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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

PDF File - 512K, Solid cancer cell lines and their genetic characteristics.

Arsenic trioxide (ATO), one of the oldest remedies used in traditional medicine, was recently rediscovered as an anticancer drug and approved for treatment of relapsed acute promyelocytic leukemia. However, its activity against nonhematologic cancers is rather limited so far. Here, we show that inhibition of ATO-mediated EGF receptor (EGFR) activation can be used to potently sensitize diverse solid cancer types against ATO. Thus, combination of ATO and the EGFR inhibitor erlotinib exerted synergistic activity against multiple cancer cell lines. Subsequent analyses revealed that this effect was based on the blockade of ATO-induced EGFR phosphorylation leading to more pronounced G2–M arrest as well as enhanced and more rapid induction of apoptosis. Comparable ATO-sensitizing effects were also found with PI3K/AKT and mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, suggesting an essential role of the EGFR-mediated downstream signaling pathway in cancer cell protection against ATO. H2AX staining and comet assay revealed that erlotinib significantly increases ATO-induced DNA double-strand breaks (DSB) well in accordance with a role of the EGFR signaling axis in DNA damage repair. Indeed, EGFR inhibition led to downregulation of several DNA DSB repair proteins such as Rad51 and Rad50 as well as reduced phosphorylation of BRCA1. Finally, the combination treatment of ATO and erlotinib was also distinctly superior to both monotreatments against the notoriously therapy-resistant human A549 lung cancer and the orthotopic p31 mesothelioma xenograft model in vivo. In conclusion, this study suggests that combination of ATO and EGFR inhibitors is a promising therapeutic strategy against various solid tumors harboring wild-type EGFR. Mol Cancer Ther; 12(6); 1073–84. ©2013 AACR.

Supplementary Figure 1: Tumor burden and inflammation in IL-10 KO mice. Supplementary Figure 2: LOH, MSI and CIMP in IL-10 KO mice. Supplementary Figure 3: DNA damage and apoptosis in IL-10KO mice. Supplementary Figure 4: Nrf2 is increased in IL-10 KO small and large intestinal organoids.

Supplementary Methods

Introduction. The incidence of post-traumatic stress disorder (PTSD) is increasing due to war in Ukraine is of growing importance as this disorder is a serious risk factor for alcohol/drug abuse, depression, suicide and functional dysregulation of internal organs, in particular cardiovascular, endocrine, immune systems. PTSD can also lead to social inadaptation in family and at work. This makes PTSD a very important social problem in Ukraine that causes significant economic damage, since most of the PTSD patients are young and in working age. Objectives. The purpose of this paper is to describe the rationale, key elements of design, methodology and future prospects of the ongoing project “Exploratory study of health related physiological and biochemical parameters to identify simple, robust and relevant biomarkers for therapeutic interventions for PTSD patients”. Methods. Short time ECG records (5 minutes in supine position and 6 minutes in orthostatic test) recorded by “CARDIOLAB” (Kharkiv, Ukraine) digital ECG device with software to analyze HRV was used to calculate time-domain and frequency-domain parameters of HRV. Results. The pattern of HRV changes indicates suppression of parasympathetic activity, low overall spectral power and dominance of sympathetic branch of autonomic nervous system in participants with PTSD. Conclusions. PTSD has no effective treatment so far and the best clinical practices rarely reach efficacy of 50%. The realization of the exploratory clinical study described in this paper will provide insights in the mechanisms underlying systemic consequences of PTSD and will build the evidence enabling clinical trial(s) for studying treatment intervention for patients with PTSD.

Road markers are exposed to various chemicals and particles. The aim of this study was to determine whether road worker exposure induceschromosomal damage which is indicative for increased cancer risks. Micronucleus (MN) cytome assays were thus conducted with exfoliated nasal and buccal cells collected from 42 workers and 42 matched controls. The frequencies of MN (reflecting chromosomal aberrations), nuclear buds (NBuds; reflecting gene amplifications) and binucleated cells (BN; reflecting disturbed mitosis) were scored. Further, the rates of nuclear anomalies indicative of acute cytotoxicity (condensed chromatin, karyorrhexis, karyolysis, pyknosis) were evaluated. Data demonstrated marked induction of MN, NBuds, and BN by 1.34-fold, 1.24-fold and 1.14-fold in buccal cells. In nasal cells, only MN frequencies were elevated, 1.23-fold. These effects were paralleled by increased rates of condensed chromatin, karyorrhexis and karyolysis in both cell types. The effects were more pronounced in individuals who had worked for more than 10 years while smoking did not produce synergistic responses. This is the first investigation concerning the induction of genetic damage in road markers and the results are suggestive for enhanced cancer risks. It is conceivable that exposure to silica dust (known to induce cancer and genetic damage) and/or benzoyl peroxide which forms reactive radicals may be associated with the observed genetic damage in road workers. Further investigations of the cancer risks of these workers are warranted.

