Fpg and hOGG1-sensitive sites in the comet assay. Based on a literature search we selected the photosensitiser Ro19-8022 plus light, KBrO3 , 4-nitroquinoline-1-oxide, 2 Cr2 O7 and ferric nitrilotriacetate as possible assay controls. A subsequent assessment of these compounds for generating cryopreserved assay controls in mononuclear blood cells showed that Ro19-8022 plus light, KBrO3 and 4-nitroquinoline-1-oxide provided suitable assay controls. We recommend these compounds as comet assay controls for oxidatively damaged DNA. [ABSTRACT FROM AUTHOR]

Carbon nanotube (CNT) is a nanomaterial that has received interest because of its high-tensile strength and low weight. Although CNTs differ substantially in physico-chemical properties, they share high aspect ratio which resembles that of asbestos and other fibers causing lung cancer and mesothelioma. One type of multi-walled CNTs (i.e. MWCNT-7) has been classified as possibly carcinogenic to humans by IARC (Group 2B) based on experimental animal data, whereas other types of MWCNTs and single-walled CNTs (SWCNT) could not be classified due to lack of data from epidemiologic studies and insufficient mechanistic evidence. Damage to DNA is considered to be a key mechanistic step in the development of fiber-induced cancer. Thus, the genotoxic potential can be a cornerstone in the evaluation of hazards of CNTs. The present study used a weight of evidence (WoE) analysis to evaluate the genotoxicity of different types of CNTs. Genotoxicity endpoints close to cancer (mutations and chromosome aberrations) and animal models had highest weight in the WoE analysis. Eight CNT materials out of 130, which had been assessed in several studies, were evaluated in the WoE analysis. The results demonstrated that MWCNT-7 has strongest WoE for a genotoxic hazard among the MWCNTs. Two types of SWCNTs have a similar WoE for genotoxicity as MWCNT-7. Several reference materials from the Joint Research Centre have less WoE for genotoxicity. The WoE analysis demonstrates a difference in genotoxicity for CNTs, but further research is required to unravel the physico-chemical characteristics that govern the differences in genotoxic hazard. [ABSTRACT FROM PUBLISHER]

The development of products containing carbon nanotubes (CNTs) is a major achievement of nanotechnology, although concerns regarding risk of toxic effects linger if the hazards associated with these materials are not thoroughly investigated. Exposure to CNTs has been associated with depletion of antioxidants, increased intracellular production of reactive oxygen species and pro-inflammatory signaling in cultured cells with primary function in the immune system as well as epithelial, endothelial and stromal cells. Pre-treatment with antioxidants has been shown to attenuate these effects, indicating a dependency of oxidative stress on cellular responses to CNT exposure. CNT-mediated oxidative stress in cell cultures has been associated with elevated levels of lipid peroxidation products and oxidatively damaged DNA. Investigations of oxidative stress endpoints in animal studies have utilized pulmonary, gastrointestinal, intravenous and intraperitoneal exposure routes, documenting elevated levels of lipid peroxidation products and oxidatively damaged DNA nucleobases especially in the lungs and liver, which to some extent occur concomitantly with altered levels of components in the antioxidant defense system (glutathione, superoxide dismutase or catalase). CNTs are biopersistent high aspect ratio materials, and some are rigid with lengths that lead to frustrated phagocytosis and pleural accumulation. There is accumulating evidence showing that pulmonary exposure to CNTs is associated with fibrosis and neoplastic changes in the lungs, and cardiovascular disease. As oxidative stress and inflammation responses are implicated in the development of these diseases, converging lines of evidence indicate that exposure to CNTs is associated with increased risk of cardiopulmonary diseases through generation of a pro-inflammatory and pro-oxidant milieu in the lungs. [ABSTRACT FROM AUTHOR]

• The Fpg-modified comet assay is a sensitive technique for measurement of DNA damage. • Levels of Fpg-sensitive sites detected in human cells appears to differ between labs. • Calibration using ionizing radiation reduces heterogeneity in DNA damage between labs. • Lesions/106 base pair is the most informative comet assay descriptor. The comet assay is a simple technique for measurements of low levels of DNA damage and repair in single cells. However, there is variation in background levels of DNA damage in peripheral blood mononuclear cells (PBMCs). This variation has been documented by inter-laboratory ring-trials where identical samples have been analysed in different laboratories using the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. The coefficient of variation of background levels of Fpg-sensitive sites was 128 % in the first inter-laboratory validation trial called European Standards Committee on Oxidative DNA Damage. The variation was reduced to 44 % by the end of the project. Subsequent ring-trials by the European Comet Assay Validation Group showed similar inter-laboratory variation in Fpg-sensitive sites in PBMCs (45 %). The lowest inter-laboratory variation in Fpg-sensitive sites in PBMCs was 12 % when using calibration to standardize comet assay descriptors. Introduction of standard comet assay procedures was surprisingly unsuccessful as certain laboratories experienced technical problems using unaccustomed assay conditions. This problem was alleviated by using flexible assay standard conditions rather than a standard protocol in a ring-trial by the hCOMET group. The approach reduced technical problems, but the inter-laboratory variation in Fpg-sensitive sites was not reduced. The ring-trials have not pinpointed specific assay steps as major determinants of the variation in DNA damage levels. It is likely that small differences in several steps cause inter-laboratory variation. Although this variation in reported DNA damage levels causes concern, ring-trials have also shown that the comet assay is a reliable tool in biomonitoring studies. [ABSTRACT FROM AUTHOR]

