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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]

• 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]

There is concern about human exposure to nanoplastics from intentional use or degradation of plastics in the environment. This review assesses genotoxic effects of nanoplastics, defined as particles with a primary size of less than 1000 nm. The majority of results on genotoxicity come from studies on polystyrene (PS) particles in mammalian cell cultures. Most studies have measured DNA strand breaks (standard comet assay), oxidatively damaged DNA (Fpg-modified comet assay) and micronuclei. Twenty-nine out of 60 results have shown statistically significant genotoxic effects by PS exposure in cell cultures. A statistical analysis indicates that especially modified PS particles are genotoxic (odds ratio = 8.6, 95 % CI: 1.6, 46) and immune cells seems to be more sensitive to genotoxicity than other cell types such as epithelial cells (odds ratio = 8.0, 95 % CI: 1.6, 39). On the contrary, there is not a clear association between statistically significant effects in genotoxicity tests and the primary size of PS particles, (i.e. smaller versus larger than 100 nm) or between the type of genotoxic endpoint (i.e. repairable versus permanent DNA lesions). Three studies of PS particle exposure in animals have shown increased level of DNA strand breaks in leukocytes and prefrontal cortex cells. Nanoplastics from polyethylene, propylene, polyvinyl chloride and polyethylene terephthalate have been investigated in very few studies and it is currently not possible to draw conclusion about their genotoxic hazard. In summary, there is some evidence suggesting that PS particles may be genotoxic in mammalian cells. [ABSTRACT FROM AUTHOR]

Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related to leukocyte-mediated oxidative stress. The study utilized a cross sectional design performed in 58 study participants from a larger cohort. Levels of circulating endothelial progenitor cells, defined as either late (CD34 + KDR + cells) or early (CD34 + CD133 + KDR + cells) subsets were measured using polychromatic flow cytometry. We additionally measured production of reactive oxygen species in leukocyte subsets (lymphocytes, monocytes and granulocytes) by flow cytometry using intracellular 2′,7′-dichlorofluoroscein. The measurements encompassed both basal levels of reactive oxygen species production and capacity for reactive oxygen species production for each leukocyte subset. We found that the late endothelial progenitor subset was negatively associated with levels of ultrafine particles measured within the participant residences and with reactive oxygen species production capacity in lymphocytes. Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate that exposure to fine and ultrafine particles derived from indoor sources may have adverse effects on human vascular health. [ABSTRACT FROM AUTHOR]

Particulate air pollution increases risk of cancer and cardiopulmonary disease, partly through oxidative stress. Traffic-related noise increases risk of cardiovascular disease and may cause oxidative stress. In this controlled random sequence study, 18 healthy subjects were exposed for 3 h to diesel exhaust (DE) at 276 μg/m 3 from a passenger car or filtered air, with co-exposure to traffic noise at 48 or 75 dB(A). Gene expression markers of inflammation, ( interleukin - 8 and tumor necrosis factor ), oxidative stress ( heme oxygenase ( decycling-1 )) and DNA repair ( 8-oxoguanine DNA glycosylase ( OGG1 )) were unaltered in peripheral blood mononuclear cells (PBMCs). No significant differences in DNA damage levels, measured by the comet assay, were observed after DE exposure, whereas exposure to high noise levels was associated with significantly increased levels of hOGG1-sensitive sites in PBMCs. Urinary levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine were unaltered. In auxiliary ex vivo experiments whole blood was incubated with particles from the exposure chamber for 3 h without effects on DNA damage in PBMCs or intracellular reactive oxygen species production and expression of CD11b and CD62L adhesion molecules in leukocyte subtypes. Conclusion 3-h exposure to DE caused no genotoxicity, oxidative stress or inflammation in PBMCs, whereas exposure to noise might cause oxidatively damaged DNA. [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]

• Nanoclay surface modification reduces pro-inflammatory response in both cell lines. • GO and r-GO induced a pro-inflammatory response in A549 cells. • Strong in vitro-in vivo correlation of nanomaterials-induced inflammation. • Most materials induced genotoxicity in THP-1 and only one in A549 cells. • Concordant genotoxicity between THP-1a cells and BAL cells. Nanoclays and graphene oxide nanomaterials represent a class of materials sharing similar shapes constituted of high aspect ratio platelets. The increased production of these materials for various industrial applications increases the risk of occupational exposure, consequently with elevated risk of adverse reactions and development of pulmonary diseases, including lung cancer. In this study, pro-inflammatory responses and genotoxicity were assessed in alveolar epithelial cells (A549) and activated THP-1 macrophages (THP-1a) after exposure to three nanoclays; a pristine (Bentonite) and two surface modified (benzalkonium chloride-coated Nanofil9, and dialkyldimethyl-ammonium-coated NanofilSE3000); graphene oxide (GO) and reduced graphene oxide (r-GO) nanomaterials. The pro-inflammatory response in terms of IL-8 expression was strongest in cells exposed to Bentonite, whereas surface modification resulted in decreased toxicity in both cell lines when exposed to Nanofil9 and NanofilSE3000. GO and r-GO induced a pro-inflammatory response in A549 cells, while no effect was detected with the two nanomaterials on THP-1a cells. The pro-inflammatory response was strongly correlated with in vivo inflammation in mice after intra-tracheal instillation when doses were normalized against surface area. Genotoxicity was assessed as DNA strand breaks, using the alkaline comet assay. In A549 cells, an increase in DNA strand breaks was detected only in cells exposed to Bentonite, whereas Bentonite, NanofilSE3000 and GO caused an increased level of genotoxicity in THP-1a cells. Genotoxicity in THP-1a cells was concordant with the DNA damage in bronchoalveolar lavage fluid cells following 1 and 3 days after intra-tracheal instillation in mice. In conclusion, this study shows that surface modification of pristine nanoclays reduces the inflammatory and genotoxic response in A549 and THP-1a cells, and these in vitro models show comparable toxicity to what seen in previous mouse studies with the same materials. [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]