Methamphetamine (METH) is a widely consumed psychostimulant drug; its acute toxic effects in brain and liver are well known, furthermore, there is some evidence in regard to its DNA damaging properties in humans. Therefore, we studied the impact of the drug on genomic stability in human derived hepatoma (HepG2) cells, which reflect the activation/detoxification of drugs better than other cell lines. Furthermore, experiments with human buccal derived cells (TR146) were conducted as the drug is consumed orally. Induction of DNA damage in both cell types with doses reflecting the exposure in abusers was found in single cell gel electrophoresis (SCGE) assays (which detect single and double strand breaks as well as apurinic sites). Furthermore, induction of micronuclei (formed as a consequence of structural and numerical chromosomal aberrations) and formation of nuclear buds resulting from gene amplifications was detected. Additional experiments with lesion-specific enzymes showed that the drug causes oxidation of purines and pyrimidines, indicating that its genotoxic effects may be due to oxidation of the DNA. Our findings support the assumption that the drug may cause adverse health effects (such as cancer and infertility) in long-term users which are causally related to DNA damage.

Consumption of very hot beverages and foods increases the incidence of oral and esophageal cancer but the mechanisms are not known and the critical temperature is not well defined. We realized a study with exfoliated cells from the oral cavity of individuals (n = 73) that live in an area in Iran which has the highest incidence of EC worldwide. Consumption of beverages at very high temperatures is a characteristic feature of this population. We analyzed biomarkers which are (i) indicative for genetic instability (micronuclei that are formed as a consequence of chromosomal damage, nuclear buds which are a consequence of gene amplifications and binucleated cells which reflect mitotic disturbances), (ii) markers that reflect cytotoxic effects (condensed chromatin, karyorrhectic, karyolitic and pyknotic cells), (iii) furthermore, we determined the number of basal cells which is indicative for the regenerative capacity of the buccal mucosa. The impact of the drinking temperature on the frequencies of these parameters was monitored with thermometers. We found no evidence for induction of genetic damage but an increase of the cytotoxic effects with the temperature was evident. This effect was paralleled by an increase of the cell division rate of the mucosa which was observed when the temperature exceeded 60 °C. Our findings indicate that cancer in the upper digestive tract in drinkers of very hot beverages is not caused by damage of the genetic material but by an increase of the cell division rate as a consequence of cytotoxic effects which take place at temperatures over 60 °C. It is known from earlier experiments with rodents that increased cell divisions lead to tumor promotion in the esophagus. Our findings provide a mechanistic explanation and indicate that increased cancer risks can be expected when the drinking temperature of beverages exceeds 60 °C.

Cervical cancer (CC) is the fourth most common cancer in women; the survival rates depend strongly on its early detection. The Pap test is the most frequently used diagnostic tool, but due to its limited sensitivity/specificity, additional screening tests are needed. Therefore, we evaluated the use of micronucleus (MN) assays with cervical cells for the prediction and diagnosis of CC. MN reflects structural and numerical chromosomal aberrations. A search was performed in Pubmed, Scopus, Thomson ISI and Google Scholar. Subsequently, meta-analyses were performed for different grades of abnormal findings in smears and biopsies from patients which were diagnosed with CC. Results of 21 studies in which findings of MN experiments were compared with data from Pap tests show that higher MN frequencies were found in women with abnormal cells that are indicative for increased cancer risks. MN frequency ratios increased in the order inflammation (2.1) < ASC-US and ASC-H (3.3) < LGSIL (4.4) < HGSIL (8.4). Furthermore, results are available from 17 investigations in which MN were scored in smears from patients with neoplasia. MN rates increased with the degree of neoplasia [CIN 1 (4.6) < CIN 2 (6.5) and CIN 3 (10.8)] and were significantly higher (8.8) in CC patients. Our meta-analysis indicates that the MN assay, which is easy to perform in combination with Pap tests, may be useful for the detection/prediction of CC. However, standardization (including definition of the optimal cell numbers and stains) and further validation is necessary before the MN test can be implemented in routine screening.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre‑metastatic niches, leading to their colonisation. 'S' stereoisomer 12S‑hydroxy‑5Z,8Z,10E,14Z‑eicosatetraenoic acid [12(S)‑HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)‑HETE‑treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v‑rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY‑box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription‑quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11‑7082, extended the mono‑culture experiments to co‑culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent‑induced defect' assay. 12(S)‑HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)‑HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK‑inhibitor defactinib and the NF‑κB inhibitor Bay11‑7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro‑metastatic signalling cascade. Therefore, targeting the endothelial‑specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.