Levels of oxidatively damaged cellular DNA and urinary excretion of damaged 2′-deoxyribonuclosides are widely measured in biomonitoring studies examining the role of oxidative stress induced by environmental exposures, lifestyle factors and development of disease. This has promoted efforts to harmonise measurements of oxidised guanine nucleobases by the variety of analytical approaches for DNA and urinary levels of damage, in multi-laboratory trials that are centred in Europe. The large inter-laboratory variation reported of values of oxidatively damaged DNA is reduced by harmonising assay protocols. Recent attention on optimal conditions for the comet assay may lead to better understanding of the most critical steps in procedure, which generate variation in DNA damage levels between laboratories. Measurements of urinary excretion of oxidatively generated 8-oxo-7,8-dihydro-2′-deoxyguanosine also show large differences between different methods, where chromatographic techniques generally show more reliable results than antibody-based methods. In this case, standardising calibrants is aimed at improving within technique agreement. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies reporting a baseline level of damaged DNA that was fewer than 5 lesions/106 dG showed that 21 of 29 studies reported age-associated accumulation of DNA damage. The standardized mean difference in oxidatively damaged DNA between the oldest and the youngest age groups was 1.49 (95% CI 1.03–1.95). There was no difference between age span, number of tested organs, statistical power, sex, strain, or breeding between the studies showing positive and null effects. Citation and publication bias seems to be a problem in the overall dataset, whereas it is less pronounced in the restricted dataset. There is compelling evidence for aging-associated accumulation of oxidatively damaged DNA in organs with limited cell proliferation. [Copyright &y& Elsevier]

The objective of this review was to evaluate the association between hypoxia and oxidative damage to DNA and lipids. Evaluation criteria encompassed specificity and validation status of the biomarkers, study design, strength of the association, dose-response relationship, biological plausibility, analogous exposures, and effect modification by intervention. The collective interpretation indicates persuasive evidence from the studies in humans for an association between hypoxia and elevated levels of oxidative damage to DNA and lipids. The levels of oxidatively generated DNA lesions and lipid peroxidation products depend on both the duration and severity of the exposure to hypoxia. Largest effects are observed with exposure to hypoxia at high altitude, but other factors, including ultraviolet light, exercise, exertion, and low intake of antioxidants, might contribute to the effect observed in subjects at high altitude. Most of the animal experimental models should be interpreted with caution because the assays for assessment of lipid peroxidation products have suboptimal validity. © 2008 IUBMB IUBMB Life, 60(11): 707–723, 2008 [ABSTRACT FROM AUTHOR]

Abstract: There is growing concern that air pollution exposure increases the risk of lung cancer. The mechanism of action is related to particle-induced oxidative stress and oxidation of DNA. Humans exposed to urban air with vehicle emissions have elevated levels of oxidized guanine bases in blood cells and urine. Animal experimental studies show that pulmonary and gastrointestinal exposure is associated with elevated levels of oxidized guanines in the lung and other organs. Collectively, there is evidence indicating that exposure to traffic-related air pollution particles is associated with oxidative damage to DNA and this might be associated with increased risk of cancer. [Copyright &y& Elsevier]

Oxidative damage to DNA is regarded as an important step in carcinogenesis. These lesions may arise as a consequence of exposure to xenobiotics, but are also generated as a consequence of endogenous generation of oxidizing compounds. Measurements of oxidative damage to guanines, such as 8-oxo-7, 8-dihydroguanine (8-oxodG) are increasingly being regarded as reliable biomarkers of oxidative stress and they may have a predictive value of cancer risk, although this needs to be established independently in several cohort studies. A survey of intervention studies of the ingestion of antioxidant-containing foods or tablets of antioxidants indicate that about one-third of the studies reported a protective effect in terms of lower levels of oxidative damage to DNA in white blood cells or decreased urinary excretion of 8-oxodG. Although firm conclusions cannot be reached, there appears to be links between ingestion of antioxidants, oxidative damage to DNA, and risk of cancer. [ABSTRACT FROM AUTHOR]

Abstract: Pulmonary exposure to diesel exhaust particles (DEP) has been associated with high levels of oxidized DNA in lung cells, whereas long-term oral DEP exposure appears to induce the DNA repair system with concomitant unaltered levels of oxidized DNA in the colon and liver of rats. Here we studied the generation of oxidatively damaged DNA in young wild type (WT) and oxoguanine DNA glycosylase 1 (OGG1) deficient mice after dietary exposure to 0mg/kg, 0.8mg/kg, or 8mg/kg Standard Reference Material 1650 in the feed for 21 days. The ingestion of DEP did not increase the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine and comet assay endpoints in terms of strand break, endonuclease III, and formamidopyrimidine glycosylase (FPG) in the colon, liver, and lung tissue of WT or Ogg1 −/− mice. The level of OGG1 mRNA could only be measured in WT mice and it was not increased by DEP feeding. On the contrary, the level of FPG sites was twofold higher in the liver and lung of Ogg1 −/− mice compared to the levels in the WT mice tissues. In conclusion, although Ogg1 −/− mice have high levels of oxidized guanine lesions, they do not appear to be markedly vulnerable to the genotoxicity by oral administration of DEP. [Copyright &y& Elsevier]

Abstract: Many biomonitoring studies have investigated the role of antioxidants in reducing oxidatively generated DNA damage in urine and white blood cells. A collective interpretation is difficult because many studies lack sufficient control and have unreasonably high baseline levels of oxidatively damaged DNA. In a survey of this antioxidant hypothesis, we identified 139 cross-sectional and intervention studies. Restricted selection criteria with exclusion of studies having suboptimal design or unreasonably high baseline damage level provided 85 eligible studies for analysis. Ten of the 27 cross-sectional studies reported negative correlations between antioxidants and oxidatively damaged DNA, albeit with correlation coefficients explaining less than 20% of the variance. Sixty-two intervention studies reported mixed results, which did not depend on sample size or duration of the intervention. Reduced levels of oxidatively damaged DNA in white blood cells and urine were reported in far more studies than expected by chance alone. Supplementation with antioxidant-rich foods was more effective than that with single antioxidants in lowering urinary excretion of oxidatively damage DNA. In conclusion, this survey indicates that ingestion of antioxidants may be associated with reduced level of DNA damage in white blood cells and urine of humans, albeit the effect is lower than previously expected. [Copyright &y& Elsevier]