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]

Abstract: The particulate phase of diesel engine exhaust is likely carcinogenic. However, the mechanisms of diesel exhaust particles (DEPs) induced mutagenicity/carcinogenicity are still largely unknown. We determined the mutant frequency following eight repeated 72h incubations with 37.5 or 75μg/ml DEP (NIST SRM 1650) in the FE1-Muta™Mouse lung epithelial cell line. We measured DEP-induced acellular and intracellular production of reactive oxygen species (ROS) and compared with ROS production induced by carbon black, which we have previously shown is mutagenic in this cell line [N.R. Jacobsen, A.T. Saber, P. White, P. Moller, G. Pojana, U. Vogel, S. Loft, J. Gingerich, L. Soper, G.R. Douglas, H. Wallin. Increased mutant frequency by carbon black, but not quartz, in the lacZ and cII transgenes of muta(TM)mouse lung epithelial cells, Environ. Mol. Mutagen. 48(6) (2007) 451–461]. The mutant frequency was marginally elevated in cells treated with 37.5μg/ml DEP (1.29-fold [95% CI: 0.96–1.60], p =0.08) and significantly increased in cells treated with 75μg/ml DEP (1.55-fold [95% CI: 1.23–1.87], p <0.001). ROS production from DEP was low both within cells and in acellular systems when compared to carbon black. These results show that DEP are mutagenic in a mammalian cell line in vitro and that additional pathways besides ROS production, such as those involving the presence of polycyclic aromatic hydrocarbons, likely are involved in the mutagenesis. [Copyright &y& Elsevier]

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]

Abstract: Exposure to ambient air particulate matter (PM) is associated with pulmonary and cardiovascular diseases and cancer. The mechanisms of PM-induced health effects are believed to involve inflammation and oxidative stress. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from the surface of particles, soluble compounds such as transition metals or organic compounds, altered function of mitochondria or NADPH-oxidase, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species. Resulting oxidative DNA damage may be implicated in cancer risk and may serve as marker for oxidative stress relevant for other ailments caused by particulate air pollution. There is overwhelming evidence from animal experimental models, cell culture experiments, and cell free systems that exposure to diesel exhaust and diesel exhaust particles causes oxidative DNA damage. Similarly, various preparations of ambient air PM induce oxidative DNA damage in in vitro systems, whereas in vivo studies are scarce. Studies with various model/surrogate particle preparations, such as carbon black, suggest that the surface area is the most important determinant of effect for ultrafine particles (diameter less than 100nm), whereas chemical composition may be more important for larger particles. The knowledge concerning mechanisms of action of PM has prompted the use of markers of oxidative stress and DNA damage for human biomonitoring in relation to ambient air. By means of personal monitoring and biomarkers a few studies have attempted to characterize individual exposure, explore mechanisms and identify significant sources to size fractions of ambient air PM with respect to relevant biological effects. In these studies guanine oxidation in DNA has been correlated with exposure to PM2.5 and ultrafine particles outdoor and indoor. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. Related biomarkers and personal monitoring may be useful tools for risk characterization. [Copyright &y& Elsevier]

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]

Cells are constantly exposed to oxidants from metabolic and other biochemical reactions as well as external factors, suggesting that DNA repair systems and nutritional antioxidants are important determinants for low levels of DNA damage and cancer risk. The effects of single antioxidants, as well as various vegetables, fruits and carotenoid- and polyphenolic-rich products, have been assessed with biomarkers, mainly including DNA damage in white blood cells (WBC), urinary excretion of oxidized bases and nucleosides and DNA repair capacity. The basal levels of oxidative DNA damage, and effects of the interventions have been rather variable, possibly reflecting differences in the populations, regimens, and the type of assays. In general, single dose antioxidant interventions have shown protective effects with respect to WBC DNA oxidation. Studies with continuous ingestion of antioxidants show mixed results with respect to effects on oxidative DNA damage in WBC, possibly due to various problems with design, statistical power and period effects. Studies with only male subjects appear to show consistent antioxidant effects in terms of reduced levels of oxidized pyrimidines. Investigations of oxidatively stressed subjects, e.g. HIV-infected patients or diabetics, suggest beneficial outcomes in populations with high initial levels of oxidative DNA damage. Recent research on the effect of antioxidants on DNA repair enzymes suggest effects in terms of increased removal of oxidized purines, whereas mRNA levels of the relevant DNA repair genes appears to be unaffected by an antioxidant-rich diet. In the future, care should be taken with respect to design of intervention studies and considerations of gender effect, genotypes of defence enzymes as well as DNA repair capacity. [Copyright &y& Elsevier]