An amendment to this paper has been published and can be accessed via the original article.

The purpose of this study was to investigate the effect of six months strength training with or without supplementing protein and vitamins, on chromosomal integrity of buccal cells in institutionalized elderly. One hundred seventeen women and men (65–98 years) performed either resistance training (RT), RT combined with a nutritional supplement (RTS) or cognitive training (CT) twice per week for six months. Participants’ fitness was measured using the 6 min walking, the chair rise, and the handgrip strength test. Genotoxicity and cytotoxicity parameters were investigated with the Buccal Micronucleus Cytome (BMcyt) assay. Six minutes walking and chair rise performance improved significantly, however, no changes of the parameters of the BMcyt were detected. Age and micronuclei (MN) frequency correlated significantly, for both women (r = 0.597, p = 0.000) and men (r = 0.508, p = 0.000). Squared regressions revealed a significant increase in the MN frequency of buccal cells with age (R2 = 0.466, p = 0.000). Interestingly and contrary to what was shown in blood lymphocytes, chromosomal damage in buccal cells increases until very old age, which might qualify them as a valid biomarker for aging. Unexpectedly, in this group of institutionalized elderly, resistance training using elastic bands had no effect on chromosomal damage in buccal cells., Graphical abstract Image 1, Highlights • Mutation in buccal cells increased until very old age – a new aging biomarker? • Chromosomal damage in buccal cells was age-dependent and equal for women and men. • Strength training improved fitness but not mutagenicity in buccal cells of elderly.

Tamara Grummt passed away on January 26, 2020 in Oelsnitz/Vogtland, Germany. Tamara was one of the scientific pioneers in the field of environmental toxicology, namely genotoxicity and hygiene of drinking and bathing waters. Her passing is not only a great loss to environmental research and to the global environmental toxicology community—we have lost an outstanding personality with the heart in the right place, who has become, for many of us, a wonderful friend.

Health authorities are alarmed worldwide about the increase of obesity and overweight in the last decades which lead to adverse health effects including inflammation, cancer, accelerated aging and infertility. We evaluated the state of knowledge concerning the impact of elevated body mass on genomic instability. Results of investigations with humans (39 studies) in which DNA damage was monitored in lymphocytes and sperm cells, are conflicting and probably as a consequence of heterogeneous study designs and confounding factors (e.g. uncontrolled intake of vitamins and minerals and consumption of different food types). Results of animal studies with defined diets (23 studies) are more consistent and show that excess body fat causes DNA damage in multiple organs including brain, liver, colon and testes. Different molecular mechanisms may cause genetic instability in overweight/obese individuals. ROS formation and lipid peroxidation were found in several investigations and may be caused by increased insulin, fatty acid and glucose levels or indirectly via inflammation. Also reduced DNA repair and formation of advanced glycation end products may play a role but more data are required to draw firm conclusions. Reduction of telomere lengths and hormonal imbalances are characteristic for overweight/obesity but the former effects are delayed and moderate and hormonal effects were not investigated in regard to genomic instability in obese individuals. Increased BMI values affect also the activities of drug metabolizing enzymes which activate/detoxify genotoxic carcinogens, but no studies concerning the impact of these alterations of DNA damage in obese individuals are available. Overall, the knowledge concerning the impact of increased body weight and DNA damage is poor and further research is warranted to shed light on this important issue.

Purpose Aim of the study was to find out if gallic acid (GA), a common phenolic in plant foods, prevents obesity induced DNA damage which plays a key role in the induction of overweight associated cancer. Methods Male and female C57BL6/J mice were fed with a low fat or a high fat diet (HFD). The HFD group received different doses GA (0, 2.6–20 mg/kg b.w./day) in the drinking water for 1 week. Subsequently, alterations of the genetic stability in blood and inner organs were monitored in single cell gel electrophoresis assays. To elucidate the underlying molecular mechanisms: oxidized DNA bases, alterations of the redox status, lipid and glucose metabolism, cytokine levels and hepatic NF-κB activity were monitored. Results HFD fed animals had higher body weights; increased DNA damage and oxidation of DNA bases damage were detected in colon, liver and brain but not in blood and white adipose tissue. Furthermore, elevated concentrations of insulin, glucose, triglycerides, MCP-1, TNF-α and NF-κB activity were observed in this group. Small amounts of GA, in the range of human consumption, caused DNA protection and reduced oxidation of DNA bases, as well as biochemical and inflammatory parameters. Conclusions Obese animals have increased DNA damage due to oxidation of DNA bases. This effect is probably caused by increased levels of glucose and insulin. The effects of GA can be explained by its hypoglycaemic properties and indicate that the consumption of GA-rich foods prevents adverse health effects in obese individuals. Electronic supplementary material The online version of this article (10.1007/s00394-018-1782-2) contains supplementary material, which is available to authorized users.