Abstract: Genotoxicity measured by the comet assay is expressed by different researchers using parameters that are not easy to conceptualize, except for percent tail DNA (%T) or visual score (arbitrary units). A total of 125 publications have reported genotoxicity as DNA damage (representing strand breaks, alkaline labile sites, and transient repair sites), endonuclease III (ENDOIII), or formamidopyrimidine DNA glycosylase (FPG) sensitive sites. I have recalculated the visual score so that it is expressed in the range of 0–100, similar to that of %T. Similar values were obtained for DNA damage and ENDOIII sites, regardless of whether of the data were reported as %T or visual score. Thus, these endpoints can be used interchangeably, assuming that the visual score is expressed in the 0–100 range. Pooled analysis of %T and visual score data showed that the median (25–75%) values of DNA damage, ENDOIII, and FPG sites were 8.6 (4.4–14.5), 11.0 (4.2–19.5), 7.6 (3.2–14.2), respectively. The duration of alkaline treatment and electrophoresis had no significant effect on the level of DNA damage. There was a positive correlation between age and the level of DNA damage. A sub-analysis of DNA damage obtained from European countries showed a negative correlation with latitude. In conclusion, reference values for DNA lesions measured by the comet assay are around 7–11 %T or arbitrary units. [Copyright &y& Elsevier]

Exposure to ultrafine particles (UFPs) from vehicle exhaust has been related to risk of cardiovascular and pulmonary disease and cancer, even though exposure assessment is difficult. We studied personal exposure in terms of number concentrations of UFPs in the breathing zone, using portable instruments in six 18-hr periods in 15 healthy nonsmoking subjects. Exposure contrasts of outdoor pollution were achieved by bicycling in traffic for 5 days and in the laboratory for 1 day. Oxidative DNA damage was assessed as strand breaks and oxidized purines in mononuclear cells isolated from venous blood the morning after exposure measurement. Cumulated outdoor and cumulated indoor exposures to UFPs each were independent significant predictors of the level of purine oxidation in DNA but not of strand breaks. Ambient air concentrations of particulate matter with an aerodynamic diameter of ≤ 10 µm (PM10), nitrous oxide, nitrogen dioxide, carbon monoxide, and/or number concentration of UFPs at urban background or busy street monitoring stations was not a significant predictor of DNA damage, although personal UFP exposure was correlated with urban background concentrations of CO and NO2, particularly during bicycling in traffic. The results indicate that biologic effects of UFPs occur at modest exposure, such as that occurring in traffic, which supports the relationship of UFPs and the adverse health effects of air pollution. [ABSTRACT FROM AUTHOR]

Oxidative DNA damage detected by the comet assay as formamidopyrimidine DNA glycosylase (FPG) senstitive sites, almost as a rule is reported as comet assay score rather than numerical sites in the genome, probably because the latter requires X-ray calibration. We compared the ability of five experienced and five inexperienced comet assay investigators to detect a dose-response relationship in irradiated A549 lung epithelial cell culture samples (0, 10 Gy and three samples of 5 Gy), based on an arbitrary five class scoring system. The samples were scored on three different occasions, thus allowing determination of the variation in sample scoring. All investigators qualitatively distinguished between samples in a dose-dependent manner, albeit with large variation in the slope and intercept of dose-response curves. There was a tendency that investigators with experience in scoring A549 cells had more consistent results than experienced investigators who had only scored lymphocytes or inexperienced investigators. The inexperienced investigators improved their scoring ability during the three sessions. Subsequently we showed that the variation in baseline level of FPG modifications in mononuclear blood cells of five healthy humans was lower when investigators used their individual X-ray calibration curve as compared to a common calibration curve. In conclusion, this study showed that comet assay investigators score differently when using a five class scoring system, which indicates that more consistent estimations of FPG sites in the genome are obtained by use of investigators' individual X-ray calibrations. [ABSTRACT FROM AUTHOR]

Berry extracts possess antioxidant activity in cell free systems, whereas cell culture and animal experimental systems have produced mixed outcomes. Our aim was to investigate the effects of blackcurrant juice and specifically blackcurrant anthocyanins on the steady state level of oxidative DNA damage in mononuclear blood cells (MNBC) of humans, determined as strand breaks (SB) as well as endonuclease III (Endo III) and formamidopyrimidine DNA glycosylase (Fpg) sensitive sites by the comet assay. Fifty-seven healthy humans completed a 3-week controlled parallel intervention study with three groups randomized to supplementation with blackcurrant juice, anthocyanin drink, or a control drink. The daily doses ranged from 475 to 1000 ml/d according to body weight (mean anthocyanin intakes in blackcurrant juice and anthocyanin drink groups were 397 and 365 g/d, respectively) and they were ingested during three daily meals while all volunteers were on the same strictly controlled low-flavonoid diet. Fasting venous blood samples were obtained at baseline and after 3-week of supplementation. The baseline level of oxidative DNA damage was low (e.g. less than 200 Fpg lesions per diploid cell). Fpg sensitive sites increased during the intervention within the blackcurrant juice group, whereas there were no differences between treatments in any of the DNA damage markers. In conclusion, this study shows that even large amounts of dietary antioxidants did not decrease the already low steady state levels of oxidative DNA damage in healthy adequately nourished humans. [Copyright &y& Elsevier]