Earlier studies have indicated that sucrose increases 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced aberrant crypt foci in the colon. In this study, we investigated the role of sucrose in IQ-induced genotoxicity of the colon mucosa and liver. Big Blue™ rats were fed with IQ (20 ppm in feed) and/or sucrose (3.45 or 6.85 wt.% in feed) for 3 weeks. IQ increased DNA strand breaks in the colon, whereas the mutation frequency was increased in the liver. The level of IQ-induced DNA adducts was elevated in both colon mucosa cells and liver. In the liver, high sucrose intake increased the level of DNA adducts above that of IQ and low sucrose intake. Oxidative DNA damage detected in terms of 7-hydro-8-oxo-2′-deoxyguanosine by HPLC-EC, or endonuclease III or formamidopyrimidine DNA glycosylase sensitive sites were unaltered in the colon and liver. Expression of ERCC1 and OGG1 mRNA levels were unaffected by IQ or sucrose feeding. Biomarkers of oxidative stress, including Vitamin C, malondialdehyde and protein oxidations (γ-glutamyl semialdehyde and 2-amino adipic semialdehyde) were unaltered in plasma and in liver. In conclusion, sucrose feeding increases IQ-induced genotoxicity in liver but not in colon, suggesting different mechanisms for sucrose and IQ in colon mutagenesis. [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]

• Air pollution during pregnancy associated with telomere length (TL) in newborns. • Second trimester air pollution positively associated with umbilical cord blood TL. • Third trimester air pollution inversely associated with umbilical cord blood TL. • Air pollution at home and work show similar association with umbilical cord blood TL. • No association between air pollution and TL in placenta or maternal blood. Telomere length (TL) is a biomarker of biological aging that may be affected by prenatal exposure to air pollution. The aim of this study was to assess the association between prenatal exposure to air pollution and TL in maternal blood cells (leukocytes), placenta and umbilical cord blood cells, sampled immediately after birth in 296 Danish mother-child pairs from a birth cohort. Exposure data was obtained using the high-resolution and spatial–temporal air pollution modeling system DEHM-UBM-AirGIS for PM 2.5 , PM 10 , SO 2 , NH 4 +, black carbon (BC), organic carbon (OC), CO, O 3 , NO 2 , and NO x at residential and occupational addresses of the participating women for the full duration of the pregnancy. The association between prenatal exposure to air pollutants and TL was investigated using distributed lag models. There were significant and positive associations between TL in umbilical cord blood cells and prenatal exposure to BC, OC, NO 2 , NO x , CO, and O 3 during the second trimester. TL in umbilical cord blood was significantly and inversely associated with prenatal exposure to PM 2.5 , BC, OC, SO 2 , NH 4 +, CO and NO 2 during the third trimester. There were similar inverse associations between TL from umbilical cord blood cells and air pollution exposure at the residential and occupational addresses. There were weaker or no associations between air pollution exposure and TL in placenta tissue and maternal blood cells. In conclusion, both the second and third trimesters of pregnancy are shown to be sensitive windows of exposure to air pollution affecting fetal TL. [ABSTRACT FROM AUTHOR]

Humans are exposed to plastic particles, but there are no studies on environmental plastics in cell cultures or animals. The toxicological understanding arises from model particles like polystyrene, polyethylene or non-plastic particles like food-grade titanium dioxide. The majority of studies on polystyrene particles show toxicological effects on measures of oxidative stress, inflammation, mitochondrial dysfunction, lysosomal dysfunction and apoptosis. The toxic effects in cell cultures mainly occur at high concentrations. Polyethylene particles seem to generate inflammatory reactions, whereas other toxicological effects have not been assessed. There are very few studies on effects of polystyrene particles in animal models and these have not demonstrated overt indices of toxicity. Studies in animals are the likely way for hazard assessment of micro- or nanoplastics. However, co-culture systems that mimic the complex architecture of mammalian tissues can cost-efficiently determine the hazards of micro- and nanoplastics. Future studies should include low doses of micro- and nanoplastic particles, which are more relevant in the assessment of health risk than the extrapolation of effects from high doses to realistic doses. Based on studies on model particles, environmental exposure to micro- and nanoplastic particles may be a hazard to human health. • Humans are orally exposed to micro- and nanoplastic particles via food and water. • The hazard of real-life plastic particles have only been assessed indirectly via model particles. • Polystyrene particles, primarily in nanosize, produce toxic responses in cell cultures. • There are insufficient studies to conclude anything about the toxicity of polystyrene particles in animals. • Using food-grade TiO 2 as model particle for plastic particles does not indicate strong toxic effects. [ABSTRACT FROM AUTHOR]