Results of a number of studies indicate that electroplaters have increased cancer risks as a consequence of exposure to genotoxic metals such as chromium (VI) and nickel. These effects may be due to induction of damage of the genetic material which plays a key role in the etiology of cancer, and it was found that workers in galvanization factories exhibited increased levels of DNA damage. The aim of the present study was to investigate genetic stability in workers of a bright plating factory who are exposed to chromium (Cr) and cobalt (Co). Exfoliated cells were collected from the buccal and nasal mucosa of workers (n = 42) and matched controls (n = 43) and analyzed for induction of micronuclei (MN) which are formed as a consequence of chromosomal aberrations. In addition, other nuclear anomalies namely nuclear buds (Nbuds) which are formed as a consequence of gene amplification and markers indicating different stages of cell death (condensed chromatin, karyorrhexis, karyolysis, and pyknosis) were also assessed. No evidence was noted for induction of MN, but significantly increased rates of Nbuds in cells from both, buccal and nasal mucosa, were found. Parameters which are indicative for cytotoxic effects were more pronounced in nasal cells and rose with duration of employment period. Overall, our findings indicated that no apparent chromosomal damage occurred in bright electroplaters. However, data demonstrated that acute cytotoxic effects may lead to inflammations and/or lesions in epithelia of the respiratory tract of the workers.

Some epidemiological studies indicate that mobile phones cause glioblastomas in humans. Since it is known that genomic instability plays a key role in the etiology of cancer, we investigated the effects of the universal mobile telecommunications system radiofrequency (UMTS-RF) signal, which is used in "smart" phones, on micronucleus (MN) formation and other anomalies such as nuclear buds (NBUDs) and nucleoplasmatic bridges (NPBs). MN are formed by structural and numerical aberrations, NBs reflect gene amplification and NPBs are formed from dicentric chromosomes. The experiments were conducted with human glioblastoma cell lines, which differ in regard to their p53 status, namely U87 (wild-type) and U251 (mutated). The cells were cultivated for 16h in presence and absence of fetal calf serum and exposed to different SAR doses (0.25, 0.50 and 1.00W/kg), which reflect the exposure of humans, in presence and absence of mitomycin C as former studies indicate that RF may cause synergistic effects in combination with this drug. We found no evidence for induction of MN and other anomalies. However, with the highest dose, induction of apoptosis was observed in U251 cells on the basis of the morphological features of the cells. Our findings indicate that the UMTS-RF signal does not cause chromosomal damage in glioblastoma cells; the mechanisms which lead to induction of programmed cell death will be investigated in further studies.

Obesity as a multifactorial disorder involves low-grade inflammation, increased reactive oxygen species incidence, gut microbiota aberrations, and epigenetic consequences. Thus, prevention and therapies with epigenetic active antioxidants, (-)-Epigallocatechin-3-gallate (EGCG), are of increasing interest. DNA damage, DNA methylation and gene expression ofDNA methyltransferase 1,interleukin 6, andMutL homologue 1were analyzed in C57BL/6J male mice fed a high-fat diet (HFD) or a control diet (CD) with and without EGCG supplementation. Gut microbiota was analyzed with quantitative real-time polymerase chain reaction. An induction of DNA damage was observed, as a consequence of HFD-feeding, whereas EGCG supplementation decreased DNA damage. HFD-feeding induced a higher inflammatory status. Supplementation reversed these effects, resulting in tissue specific gene expression and methylation patterns ofDNA methyltransferase 1andMutL homologue 1. HFD feeding caused a significant lower bacterial abundance. TheFirmicutes/Bacteroidetesratio is significantly lower in HFD + EGCG but higher in CD + EGCG compared to control groups. The results demonstrate the impact of EGCG on the one hand on gut microbiota which together with dietary components affects host health. On the other hand effects may derive from antioxidative activities as well as epigenetic modifications observed on CpG methylation but also likely to include other epigenetic elements.

Background: Colorectal carcinoma (CRC) is the third most common cancer worldwide. Platinum-based anticancer compounds still constitute one mainstay of systemic CRC treatment despite limitations due to adverse effects and resistance development. Trabectedin has shown promising antitumor effects in CRC, however, again resistance development may occur. In this study, we aimed to develop strategies to circumvent or even exploit acquired trabectedin resistance in novel CRC treatment regimens. Methods: Human HCT116 CRC cells were selected for acquired trabectedin resistance in vitro and characterised by cell biological as well as bioinformatic approaches. In vivo xenograft experiments were conducted. Results: Selection of HCT116 cells for trabectedin resistance resulted in p53-independent hypersensitivity of the selected subline against cisplatin. Bioinformatic analyses of mRNA microarray data suggested deregulation of nucleotide excision repair and particularly loss of the ubiquitin ligase CUL4A in trabectedin-selected cells. Indeed, transient knockdown of CUL4A sensitised parental HCT116 cells towards cisplatin. Trabectedin selected but not parental HCT116 xenografts were significantly responsive towards cisplatin treatment. Conclusions: Trabectedin selection-mediated CUL4A loss generates an Achilles heel in CRC cancer cells enabling effective cisplatin treatment. Hence, inclusion of trabectedin in cisplatin-containing cancer treatment regimens might cause profound synergism based on reciprocal resistance prevention.