• The review summarizes genotoxic effects of multi-walled carbon nanotubes. • Studies on NM-400, NM-401, NM-402, NM-403 and MWCNT-7 have been assessed. • Meta-analysis show generation of DNA strand breaks by MWCNT-7 in cell cultures. • Strong evidence of genotoxicity by MWCNT-7 in cell culture studies. • Knowledge gaps or inconclusive genotoxicity results by materials other than MWCNT-7. Carbon nanotubes (CNTs) were the first nanomaterials to be evaluated by the International Agency for Research on Cancer (IARC). The categorization as possibly carcinogenic agent to humans was only applicable to multi-walled carbon nanotubes called MWCNT-7. Other types of CNTs were not classifiable because of missing data and it was not possible to pinpoint unique CNT characteristics that cause cancer. Importantly, the European Commission's Joint Research Centre (JRC) has established a repository of industrially manufactured nanomaterials that encompasses at least four well-characterized MWCNTs called NM-400 to NM-403 (original JRC code). This review summarizes the genotoxic effects of these JRC materials and MWCNT-7. The review consists of 36 publications with results on cell culture experiments (22 publications), animal models (9 publications) or both (5 publications). As compared to the publications in the IARC monograph on CNTs, the current database represents a significant increase as there is only an overlap of 8 publications. However, the results come mainly from cell cultures and/or measurements of DNA strand breaks by the comet assay and the micronucleus assay (82 out of 97 outcomes). A meta-analysis of cell culture studies on DNA strand breaks showed a genotoxic response by MWCNT-7, less consistent effect by NM-400 and NM-402, and least consistent effect by NM-401 and NM-403. Results from other in vitro tests indicate strongest evidence of genotoxicity for MWCNT-7. There are too few observations from animal models and humans to make general conclusions about genotoxicity. [ABSTRACT FROM AUTHOR]

BACKGROUND: Particulate matter, especially ultrafine particles (UFPs), may cause health effects through generation of oxidative stress, with resulting damage to DNA and other macromolecules. OBJECTIVES: Wc investigated oxidative damagc to DNA and related repair capacity in peripheral blood mononuclear cells (PBMCs) during controlled exposure to urban air particles with assignment of numher concentration (NC) to four size modes with average diameters of 12, 23, 57, and 212 nm. DESIGN. Twenty-nine healthy adults participated in a randomized, two-factor cross-ovcr study with or without biking excrcise for 180 min and with exposure to particles (NC 6169-15362/cm³) or filtered air (NC 91-542/cm³) for 24 hr. METHODS: The levels of DNA strand breaks (SBs), oxidized purines as formamidopyrimidine DNA glycolase (FPG) sites, and activity of 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGGI) in PBMCs were measured by the Comet assay. mRNA levels of OGGI, nucleoside diphosphate linked moiety X-type motif I (NUDTI), and heme oxygenase-1 (HOl) were determined by real-time reverse transcriptase-polymerase chain reaction. RESULTS: Exposure to UFPs for 6 and 24 hr significantly increased the levels of SBs and FPG sites, with a further insignificant increase after physical exercise. The OGGI activity and expression of OGGI, NUDl, and HOl were unaltered. There was a significant dose-response relationship between NC and DNA damage, with the 57-nm mode as the major contributor to effects. Concomitant exposure to ozone, nitrogen oxides, and carbon monoxide had no influence. CONCLUSION: Our results indicate that UFPs, especially the 57-nm soot fraction from vehicle emissions, causes systemic oxidative stress with damage to DNA and no apparent compensatory up-regulation of DNA repair within 24 hr. [ABSTRACT FROM AUTHOR]

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m3) in Ogg1- / -  and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in Ogg1- / -  mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1- / -  mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1- / -  mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1- / -  mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP. [ABSTRACT FROM AUTHOR]

Carbon nanotubes (CNTs) are fiber-like nanomaterials, which are used in various applications with possible exposure to humans. The genotoxicity and carcinogenic potential of CNTs remain to be fully understood. This study assessed the genotoxicity of three different multi-walled carbon nanotubes (MWCNTs) (MWCNT-7, NM-401 and NM-403) and one single-walled carbon nanotube (SWCNT) (NM-411) in FE1-Muta™Mouse lung epithelial (MML) cells using the alkaline comet assay. With the 2',7'-dichlorodihydrofluorescein diacetate fluorescent probe, we assessed the effect of CNT-exposure on the intracellular production of reactive oxygen species (ROS). We measured the effect of a 10-week CNT exposure on telomere length using quantitative PCR. Two of the included MWCNTs (NM-401 and MWCNT-7) and the SWCNT (NM-411) caused a significant increase in the level of DNA damage at concentrations up to 40 µg/ml (all concentrations pooled, p < 0.05), but no concentration-response relationships were found. All of the CNTs caused an increase in intracellular ROS production compared to unexposed cells (ptrend < 0.05). Results from the long-term exposure showed longer telomere length in cells exposed to MWCNTs compared to unexposed cells (p < 0.01). In conclusion, our results indicated that the included CNTs cause ROS production and DNA strand breaks in FE1-MML cells. Moreover, the MWCNTs, but not the SWCNT, had an impact on telomere length in a long-term exposure scenario. [ABSTRACT FROM AUTHOR]

Many in vitro and in vivo studies have shown that exposure to carbon nanotubes (CNTs) is associated with inflammation, oxidative stress and genotoxicity, although there is a paucity of studies on these effects in the pleural cavity. In the present study, we investigated adverse outcomes of pleural exposure to multi-walled CNTs (MWCNT-7, NM-401 and NM-403) and single-walled CNTs (NM-411). Female C57BL/6 mice were exposed to 0.2 or 5 µg of CNTs by intra-pleural injection and sacrificed one-year post-exposure. Exposure to long and straight types of MWCNTs (i.e. MWCNT-7 and NM-401) was associated with decreased number of macrophages and increased number of neutrophils and eosinophils in pleural lavage fluid. Increased protein content in the pleural lavage fluid was also observed in mice exposed to MWCNT-7 and NM-401. The concentration of mesothelin was increased in mice exposed to MWCNT-7 and NM-411. Levels of DNA strand breaks and DNA oxidation damage, measured by the comet assay, were unaltered in cells from pleural scrape. Extra-pleural effects were seen in CNT exposed mice, including enlarged and pigmented mediastinal lymph nodes (all four types of CNTs), pericardial plaques (MWCNT-7 and NM-401), macroscopic abnormalities on the liver (MWCNT-7) and ovaries/uterus (NM-411). In conclusion, the results demonstrate that intra-pleural exposure to long and straight MWCNTs is associated with adverse outcomes. Certain observations such as increased content of mesothelin in pleural lavage fluid and ovarian/uterine abnormalities in mice exposed to NM-411 suggests that exposure to SWCNTs may also be associated with some adverse outcomes. [ABSTRACT FROM AUTHOR]