The European weather loach ( Misgurnus fossilis ) represents one of many European freshwater fishes in decline. Efficient monitoring is essential if conservation efforts are to be successful, but due to the species’ cryptic biology, traditional monitoring methods currently in use are inefficient, time consuming and likely prone to non-detection error. Here, we investigate the usefulness of environmental DNA (eDNA) monitoring as an alternative or supplementary method for surveying the Danish weather loach population, which is presumed to consist primarily of a single group of no more than 50 individuals. In 2008, the majority of historical Danish localities were surveyed, using traditional fishing techniques. We then applied eDNA methods to a number of these, as well as other potential localities. We successfully detected the weather loach at multiple sites in the single known remaining locality; a result that was later confirmed when local managers caught eight live specimens. Furthermore, the eDNA method indicated presence of the weather loach in another historical locality, where the species has not been observed since 1995. At the remaining localities, weather loach eDNA was not detected, providing further evidence for its absence. Importantly, the eDNA survey required less effort in person-hours and lower costs than the traditional fishing survey. This study confirms that eDNA monitoring is a valid supplement to traditional monitoring methods currently applied to monitor rare freshwater fishes. We propose that by providing reliable distribution data at lower cost and limited effort, the eDNA method can allow for increased management efficiency of endangered freshwater species such as the European weather loach. [ABSTRACT FROM AUTHOR]

A new species of the rare, deep-sea genus Sciadonus Garman, 1899 (Bythitidae) is described based on two specimens obtained by the Brazilian R/V Alpha Crucis on the continental slope off São Paulo State, Southeastern Brazil, western South Atlantic. It differs from its congeners by the combination of the following characters: body pale lacking dark pigmentation except for on female claspers; a pair of dermal tissue flaps anteriorly on lower jaw; pelvic-fin rays present; precaudal vertebrae 39 or 40 and total vertebrae 74 or 75. The key to the species of Sciadonus is updated. A discussion of the presence and differentiation between troglomorphic and miniature characteristics among the species in the aphyonid clade is provided and compared with other bythitids. • A new species of the rare genus Sciadonus is discovered from Brazilian waters. • Highly specialized reproductive apparatus enables internal fertilization and viviparity in a deep-sea fish. • The depletion of sunlight resulted on convergent evolution of troglomorphic traits in deep-sea fishes and cave fishes. • Human activities of oil and natural gas exploration, fisheries and littering may be impacting rare deep-sea species. [ABSTRACT FROM AUTHOR]

Several chemical mutagens and carcinogens, including polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs, are adsorbed to the surface of diesel exhaust particles (DEP). DEP can induce formation of reactive oxygen species and cause oxidative DNA damage as well as bulky carcinogen DNA adducts. Lung tissue is a target organ for DEP induced cancer following inhalation. Recent studies have provided evidence that the lung is also a target organ for DNA damage and cancer after oral exposure to other complex mixtures of PAHs. The genotoxic effect of oral administration of DEP was investigated, in terms of markers of DNA damage, mutations and repair, in the lung of Big Blue® rats fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. There was no significant increase in the mutation frequency in the cII gene. However, an increase of DNA damage measured as DNA strand breaks (comet assay) and bulky DNA adducts (32P post labeling) was observed. The level of DNA strand breaks increased significantly at all dose levels while the level of DNA adducts increased significantly only at the intermediate dose levels. Similarly, the number of oxidized DNA bases measured as endonuclease III and fapyguanine glycosylase (FPG) sensitive sites increased at the intermediate dose levels. The induction of DNA damage by DEP exposure did not increase the expression of the repair genes OGG1 and ERCC1 at the mRNA level. The present study indicates that the lung is a target organ for primary DNA damage following oral exposure to DEP. DNA damage was induced following exposure to relatively low levels of DEP, but under the conditions used in the present experiment DNA damage did not result in an increased mutation rate. [Copyright &y& Elsevier]