This chapter gives an overview on the use of micronucleus (MN) assays in occupational studies involving exposure to different groups of chemical genotoxins. Currently available data show that experiments with lymphocytes detect genetic damage in workers caused by different groups of carcinogens. It is well documented that MN formation in lymphocytes is a valid marker for the detection of cancer risks in humans, this is probably also true for exfoliated buccal cells. Only a few studies with nasal and bladder cells have been realized with chemically exposed workers; the results are promising and indicate that the former approach may be useful for biomonitoring genotoxic effects of inhalative exposures, while the latter detects the effects of genotoxic chemicals that cause cancer in the urothelial tract. Taken together, the evaluation of the data shows that experiments with lymphocytes are currently the most widely used approach in occupational studies and that the number of investigations increased worldwide substantially over the last few years; the latter conclusion is also true for experiments with exfoliated buccal cells, which have the advantage that samples can be collected with non-invasive methods and that no cultivation of the cells is required.

Tobacco smoke contains numerous genotoxic carcinogens (e.g. polycyclic aromatic hydrocarbons, heavy metals, tobacco-specific nitroso-compounds, aromatic amines) and causes more than 7 million deaths globally per year. In addition to the lungs, tumor induction was found in several other organs of smokers, in particular in the bladder, larynx and pharynx. In vitro studies with mammalian cell lines and experiments with laboratory rodents found that individual smoke constituents and also smoke condensates cause the formation of micronuclei (MN) and other biomarkers of DNA damage. Human studies with cigarette smokers detected MN induction in lymphocytes and also in exfoliated cells from the mouth, bladder and cervix, but clear positive results were confined to heavy smokers. Furthermore, positive results were observed with water pipe smokers (two studies), while negative results were reported in a study with tobacco free electronic cigarettes. Data concerning cigar and pipe smoking are not available at present. There is some evidence of potential synergistic effects of smoking in combination with occupational exposures to other genotoxins, but more investigations are required to draw firm conclusions. Also combination effects with alcohol consumption and the impact of age and body weight on MN formation have only been poorly investigated.

Epidemiological findings indicate that the chewing of tobacco, betel (with and without tobacco) and khat leaves causes cancer and preneoplastic lesions (leukoplakia) in the oral cavity, while no data are available concerning the effects of coca leaves. The results of human studies show that these habits (except the consumption of coca leaves) cause the formation of micronuclei (MN) in exfoliated buccal mucosa cells. Taken together, the results show that MN reflect the cancer risks of this widespread chewing habit. In vitro and animal experiments with extracts of betel quid and tobacco support the assumption that chewing causes damage of the genetic material. It is likely that alkaloids that are contained in these plants, such as cathinione in khat, arecoline and structurally related compounds in betel quid, account for their genotoxic properties. Furthermore, specific nitrosamines were identified in betel quid and also in chewing tobacco which may play a role in regard to MN formation in the buccal cavity.

Epidemiological findings indicate that exposure to high frequency electromagnetic fields (HF-ELF), which are used for mobile telecommunications, and also to extremely low frequency magnetic fields (from usage of electric current) may cause cancer in humans. Evaluation of the literature about induction of micronuclei shows that the results of in vitro experiments are controversial and depend strongly on the type of indicator cells. However, the results of meta-analyses of studies with human buccal cells and lymphocytes indicate that both forms of radiation cause micronuclei formation, which is a biomarker of numerical and/or structural chromosomal aberrations. Since the methodological quality of many human studies on electromagnetic fields is poor, further high-quality investigations are warranted.

Numerous studies indicate that damage of the genetic material plays a crucial role in a variety of human diseases, in particular in the etiology of cancer. Human, animal and also in vitro studies provide evidence that coffee and specific constituents (e.g. chlorogenic acids, N-methylpyridinium, caffeine and diterpenoids) prevent DNA damage. Among the most relevant molecular mechanisms which have been identified so far are prevention of inflammations and of oxidative damage of the genetic material as well as protection against genotoxic carcinogens found in human foods (heterocyclic aromatic amines, polycyclic aromatic hydrocarbons and mycotoxins). These effects are caused by induction of antioxidant and drug detoxifying (phase II) enzymes. It is possible that cancer protective properties of coffee (e.g. prevention of hepatocellular carcinoma) as well as other beneficial health effects (improvement of male fertility and prevention of neurodegenerative disorders) are due to the antimutagenic properties of coffee, but further investigations are required to elucidate these associations.