The ubiquitous pollution of plastic particles in most environmental matrices leads to concern about any potential adverse effects on human health. Most studies on the toxicological effect of nanoplastics has focused on standard particles of polystyrene. In reality humans are exposed to a large variety of different types and sizes of plastic material via oral intake and inhalation. In this study, we investigated the effect of polyethylene terephthalate (PET) nanoplastic particles from ground food containers from a supermarket. The aim was to investigate a possible link between exposure to PET nanoplastics and genotoxic response in a cell model of the human airway epithelial (A549) cells. Further, we investigated the combined effect of PET and chemicals known to alter the cellular redox state, as a model of partially compromised antioxidant defense system. DNA damage was assessed by the alkaline comet assay. The ground PET nanoplastics have a mean hydrodynamic diameter of 136 nm in water. The results showed that PET exposure led to increased reactive oxygen species production (approximately 30 % increase compared to unexposed cells). In addition, exposure to PET nanoplastic increased the level of DNA strand breaks (net increase = 0.10 lesions/106 base pair, 95 % confidence interval: 0.01, 0.18 lesions/106 base pair). Pre- or post-exposure to hydrogen peroxide or buthionine sulfoximine did not lead to a higher level of DNA damage. Overall, the study shows that exposure to PET nanoplastics increases both intracellular reactive oxygen production and DNA damage in A549 cells. • The hydrodynamic particle sizes of ground nanoplastics from PET food containers were 136–167 nm. • PET nanoplastic particles caused intracellular ROS production, both and in combination with pre-exposure to known oxidants. • PET nanoplastic particles, 125 µg/ml, caused DNA damage with a net increase of 0.10 lesions/106 bp. [ABSTRACT FROM AUTHOR]

• Particles were collected in diesel-powered trains and firefighter training location. • Particles generated intracellular reactive oxygen species in A549 cells. • Long-term exposure to particles promoted shortening in telomeres in A549 cells. • Shorter telomeres were observed in mononuclear blood cells of particle-exposed humans. Particulate matter (PM) from combustion processes has been associated with oxidative stress to DNA, whereas effects related to telomere dysfunction are less investigated. We collected air-borne PM from a passenger cabin of a diesel-propelled train and at a training facility for smoke diving exercises. Effects on oxidative stress biomarkers, genotoxicity measured by the comet assay and telomere length in PM-exposed A549 cells were compared with the genotoxicity and telomere length in peripheral blood mononuclear cells (PBMCs) from human volunteers exposed to the same aerosol source. Although elevated levels of DNA strand breaks and oxidatively damaged DNA in terms of Fpg-sensitive sites were observed in PBMCs from exposed humans, the PM collected at same locations did not cause genotoxicity in the comet assay in A549 cells. Nevertheless, A549 cells displayed telomere length shortening after four weeks exposure to PM. This is in line with slightly shorter telomere length in PBMCs from exposed humans, although it was not statistically significant. In conclusion, the results indicate that genotoxic potency measured by the comet assay of PM in A549 cells may not predict genotoxicity in exposed humans, whereas telomere length measurements may be a novel indicator of genotoxic stress in cell cultures and humans. [ABSTRACT FROM AUTHOR]

The comet assay offers the opportunity to measure both DNA damage and repair. Various comet assay based methods are available to measure DNA repair activity, but some requirements should be met for their effective use in human biomonitoring studies. These conditions include i) robustness of the assay, ii) sources of inter- and intra-individual variability must be known, iii) DNA repair kinetics should be assessed to optimize sampling timing; and iv) DNA repair in accessible surrogate tissues should reflect repair activity in target tissues prone to carcinogenic effects. DNA repair phenotyping can be performed on frozen and fresh samples, and is a more direct measurement than genomic or transcriptomic approaches. There are mixed reports concerning the regulation of DNA repair by environmental and dietary factors. In general, exposure to genotoxic agents did not change base excision repair (BER) activity, whereas some studies reported that dietary interventions affected BER activity. On the other hand, in vitro and in vivo studies indicated that nucleotide excision repair (NER) can be altered by exposure to genotoxic agents, but studies on other life style related factors, such as diet, are rare. Thus, crucial questions concerning the factors regulating DNA repair and inter-individual variation remain unanswered. Intra-individual variation over a period of days to weeks seems limited, which is favourable for DNA repair phenotyping in biomonitoring studies. Despite this reported low intra-individual variation, timing of sampling remains an issue that needs further investigation. A correlation was reported between the repair activity in easily accessible peripheral blood mononuclear cells (PBMCs) and internal organs for both NER and BER. However, no correlation was found between tumour tissue and blood cells. In conclusion, although comet assay based approaches to measure BER/NER phenotypes are feasible and promising, more work is needed to further optimize their application in human biomonitoring and intervention studies. [ABSTRACT FROM AUTHOR]

Candle burning produces a large amount of particles that contribute substantially to the exposure to indoor particulate matter. The exposures to various types of combustion particles, such as diesel exhaust particles, have been associated with increased risk of lung cancer by mechanisms that involve oxidative stress, inflammation and genotoxicity. The aim of this study was to compare pulmonary effects of candle light combustion particles (CP) with two benchmark diesel exhaust particles (A-DEP and SRM2975). Intratracheal (i.t.) instillation of CP (5 mg/kg bodyweight) in C57BL/6n mice produced a significant influx of alveolar macrophages and polymorphonuclear leukocytes and increased concentrations of proteins and lactate dehydrogenase activity in bronchoalveolar fluid. Lower levels of these markers of inflammation and cytotoxicity were observed after i.t. instillation of the same dose of A-DEP or SRM2975. The i.t. instillation of CP did not generate oxidative damage to DNA in lung tissue, measured as DNA strand breaks and human 8-oxoguanine glycosylase-sensitive sites by the comet assay. The lack of genotoxic response was confirmed in lung epithelial (A549) cells, although the exposure to CP increased intracellular levels of reactive oxygen species. In conclusion, pulmonary exposure to particles from burning candles is associated with inflammation and cytotoxicity in the lungs. [ABSTRACT FROM AUTHOR]