Exposure to ambient air pollution has been associated with cancer. Ambient air contains a complex mixture of toxics, including particulate matter (PM) and benzene. Carcinogenic effects of PM may relate both to the content of PAH and to oxidative DNA damage generated by transition metals, benzene, metabolism and inflammation.By means of personal monitoring and biomarkers of internal dose, biologically effective dose and susceptibility, it should be possible to characterize individual exposure and identify air pollution sources with relevant biological effects. In a series of studies, individual exposure to PM2.5, nitrogen dioxide (NO2) and benzene has been measured in groups of 40–50 subjects. Measured biomarkers included 1-hydroxypyrene, benzene metabolites (phenylmercapturic acid (PMA) and trans-trans-muconic acid (ttMA)), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in urine, DNA strand breaks, base oxidation, 8-oxodG and PAH bulky adducts in lymphocytes, markers of oxidative stress in plasma and genotypes of glutathione transferases (GSTs) and NADPH:quinone reductase (NQO1).With respect to benzene, the main result indicates that DNA base oxidation is correlated with PMA excretion. With respect to exposure to PM, biomarkers of oxidative damage showed significant positive association with the individual exposure. Thus, 8-oxodG in lymphocyte DNA and markers of oxidative damage to lipids and protein in plasma associated with PM2.5 exposure. Several types of DNA damage showed seasonal variation. PAH adduct levels, DNA strand breaks and 8-oxodG in lymphocytes increased significantly in the summer period, requiring control of confounders. Similar seasonal effects on strand breaks and expression of the relevant DNA repair genes ERCC1 and OGG1 have been reported.In the present setting, biological effects of air pollutants appear mainly related to oxidative stress via personal exposure and not to urban background levels. Future developments include personal time-resolved monitors for exposure to ultrafine PM and PM2.5, use of GPS, as well as genomics and proteomics based biomarkers. [Copyright &y& Elsevier]

We investigated the interaction of BZ and lipolysaccharide (LPS), a well-known inflammation-promoting agent, in wild-type and inducible nitrogen oxide synthase (iNOS) knockout mice. BZ generated DNA strand breaks (SB) in the liver of both wild-type and iNOS-deficient mice. In the bone marrow (BM) BZ and LPS generated SB only in wild-type mice. The effects were additive, suggesting that both a redox cycling and an iNOS-dependent pathway may be involved. Formamidopyrimidine DNA glycosylase sensitive sites were elevated by BZ in the BM in both types of mice, whereas endonuclease III sensitive sites were not affected by any treatment. Since BZ is associated with leukemia in humans, it suggests that oxidative DNA base damage rather than SB may be important in the development of leukemia. [Copyright &y& Elsevier]

Environmental variation across the range of wild species can lead to local adaptations. The Baltic Sea was formed when the Fenno-Scandian ice sheet retreated around 12 thousand years ago, creating a new brackish water habitat colonised by both marine and freshwater fish species. The European perch (Perca fluviatilis) is a predatory freshwater fish with a large geographical distribution across Eurasia, where it inhabits a wide range of environmental niches. In the Baltic Sea region it has even developed a specialised brackish water perch variant that can tolerate environmental salinity levels, which are lethal to freshwater perch. However, very little is known about the underlying mechanisms facilitating the colonisation and adaptation of perch to the Baltic Sea. Here, we use Genotyping-By-Sequencing data from six freshwater and six brackish water localities to disclose the evolutionary relationship between freshwater and brackish water perch. Our results show that the brackish water perch occurs in multiple distinct genetic clusters. We find that gene flow between brackish water perch with full access to the sea likely led to lower levels of differentiation and higher diversity than in freshwater perch. Selection analyses suggest that genomic adaptation played a role in the colonisation of the Baltic Sea and that the top three regions under selection harbour salinity tolerance genes. We complete by discussing the implications of our findings for management of brackish water perch in the western Baltic sea. [Display omitted] • GBS data from 12 perch populations, six with brackish and six with freshwater origin. • Brackish water populations were found in all three major genetic clades. • Top three regions under selection harboured salinity tolerance genes. [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]

Mining rare earth elements (REEs) can release large amounts of metal(loid)-rich dust, which can pose significant health risks to local residents. However, compared to other types of particulates, toxicity of mining dust has been largely overlooked. To provide experimental evidence on toxicity of REE mine dust, the study assessed the oxidative stress potential and genotoxicity of inhalable particles collected in a REE mining area, and associated toxicological response with source compositions. Both source types (i.e., mine and tailing area) and distances from source (i.e., industrial and residential areas) were considered when selecting the 44 sampling sites. The particle samples contained 2.3–3.5 folds higher concentrations of tested metal(loid)s than background concentrations in soil. Specially, elevated Fe, REEs, Cd, Pb were found. In spite of low cytotoxicity in lung epithelial A549 cells, there was increased cellular ROS production by of particle exposure. Samples with higher mining-originated source contributions (Provenance Index <0.3) had higher cellular ROS production (1.72 fold, 95%CI: 1.66–1.79 fold) than samples with lower mining contributions (1.58 fold, 95%CI: 1.52–1.65 fold). The factors soil (~46%), mine (~22%), and heavy metal (~20%) sources were recognized by source apportionment analysis as the main contributors to cellular ROS production; importantly, mine and heavy metal sources counted more in industrial samples. While samples generated genotoxicity, there were no differences in DNA damage between the location groups of sampling. Collectively, the results indicate that particles in mining areas may cause ROS production and DNA damage in lung cells depending on mine dust. Coupled with the long-range transportation potential of mine dust, safety measures on open pit and dust disposal sites should be adopted. Unlabelled Image • Particles around rare earth mine and tailing areas were rich in metallic components. • The particles induced cellular ROS production and genotoxicity. • Particles collected in industrial areas induced more cellular ROS than in residential areas. • Soil , mine and heavy metal sources were the main contributors to ROS. [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]