About 40 million workers are occupationally exposed to crystalline silica (CS) which was classified as a human carcinogen by the IARC. It is assumed that damage of the genetic material via inflammation and reactive oxygen species by CS lead to formation of malignant cells. We conducted a systematic literature search to find out if inhalation of CS containing dusts at workplaces causes damage of the genetic material. Thirteen studies were found eligible for this review, in most of them (n = 9) micronuclei (MN) which reflect structural/numerical chromosomal aberrations were monitored in lymphocytes and/or in exfoliated buccal cells. In 5 investigations DNA damage was measured in blood cells in single cell gel electrophoresis (comet) experiments. Frequently studied groups were potters, stone cutters, miners and construction workers. Results of meta-analyses show that exposure to CS causes formation of MN and DNA breaks, the overall ratio values were in exposed workers 2.06- and 1.96-fold higher than in controls, respectively. Two studies reported increased levels of oxidized guanine, and higher levels of DNA adducts with malondialdehyde indicating that exposure to CS leads to oxidative damage. The exposure of the workers to CS was quantified only in two studies, information concerning the size and chemical structures of the particles is lacking in most investigations. Therefore, it is not possible to use the results to derive occupational exposure limits of workers to CS which vary strongly in different countries. Nevertheless, the evaluation of the current state of knowledge shows that biomonitoring studies in which damage of the genetic material is measured in CS exposed workers can contribute to assess adverse health effects as consequence of DNA instability in specific occupations. Refereed/Peer-reviewed

During the harvest period, tobacco workers are exposed to nicotine and it is known that absorption of the alkaloid via the leaves causes green tobacco sickness (GST). We investigated if GST and its symptoms are associated with DNA damage and alterations of the redox status. DNA damage was measured in lymphocytes of tobacco workers and controls (n = 40/group) in single cell gel electrophoresis assays. Exposure to nicotine was determined by plasma cotinine measurements, alterations of the redox status by quantification of the total antioxidant capacity (TEAC) and of thiobarbituric acid reactive substances (TBARS). The symptoms of GTS included nausea, abdominal cramps, headache, vomiting and dizziness, and 50% of the workers had more than one symptom. Cotinine levels were enhanced in the workers (111 ng/mL); furthermore, the extent of DNA damage was ca. 3-fold higher than in the controls. This effect was more pronounced in participants with GST compared to healthy nicotine exposed workers and increased in individuals with specific symptoms (range 22–36%). TBARS levels did not differ between workers and unexposed controls, while TEAC values were even increased (by 14.3%). Contact with nicotine present in tobacco leaves causes GTS and leads to damage of the DNA; this effect is more pronounced in workers with GTS symptoms and is associated with alterations of the redox status. Damage of the genetic material which was found in the workers may lead to adverse long-term effects that are caused by genomic instability such as cancer and accelerated ageing.

Aim of this study was the investigation of the genotoxic properties of XLR-11 [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone, a widely consumed synthetic cannabinoid (SC), and of the benzoyl indole RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone). We characterized the DNA-damaging properties of these drugs in different experimental systems. No evidence for induction of gene mutations was detected in bacterial (Salmonella/microsome) tests, but clear dose-dependent effects were found in in vitro single cell gel electrophoresis (SCGE) assays with human lymphocytes and with buccal- and lung-derived human cell lines (TR-146 and A-549). These experiments are based on the determination of DNA migration in an electric field and enable the detection of single- and double-strand breaks and apurinic sites. Furthermore, we found that both drugs induce micronuclei which are formed as a consequence of chromosomal aberrations. The lack of effects in SCGE experiments with lesion-specific enzymes (FPG, Endo III) shows that the DNA damage is not caused by formation of oxidatively damaged bases; experiments with liver enzyme homogenates and bovine serum albumin indicate that the drugs are not converted enzymatically to DNA-reactive intermediates. Furthermore, results with buccal- and lung-derived human cells show that gaseous treatment of the cells under conditions which reflect the exposure situation in drug users may cause damage of the genetic material in epithelia of the respiratory tract. Since DNA instability is involved in the etiology of cancer, these findings can be taken as an indication that consumption of the SCs may cause tumors in the respiratory tract of consumers.