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful. • Inter-laboratory differences in comet assay data are only partly understood. • Ionising radiation is ideal for standardisation but doses must be accurate. • In a ring trial we calibrated radiation units in 8 labs and found major differences. • Systematic corrections for protocol variations reduced inter-laboratory differences. • This is a step forward in understanding and controlling comet assay variations. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the effects of cationic micelle and liposome drug delivery systems on liver and lung cells in a toxicological in vitro screening model, with observations on cytotoxicity and genotoxicity. A screening battery was established for assessment of a broad range of parameters related to adverse effects. Clear concentration response effects were observed related to impairment of mitochondrial function, membrane integrity and oxidative stress markers, but no effect was observed on genotoxicity. The adverse effects were highest for the liposomes. The High Content Screening seems optimal for initial screening of adverse effects, and combined with standard cytotoxicity measurements initial screening can be performed for predictive toxicological screening. [ABSTRACT FROM AUTHOR]

The comet assay is used to measure DNA damage induced by chemical and physical agents. High concentrations of test agents may cause cytotoxicity or cell death, which may give rise to false positive results in the comet assay. Systematic studies on genotoxins and cytotoxins (i.e. non-genotoxic poisons) have attempted to establish a threshold of cytotoxicity or cell death by which DNA damage results measured by the comet assay could be regarded as a false positive result. Thresholds of cytotoxicity/cell death range from 20% to 50% in various publications. Curiously, a survey of the latest literature on comet assay results from cell culture studies suggests that one-third of publications did not assess cytotoxicity or cell death. We recommend that it should be mandatory to include results from at least one type of assay on cytotoxicity, cell death or cell proliferation in publications on comet assay results. A combination of cytotoxicity (or cell death) and proliferation (or colony forming efficiency assay) is preferable in actively proliferating cells because it covers more mechanisms of action. Applying a general threshold of cytotoxicity/cell death to all types of agents may not be applicable; however, 25% compared to the concurrent negative control seems to be a good starting value to avoid false positive comet assay results. Further research is needed to establish a threshold value to distinguish between true and potentially false positive genotoxic effects detected by the comet assay. • Cytotoxicity or cell death may give false positive results in the comet assay. • About one-third of comet assay publications do not assess cytotoxicity or cell death. • At least a cytotoxicity, cell death or proliferation marker, should be mandatory. • A combination of cytotoxicity/cell death and proliferation/CFE markers is preferable. • A threshold of 25% affected cells is a good starting point to avoid false positive results. [ABSTRACT FROM AUTHOR]

Neuronal genotoxic insults from oxidative stress constitute a putative molecular link between stress and depression on the one hand, and cognitive dysfunction and dementia risk on the other. Oxidative modifications to DNA are repaired by specific enzymes; a process that plays a critical role for maintaining genomic integrity. The aim of the present study was to characterize the pattern of cerebral DNA repair enzyme regulation after stress through the quantification of a targeted range of gene products involved in different types of DNA repair. 72 male Sprague–Dawley rats were subjected to either restraint stress (6 h/day) or daily handling (controls), and sacrificed after 1, 7 or 21 stress sessions. The mRNA expression of seven genes ( Ogg1, Ape1, Ung1, Neil1, Xrcc1, Ercc1, Nudt1 ) involved in the repair of oxidatively damaged DNA was determined by quantitative real time polymerase chain reaction in the prefrontal cortex (PFC) and hippocampus (HC). DNA repair gene expression in PFC exhibited a general trend towards an induction after acute stress and a decrease after subchronic exposure compared to control animals. After chronic stress, a normalization towards control levels was observed. A similar pattern was seen in HC, but with overall smaller effects and without the induction after acute stress. Nuclear DNA damage from oxidation as measured by the comet assay was unaffected by stress in both regions. We conclude that psychological stress have a dynamic influence on brain DNA repair gene expression; however, since we were unable to identify concurrent changes in DNA damage from oxidation, the down-stream consequences of this regulation, if any, remains unclear. [ABSTRACT FROM AUTHOR]

Accumulating evidences indicate that pulmonary exposure to carbon nanotubes (CNTs) is associated with increased risk of lung diseases, whereas the effect on the vascular system is less studied. We investigated vascular effects of 2 types of multiwalled CNTs (MWCNTs) in apolipoprotein E−/− mice, wild-type mice, and cultured cells. The ApoE−/− mice had accelerated plaque progression in aorta after 5 intracheal instillations of MWCNT (25.6 μg/mouse weekly for 5 weeks). The exposure was associated with pulmonary inflammation, lipid peroxidation, and increased expression of inflammatory, oxidative stress, DNA repair, and vascular activation response genes. The level of oxidatively damaged DNA in lung tissue was unaltered, probably due to increased DNA repair capacities. Despite upregulation of inflammatory genes in the liver, effects on systemic cytokines and lipid peroxidation were minimal. The exposure to MWCNTs in cultured human endothelial cells increased the expression of cell adhesion molecules (ICAM1 and VCAM1). In cocultures, there was increased adhesion of monocytes to endothelial cells after exposure to MWCNT. The exposure to both types of MWCNT was also associated with increased lipid accumulation in monocytic-derived foam cells, which was dependent on concomitant oxidative stress because the antioxidant N-acetylcysteine inhibited the lipid accumulation. Collectively, our results indicate that exposure to MWCNT is associated with accelerated progression of atherosclerosis, which could be related to both increased adherence of monocytes onto the endothelium and oxidative stress-mediated transformation of monocytes to foam cells. [ABSTRACT FROM PUBLISHER]