• The comet assay is widely used in human biomonitoring to measure DNA damage. • Variation in comet assay results between laboratories has been a problem. • Specific steps in the assay have been identified as causes of this variation. • Including reference standards in experiments helps to control variation. • We give recommendations for improving the reliability of the assay. The comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage – strand breaks, as well as oxidised bases; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured; the duration of alkaline unwinding; the duration of electrophoresis and the voltage gradient applied; and the method used to score the comets. Including reference standards in each experiment allows experimental variability to be monitored – and if variation is not extreme, results can be normalised using reference standard values. Reference standards are also essential for inter-laboratory comparison. Finally, we offer recommendations which, we believe, will limit variability and increase the usefulness of this assay in molecular epidemiology. [ABSTRACT FROM AUTHOR]

Abstract This study investigated the capacity of an anthocyanin-rich fraction (ACN-RF) from blueberry, single anthocyanins (cyanidin, delphinidin and malvidin-3-glucoside; Cy, Dp and Mv-3-glc) and related metabolites (protocatechuic, gallic and syringic acid; PrA, GA and SA) to resolve an inflammation-driven adhesion of monocytes (THP-1) on endothelial cell (HUVECs) and secretion of cell adhesion molecules E-selectin and vascular cell adhesion molecule 1 (VCAM-1). The adhesion of THP-1 to HUVECs was induced by tumour necrosis factor α (TNF-α, 100 ng mL−1). Subsequently, ACN-RF, single ACNs and metabolites (from 0.01 to 10 μg mL−1) were incubated for 24 h. The adhesion was measured in a fluorescence spectrophotometer. E-selectin and VCAM-1 were quantified by ELISA. No toxicological effects were observed for the compounds and the doses tested. ACN-RF and Mv-3-glc reducedTHP-1 adhesion at all the concentrations with the maximum effect at 10 μg/ml (−60.2% for ACNs and-33.9% for Mv-3-glc). Cy-3-glc decreased the adhesion by about 41.8% at 10 μg mL−1, while PrA and GA reduced the adhesion of THP-1 to HUVECs both at 1 and at 10 μg mL−1 (−29.5% and −44.3% for PrA, respectively, and −18.0%and −59.3% for GA, respectively). At the same concentrations a significant reduction of E-selectin, but notVCAM-1 levels, was documented. No effect was observed following Dp-3-glc and SA supplementation. Overall, ACNs and metabolites seem to resolve, in a dose-dependent manner, the inflammation-driven adhesion of THP-1 to HUVECs by decreasing E-selectin concentrations. Interestingly, Mv-3-glc was active at physiologically relevant concentrations. Graphical abstract Anthocyanins and their metabolites are able to directly and/or indirectly modulate monocytes adhesion to endothelial cells through different pathway. They can act against oxidative stress by inducing Nrf2 expression and the activation of antioxidant and detoxifying enzymes able to counteract ROS production. Furthermore, anthocyanins are able to inhibit NF-kB pathway and suppress the production of numerous proinflammatory cytokines (e.g. TNF-α) and adhesion molecules (e.g. VCAM-1). Moreover, anthocyanins are potent inducers of vasomotor factors (i.e. NO) and repressors of vasoconstrictors (e.g. ET) and procoagulant factors (e.g. PAI). Legend: ROS (Reactive oxygen species); Nrf2 (Nuclear factor erythroid 2–related factor 2); GST (Glutathione S-transferase); SOD (Superoxide dismutase); GPx (Glutathione peroxidase); NF-kB (Nuclear factor kappa B); TNF-α (Tumor necrosis factor-alpha); IL-1β (Interleukin-1beta); IL-6 (Interleukin-6); VCAM-1 (Vascular Cell Adhesion Molecule 1); ICAM-1 (Intracellular Adhesion Molecule-1); E-selectin (endothelial-selectin); NO (Nitric oxide); EDHF (Endothelium-derived hyperpolarizing factor); ET-1 (Endothelin-1); TXA2 (Thromboxane A2); PAI-1 (Plasminogen activator inhibitor-1); PAF (Platelet-Activating Factor). Image 1 Highlights • Inflammation plays a central role in the atherosclerosis. • Polyphenols may counteract inflammation and atherosclerotic process. • Anthocyanins and metabolites resolved the adhesion process. • Anthocyanins and metabolites lowered E-selectin levels. • The effects were observed mainly at supraphysiological concentrations. [ABSTRACT FROM AUTHOR]