Acid Black 10B (AB10B) is widely used for the production of textiles, leather and prints. It is a representative of azo dyes and it is well documented that some of these compounds are mutagenic per se, and that cleavage products (in particular aromatic amines) may cause damage of the genetic material and cancer. Since no toxicological data on AB10B have been published, we evaluated its mutagenic activity in Salmonella/microsome assays and studied its acute toxic and genotoxic properties in a human derived liver cell line (HepG2) which retained the activities of drug metabolizing enzymes. The compound did not cause cytotoxicity (MTT assay), but clear genotoxic effects were detected in pro- and eukaryotic indicator cells. Dose dependent induction of his+ revertants was seen in strain TA98 which detects frameshift mutations without metabolic activation; a more pronounced effect was seen in its derivative YG1024 which overexpresses N-acetyltransferase. Induction of single/double strand breaks by Comet assay was detected with concentrations > 0.125 mg/mL in liver derived cells; as well as increased rates for micronucleus (reflecting structural and numeric chromosomal aberrations) and nuclear buds which are a consequence of gene amplifications were seen with a higher dose (2.0 mg/mL) (p

Cannabidiol (CBD) and cannabidivarin (CBDV) are natural cannabinoids which are consumed in increasing amounts worldwide in cannabis extracts, as they prevent epilepsy, anxiety, and seizures. It was claimed that they may be useful in cancer therapy and have anti-inflammatory properties. Adverse long-term effects of these drugs (induction of cancer and infertility) which are related to damage of the genetic material have not been investigated. Therefore, we studied their DNA-damaging properties in human-derived cell lines under conditions which reflect the exposure of consumers. Both compounds induced DNA damage in single cell gel electrophoresis (SCGE) experiments in a human liver cell line (HepG2) and in buccal-derived cells (TR146) at low levels (≥ 0.2 µM). Results of micronucleus (MN) cytome assays showed that the damage leads to formation of MNi which reflect chromosomal aberrations and leads to nuclear buds and bridges which are a consequence of gene amplifications and dicentric chromosomes. Additional experiments indicate that these effects are caused by oxidative base damage and that liver enzymes (S9) increase the genotoxic activity of both compounds. Our findings show that low concentrations of CBD and CBDV cause damage of the genetic material in human-derived cells. Furthermore, earlier studies showed that they cause chromosomal aberrations and MN in bone marrow of mice. Fixation of damage of the DNA in the form of chromosomal damage is generally considered to be essential in the multistep process of malignancy, therefore the currently available data are indicative for potential carcinogenic properties of the cannabinoids. Electronic supplementary material The online version of this article (10.1007/s00204-018-2322-9) contains supplementary material, which is available to authorized users.

Aim of this study was to clarify if extension of the work phase has an impact on DNA- stability, telomere lengths and inflammatory markers. We conducted an intervention trial with office workers (n = 24) and carpenters (n = 10), who changed their working schedule from 8 to 12 h per day over a period of 3 months. The work of both groups involved only moderate physical activity. We found no evidence for induction of double strand breaks (measured in γH2AX assays) and relative telomere lengths (relTL_36B4 and ALB) in lymphocytes in the two study groups. Furthermore, no overall changes of the levels of C-reactive protein (CRP), interleukin-6 (IL-6) and thiobarbituric acid reactive substances (TBARS) in plasma were detected. However, we found in agreement with earlier investigations a moderate (not significant) increase of the CRP levels with age. Furthermore, significant higher CRP concentrations (P = 0.03) were detected in young individuals (21–30 years) as a consequence of the extended working period. Taken together our findings indicate that prolongation of the working hours has no pronounced impact on DNA stability, telomere shortening and inflammatory markers; but the increase of the CRP concentrations in young workers may be indicative for adverse health effects in this subgroup.

Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malignant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is supported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activation of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems., PLoS ONE, 13 (4), ISSN:1932-6203

Synthetic cannabinoids (SCs) cause similar effects as cannabis and are sold in herbal mixtures. Recent investigations indicate that some of these drugs possess genotoxic properties. Therefore, we tested representatives of two groups, namely, aminoalkylindoles (AM-2201 and UR-144) and 1-alkylindazoles (5F-AKB-48 and AM-2201-IC) in single cell gel electrophoresis and micronucleus (MN) assays with human lymphocytes and in Salmonella/microsome assays. All drugs except AM-2201 caused DNA-migration, the LOELs were between 50 and 75 µM. Furthermore, all SCs caused inhibition of cell division and significant induction of MN which reflect structural and numerical chromosomal aberrations. The LOEL values were 50 µM for UR-144 and 5-AKB-48 and 75 µM for the other drugs. Also the levels of nucleoplasmatic bridges which are formed from dicentric chromosomes were elevated under identical conditions while the frequencies of nuclear buds were not affected. These findings show that representatives of both groups cause chromosomal damage while the negative results in Salmonella assays (in strains TA98, TA100, TA1535, TA1537 and TA102) in absence and presence of metabolic activation indicate that they do not induce gene mutations. Taken together, these findings indicate that SCs may cause adverse health effects in users as a consequence of damage of the genetic material.