This study compared the oxidative stress level and vasomotor dysfunction after exposure to urban dust, diesel exhaust particles (DEP) or single-walled carbon nanotubes (SWCNT). DEP and SWCNT increased the production of reactive oxygen species (ROS) in cultured endothelial cells and acellullarly, whereas the exposure to urban dust did not generate ROS. The apoE-/- mice, which were exposed twice to 0.5 mg/kg of the particles by intratracheal (i.t.) instillation, had unaltered acetylcholine-elicited vasorelaxation in aorta segments. There was unaltered pulmonary expression level of Vcam-1, Icam-1, Hmox-1 and Ogg1. The levels of oxidatively damaged DNA were unchanged in lung tissue. The exposure to SWCNT significantly increased the expression of Ccl-2 in the lung tissue of the mice. The exposure to DEP and SWCNT was associated with elevated ROS production in cultured cells, whereas i.t. instillation of the same particles had no effect on biomarkers of pulmonary oxidative stress and dilatory dysfunction in the aorta. [ABSTRACT FROM AUTHOR]

Abstract: Exposure to particles has been suggested to generate hepatosteatosis by oxidative stress mechanisms. We investigated lipid accumulation in cultured human hepatocytes (HepG2) and rat liver after exposure to four different carbon-based particles. HepG2 cells were exposed to particles for 3h and subsequently incubated for another 18h to manifest lipid accumulation. In an animal model of metabolic syndrome we investigated the association between intake of carbon black (CB, 14nm) particles and hepatic lipid accumulation, inflammation and gene expression of Srebp-1, Fasn and Scd-1 involved in lipid synthesis. There was a concentration-dependent increase in intracellular lipid content after exposure to CB in HepG2 cells, which was only observed after co-exposure to oleic/palmitic acid. Similar results were observed in HepG2 cells after exposure to diesel exhaust particles, fullerenes C60 or pristine single-walled carbon nanotubes. All four types of particles also generated oxidatively damaged DNA, assessed as formamidopyrimidine DNA glycosylase (FPG) sensitive sites, in HepG2 cells after 3h exposure. The animal model of metabolic syndrome showed increased lipid load in the liver after one oral exposure to 6.4mg/kg of CB in lean Zucker rats. This was not associated with increased iNOS staining in the liver, indicating that the oral CB exposure was associated with hepatic steatosis rather than steatohepatitis. The lipid accumulation did not seem to be related to increased lipogenesis because there were unaltered gene expression levels in both the HepG2 cells and rat livers. Collectively, exposure to particles is associated with oxidative stress and steatosis in hepatocytes. [Copyright &y& Elsevier]

Abstract: The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view. [Copyright &y& Elsevier]

Abstract: Toxicological effects of wood smoke particles are less investigated than traffic-related combustion particles. We investigated the effect of wood smoke particles, generated by smouldering combustion conditions, on human umbilical endothelial cells (HUVECs) co-cultured with or without monocytic THP-1 cells. Standard reference material (SRM) 2975 diesel exhaust particles were used as benchmark particles. Wood smoke particles at 50μg/ml or 100μg/ml caused adhesion of THP-1 monocytes onto HUVECs in co-cultures, whereas SRM2975 had no such effect. Both types of particles from 1μg/ml increased VCAM-1 expression on HUVECs in mono-cultures. However, only the exposure to wood smoke particles was associated with increased expression of TNF and IL8 mRNA in THP-1 cells. We found no effect on the intracellular production of reactive oxygen species by the fluorescent probe DCFH-DA, whereas especially the wood smoke particles caused increased level of DNA strand breaks and oxidised guanines at concentrations with low cytotoxicity. In conclusion, our results indicate that the adherence of monocytes on endothelial cells in wood smoke particle exposed cultures depend on activation of both cell types. [Copyright &y& Elsevier]

Abstract: Oxidatively damaged DNA base lesions are considered to be mainly repaired by 8-oxoguanine DNA glycosylase (OGG1) mediated pathways. We investigated the effect of the OGG1 Ser326Cys polymorphism on the level and repair of oxidatively damaged DNA in mononuclear blood cells (MNBC) by means of the comet assay. We collected blood samples from 1,019 healthy subjects and genotyped for the OGG1 Ser326Cys polymorphism. We found 49 subjects homozygous for the variant genotype (Cys/Cys) and selected same numbers of age-matched subjects with the heterozygous (Ser/Cys) and homozygous wild-type genotype (Ser/Ser). Carriers of the Cys/Cys genotype had higher levels of formamidopyrimidine DNA glycosylase (FPG) sensitive sites in MNBC (0.31 ± 0.03 lesions/106 bp) compared to Ser/Ser (0.19 ± 0.02 lesions/106 bp, P<0.01). The level of hOGG1 sensitive sites in MNBC from the Ser326Cys carriers (0.19 ± 0.16 lesions/106 bp) was also higher compared to the Ser/Ser genotype (0.11 ± 0.09 lesions/106 bp, P<0.05). Still, there was no genotype-related difference in DNA repair incision activity of MNBC extracts on nucleoids with oxidatively damaged DNA induced by Ro19-8022/white light (P=0.20). In addition, there were no differences in the expression of OGG1 (P=0.69), ERCC1 (P=0.62), MUTYH (P=0.85), NEIL1 (P=0.17) or NUDT1 (P=0.48) in whole blood. Our results indicate that the OGG1 Ser326Cys polymorphism has limited influence on the DNA repair incisions by extracts of MNBC, whereas the apparent increased risk of cancer in subjects with the Cys/Cys genotype may be because of higher levels of oxidatively damaged DNA. [Copyright &y& Elsevier]

Abstract: Synthetically produced perfluorinated chemicals (PFCs) are widely used in industrial products because of their anti-wetting and surfactant properties. PFCs are suspected carcinogens and a possible mechanism of action is generation of oxidative stress. We have investigated the potential of five different PFCs to generate reactive oxygen species (ROS) and to induce oxidative DNA damage in HepG2 cells. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) increased the intracellular ROS production by 1.52-fold (95% CI, 1.37–1.67) and 1.25-fold (95% CI, 1.10–1.40), respectively. However, the increase in ROS production was not concentration-dependent and the compounds did not generate DNA damage that could be detected by the alkaline comet assay as strand breakage and alkali-labile sites or formamidopyrimidine-DNA-glycosylase (FPG) sites. Perfluorobutane sulfonate (PFBS) and perfluorohexanoic acid (PFHxA) did not generate ROS or DNA damage. Only the exposure to perfluorononanoic acid (PFNA) caused a modest increase in DNA damage at a cytotoxic concentration level, which was detected as lactate dehydrogenase (LDH) release into the cell medium. This was not related to ROS generation. Collectively, these results indicate that PFCs induce only modest effects in terms of ROS production and DNA damage in a cell line representing the human liver. [Copyright &y& Elsevier]