Abstract Biomass assesment of fish stocks is a difficult task that often involves costly fisheries trawl surveys. Trawl surveys rarely return replicate samples, as trawling attempts are expensive and difficult to reproduce. Furthermore, traditional benthic trawling is often detrimental to habitats and the organisms associated with the sea bottom. The rapidly developing field of environmental DNA (eDNA) analysis offers a new approach to non-invasive monitoring of fish. In the present study, we develop and test species-specific primers and probes for qPCR detection of eDNA from Atlantic herring (Clupea harengus), Atlantic cod (Gadus morhua), European flounder (Platichthys flesus), European plaice (Pleuronectes platessa), and Atlantic mackerel (Scomber scombrus) in the Baltic Sea. A recently published qPCR system was applied for European eel (Anguilla anguilla). Filtered water samples were collected during a stratified benthic trawl survey, enabling parallel comparisons of eDNA concentrations with biomass caught by trawling. No significant correlation was found between eDNA concentrations and the biomass of fish caught by the trawl, although an association was observed between the measured concentrations of eDNA and the known distributions and main abundances of cod, herring, plaice and flounder. This indicates that while eDNA concentrations may not be directly comparable to results from existing methods – likely because aquatic eDNA concentrations are not controlled by the same combinations of factors as e.g. trawl biomass catch – eDNA analysis could prove a useful supplement for monitoring fish stocks in the future. Graphical abstract Unlabelled Image Highlights • Species-specific qPCR assays were developed for detection of environmental DNA from six commercially important marine fishes. • All six primer- probe sets were proven to be species-specific in the North Sea and Baltic Sea. • High levels of environmental DNA were found in areas with large catches of cod, herring, plaice and flounder. • No significant correlations were found between trawled catch size and the environmental DNA quantity as measured in filtered water samples. [ABSTRACT FROM AUTHOR]

Abstract Animal studies have shown that titanium dioxide (TiO 2) exposure affects arterial vasomotor function, whereas little is known about the effects in arteries from humans. This study investigated vasomotor responses after direct exposure of human subcutaneous arteries to food-grade TiO 2 (E171) (14 or 140 μg/ml) for 30 min and 18 h. Vasomotor responses to bradykinin, 5-hydroxytryptamine (5-HT), sarafotoxin 6c (S6c) and nitroglycerin were recorded in wire-myographs. Vasoconstrictor responses to 5-HT were increased in arteries exposed to E171 for 18 h (P < 0.05). Furthermore, an increase in S6c responses was seen in low concentration E171 exposed arteries (30 min exposure; P < 0.05). The vasorelaxation response to nitroglycerin was increased in low concentration E171 exposed arteries (30 min exposure; P < 0.05). Vasorelaxation responses to bradykinin were unaffected after treatment with E171. There was no difference in gene expression levels of intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, 5-hydroxytryptamine receptor 1B, 5-hydroxytryptamine receptor 2A, endothelin receptor A and endothelin receptor B in E171 exposed arteries after exposure to TiO 2 for 30 min or 18 h. In conclusion, this study shows that the same type of vasomotor dysfunction is found in artery segments of rats and humans following ex vivo exposure to E171. Highlights • Exposure of human arteries to E171 resulted in increased vasocontraction response. • There were unaltered expressions of 5-hydroxytryptamine or sarafotoxin 6c receptors. • The exposure did not change the expression of cell adhesion molecules in arteries. • Exposure to E171 increased the endothelial-independent vasorelaxation response. [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]

The single cell gel electrophoresis technique is based on the measurement of DNA migration in an electric field and enables to investigate via determination of DNA-damage the impact of foods and their constituents on the genetic stability. DNA-damage leads to adverse effects including cancer, neurodegenerative disorders and infertility. In the last 25 years approximately 90 human intervention trials have been published in which DNA-damage, formation of oxidized bases, alterations of the sensitivity towards reactive oxygen species and chemicals and of repair functions were investigated with this technique. In approximately 50% of the studies protective effects were observed. Pronounced protection was found with certain plant foods (spinach, kiwi fruits, onions), coffee, green tea, honey and olive oil. Also diets with increased contents of vegetables caused positive effects. Small amounts of certain phenolics (gallic acid, xanthohumol) prevented oxidative damage of DNA; with antioxidant vitamins and cholecalciferol protective effects were only detected after intake of doses that exceed the recommended daily uptake values. The evaluation of the quality of the studies showed that many have methodological shortcomings (lack of controls, no calibration of repair enzymes, inadequate control of the compliance and statistical analyses) which should be avoided in future investigations. • The article summarizes the results of dietary single cell gel electrophoresis studies with humans. • The findings were obtained in intervention trials with complex diets, beverages and food constituents. • Many investigations have methodological shortcomings. • Suggestions for improved study designs and adequate statistical analyses are provided. [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]

Exposure to high aspect ratio nanomaterials, such as multi-walled carbon nanotubes (MWCNTs) may be associated with increased risk of atherosclerosis, pulmonary disease, and cancer. In the present study, we investigated the cardiovascular and pulmonary health effects of 10 weeks of repeated oral or pulmonary exposures to MWCNTs (4 or 40 μg each week) in Apolipoprotein E -deficient ( ApoE −/− ) mice fed a Western-type diet. Intratracheal instillation of MWCNTs was associated with oxidative damage to DNA in lung tissue and elevated levels of lipid peroxidation products in plasma, whereas the exposure only caused a modest pulmonary inflammation in terms of increased numbers of lymphocytes in bronchoalveolar lavage fluid. Ultrasound imaging in live animals revealed an increase in the inner and outer wall thickness of the aortic arch at 10 weeks after pulmonary exposure to MWCNTs, which may suggest artery remodelling. However, we did not find accelerated plaque progression in the aorta or the brachiocephalic artery by histopathology. Furthermore, repeated oral exposure to MWCNTs did not cause changes in the composition of gut microbiota of exposed mice. Collectively, this study indicates that repeated pulmonary exposure to MWCNTs was associated with oxidative stress, whereas cardiovascular effects encompassed remodelling of the aorta wall. [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]