Abstract: Genotoxic effects of traffic-generated particulate matter (PM) are well described, whereas little data are available on PM from combustion of biomass and wood, which contributes substantially to air pollution world wide. The aim of this study was to compare the genotoxicity of wood smoke particulate matter (WSPM), authentic traffic-generated particles, mineral PM and standard reference material (SRM2975) of diesel exhaust particles in human A549 lung epithelial and THP-1 monocytic cell lines. DNA damage was measured as strand breaks (SB) and formamidopyrimidine DNA glycosylase (FPG) sites by the comet assay, whereas cell cytotoxicity was determined as lactate dehydrogenase release. The exposure to WSPM generated SB and FPG sites in both cell lines at concentrations from 2.5 or 25μg/ml, which were not cytotoxic. Compared to all other studied particles, WSPM generated greater responses in terms of both SB and FPG sites. Organic extracts of WSPM and SRM2975 elicited higher levels of SB than native and washed PM at 25 and 100μg/ml, whereas assay saturation precluded reliable assessment of FPG sites. During a 6h post-exposure period, in which the medium with PM had been replaced by fresh medium, 60% of the DNA lesions generated by WSPM were removed. In conclusion, WSPM generated more DNA damage than traffic-generated PM per unit mass in human cell lines, possibly due to the high level of polycyclic aromatic hydrocarbons in WSPM. This suggests that exposure to WSPM might be more hazardous than PM collected from vehicle exhaust with respect to development of lung cancer. [Copyright &y& Elsevier]

Abstract: Research indicates that exposure to hypoxia is associated with oxidative stress. In this investigation, healthy subjects were exposed to hypoxia by inhalation of 10% oxygen for 2h (corresponding to 5500m above sea level). The levels of strand breaks and oxidatively damaged purine bases, measured by the comet assay, and the expression of genes involved in DNA repair of oxidatively damaged DNA were investigated in mononuclear blood cells (MNBC) at baseline, after 2h of hypoxia, 2h of reoxygenation, and 1 day and 8 days after the exposure. The level of strand breaks and oxidized purine bases in MNBC increased following both the 2h of hypoxia and the 2h reoxygenation period, whereas this effect was not observed in unexposed subjects. The expressions of oxoguanine DNA glycosylase 1 (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), nei endonuclease VIII-like 1 (NEIL1), and mutY homolog (MUTYH) were unaltered throughout the experiment in both groups of subjects, indicating that DNA repair genes are not up-regulated by the hypoxia and reoxygenation treatment. Taken together, this report shows that inhalation of 10% oxygen for 2h is associated with increased number of oxidized DNA lesions in MNBC, but acute hypoxia may not inflict upon the regulation of genes involved in repair of oxidized DNA. [Copyright &y& Elsevier]

Abstract: Epidemiological studies have shown that exposure to air pollution particles is associated with cardiovascular diseases, whereas the role in the initiation of atherosclerosis is unresolved. Atherosclerosis is considered to be an inflammatory disease that also involves oxidative stress. Here we investigated effects of oxidative stress elicited by diesel exhaust particles (DEP) in the aorta, liver, and lung of dyslipidemic ApoE −/− mice at the age when visual plaques appear in the aorta (11–13 weeks). DEP was administrated by intraperitoneal injection (0, 50, 500 and 5000μg DEP/kg bodyweight) in order to omit vascular effects secondary to pulmonary inflammation. The mice were killed either 6 or 24h after the administration. Inflammation was measured as the expression of inducible nitric oxide synthase (iNOS) and serum nitric oxide and DNA damage was measured by the comet assay. The expression of iNOS mRNA was increased in the liver 6h after the administration. The level of oxidized purine bases, determined as formamidopyrimidine DNA glycosylase sites was increased by 67% (95% CI: 11–124%) in the liver after 24h in the mice administrated with only 50μg/kg bodyweight. However, there was no indication of systemic inflammation determined as the serum concentration of nitric oxide and iNOS expression, and DNA damage was not increased in the aorta. These observations indicate that intraperitoneal DEP injection does not induce inflammation or oxidatively damaged DNA in the lung and aorta, whereas a direct effect in terms of inflammation and oxidized DNA was observed in the liver of dyslipidemic ApoE −/− mice. [Copyright &y& Elsevier]

Recent research suggests that high-altitude hypoxia may serve as a model for prolonged oxidative stress in healthy humans. In this study, we investigated the consequences of prolonged high-altitude hypoxia on the basal level of oxidative damage to nuclear DNA in muscle cells, a major oxygen-consuming tissue. Muscle biopsies from seven healthy humans were obtained at sea level and after 2 and 8 weeks of hypoxia at 4100 m.a.s.l. We found increased levels of strand breaks and endonuclease III-sensitive sites after 2 weeks of hypoxia, whereas oxidative DNA damage detected by formamidopyrimidine DNA glycosylase (FPG) protein was unaltered. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1), determined by quantitative RT-PCR of mRNA levels did not significantly change during high-altitude hypoxia, although the data could not exclude a minor upregulation. The expression of heme oxygenase-1 (HO-1) was unaltered by prolonged hypoxia, in accordance with the notion that HO-1 is an acute stress response protein. In conclusion, our data indicate high-altitude hypoxia may serve as a good model for oxidative stress and that antioxidant genes are not upregulated in muscle tissue by prolonged hypoxia despite increased generation of oxidative DNA damage. [Copyright &y& Elsevier]

• 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. [ABSTRACT FROM AUTHOR]