Inhalation is the main pathway of ZnO exposure in the occupational environment but only few studies have addressed toxic effects after pulmonary exposure to ZnO nanoparticles (NP). Here we present results from three studies of pulmonary exposure and toxicity of ZnO NP in mice. The studies were prematurely terminated because interim results unexpectedly showed severe pulmonary toxicity. High bolus doses of ZnO NP (25 up to 100 μg; ≥1.4 mg/kg) were clearly associated with a dose dependent mortality in the mice. Lower doses (≥6 μg; ≥0.3 mg/kg) elicited acute toxicity in terms of reduced weight gain, desquamation of epithelial cells with concomitantly increased barrier permeability of the alveolar/blood as well as DNA damage. Oxidative stress was shown via a strong increase in lipid peroxidation and reduced glutathione in the pulmonary tissue. Two months post-exposure revealed no obvious toxicity for 12.5 and 25 μg on a range of parameters. However, mice that survived a high dose (50 μg; 2.7 mg/kg) had an increased pulmonary collagen accumulation (fibrosis) at a similar level as a high bolus dose of crystalline silica. The recovery from these toxicological effects appeared dose-dependent. The results indicate that alveolar deposition of ZnO NP may cause significant adverse health effects. [ABSTRACT FROM AUTHOR]

Carbon black exposure causes oxidative stress, inflammation and genotoxicity. The objective of this systematic review was to assess the contributions of primary (i.e. direct formation of DNA damage) and secondary genotoxicity (i.e., DNA lesions produced indirectly by inflammation) to the overall level of DNA damage by carbon black. The database is dominated by studies that have measured DNA damage by the comet assay. Cell culture studies indicate a genotoxic action of carbon black, which might be mediated by oxidative stress. Many in vivo studies originate from one laboratory that has investigated the genotoxic effects of Printex 90 in mice by intra-tracheal instillation. Meta-analysis and pooled analysis of these results demonstrate that Printex 90 exposure is associated with a slightly increased level of DNA strand breaks in bronchoalveolar lavage cells and lung tissue. Other types of genotoxic damage have not been investigated as thoroughly as DNA strand breaks, although there is evidence to suggest that carbon black exposure might increase the mutation frequency and cytogenetic endpoints. Stratification of studies according to concurrent inflammation and DNA damage does not indicate that carbon black exposure gives rise to secondary genotoxicity. Even substantial pulmonary inflammation is at best only associated with a weak genotoxic response in lung tissue. In conclusion, the review indicates that nanosized carbon black is a weak genotoxic agent and this effect is more likely to originate from a primary genotoxic mechanism of action, mediated by e.g., oxidative stress, than inflammation-driven (secondary) genotoxicity. [ABSTRACT FROM AUTHOR]

European plaice (Pleuronectes platessa) is a key species in commercial fisheries in the North Sea, Skagerrak, Kattegat, and Baltic Sea. The reformed European Union Common Fisheries Policy includes the possibility of exemptions from the landing obligation for "species for which scientific evidence demonstrates high survival rates". Discard survival from set-net fisheries is poorly studied. Trials were conducted on two commercial fishing vessels over seven trips from November to February 2017–2018 in the Baltic Sea. The nylon trammel nets had a nominal bar size for the inner/outer wall of 75/350 mm and 85/400 mm. Soaking time was 23–47 h, water depth 7–18 m, and deck temperature was −0.1–6.0 °C. Following commercial practice, the trammel nets were hauled back onto the vessel, after which netting and fish passed through a net hauler onto a steel sorting table where the entire fish catch were manually untangled by the fishers and plaice collected by scientists. We used a storage system for housing the captured fish inside fishing harbours during observations. Catch-damage-index (CDI) and Reflex Action Mortality Predictor (RAMP) scores were used to assess fish condition immediately after capture and at the end of observation periods. All plaice below 40 cm were sampled with a total number of 118 individuals from 13 fleets (several nets joined together). The fish were assessed for short-term survival for 4–10 days with full survival (100%). The majority of fish exhibited no reflex impairments. Minor bruises, fraying, and net marks were frequently observed on captured fish. The overall condition of the fish did not change during observation periods. • High short-term survival rate in plaice from trammel net fisheries during winter season. • Development of methodology for set-net discard survival studies. • Low Reflex Action Mortality Predictor (RAMP) in trammel net fished plaice. [ABSTRACT FROM AUTHOR]

Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24 h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3 h, but not 24 h. Intracellular level of reduced glutathione was increased after 24 h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction. [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]