A potential contribution of H. pylori contamination to low-grade inflammation, oxidative stress (OS) and insulin resistance as well as correlations between these parameters in asymptomatic sedentary males was analysed. We enrolled 30 apparently healthy asymptomatic young subjects (18 H. pylori negative and 12 positive) and measured whole blood glucose, glycated haemoglobin, insulin, C-peptide, cortisol, aldosterone, testosterone, thyroid stimulating hormone, C-reactive protein, interleukins 6 and 10, TNF-alpha and comet assay. As markers of OS, we used urine levels of iso-PGF

Patients with inflammatory bowel disease (IBD) have a higher risk of developing colitis-associated-cancer (CAC); however, the underlying processes of disease progression are not completely understood. Here, the molecular processes of inflammation-driven colon carcinogenesis were investigated using IL10-deficient mice (IL10 KO). IL10 KO mice were euthanized after development of colitis and dysplasia. IHC was performed for markers of colitis-induced DNA damage (CIDD): oxidative DNA lesions (8-oxoG), double-strand breaks (DSB; γH2AX). and DSB repair. MSI, LOH (Trp53, Apc), and global methylation (CIMP) were assessed on microdissected tissue. Comet assay for DNA damage, immunofluorescence, and immunoblotting were performed on intestinal organoids from wild-type (WT) and IL10 KO mice. Sequential biopsies and surgical specimens from IBD and CAC patients were used for IHC analysis. Severity of inflammation correlated with number of dysplasia. 8-oxoG and γH2AX-positive cells were significantly increased in inflamed and dysplastic areas along with activation of DSB repair. The amount of positively stained cells strongly correlated with degree of inflammation (8-oxoG: R = 0.923; γH2AX: R = 0.858). Neither CIMP, MSI nor LOH was observed. Enhanced DSBs in IL10 KO organoids were confirmed by comet assay and increased expression of γH2AX. Human clinical specimens exhibited significantly higher γH2AX and 8-oxoG in IBD, dysplasia, and CAC compared with normal mucosa. These data indicate that inflammation-driven colon carcinogenesis in IL10 KO mice and IBD patients is associated with oxidative DNA damage and overt presence of DSB. Mol Cancer Res; 16(4); 634–42. ©2018 AACR.

Obesity is associated with low-grade inflammation, increased ROS production and DNA damage. Supplementation with antioxidants might ameliorate DNA damage and support epigenetic regulation of DNA repair. C57BL/6J male mice were fed a high-fat (HFD) or a control diet (CD) with and without vitamin E supplementation (4.5 mg/kg body weight (b.w.)) for four months. DNA damage, DNA promoter methylation and gene expression of Dnmt1 and a DNA repair gene (MLH1) were assayed in liver and colon. The HFD resulted in organ specific changes in DNA damage, the epigenetically important Dnmt1 gene, and the DNA repair gene MLH1. Vitamin E reduced DNA damage and showed organ-specific effects on MLH1 and Dnmt1 gene expression and methylation. These results suggest that interventions with antioxidants and epigenetic active food ingredients should be developed as an effective prevention for obesity—and oxidative stress—induced health risks.

Low grade inflammation due to Helicobacter pylori infection is likely causative factor for insulin resistance in infected patients(1). The study analyzes effects of H. pylori contamination on low-grade inflammation and oxidative stress in asymptomatic sedentary males. We enrolled 30 apparently healthy asymptomatic young subjects (18 H. pylori negative and 12 positive). We studied whole blood glucose, glycated hemoglobin, insulin, C-peptide, cortisol, aldosterone, testosterone, thyroid stimulating hormone, C-reactive protein, interleukins 6 and 10, TNF-alpha, comet-assay, urine levels of 1,4-dihydroxynonane mercapturic acid (DHN-MA) and F2-isoprostanes. The results showed two-fold elevation of fasting insulin level and HOMA index in H. pylori –positive subjects (p H. pylori -triggered metabolic changes include development of insulin resistance in some of the H. pylori -positive subjects, however inflammatory and OS related changes are limited to the gut-liver axis with a little systemic impact.

International audience; A potential contribution of H. pylori contamination to low-grade inflammation, oxidative stress (OS) and insulin resistance as well as correlations between these parameters in asymptomatic sedentary males was analysed. We enrolled 30 apparently healthy asymptomatic young subjects (18 H. pylori negative and 12 positive) and measured whole blood glucose, glycated haemoglobin, insulin, C-peptide, cortisol, aldosterone, testosterone, thyroid stimulating hormone, C-reactive protein, interleukins 6 and 10, TNF-alpha and comet assay. As markers of OS, we used urine levels of iso-PGF2-α and 1,4-dihydroxynonane mercapturic acid (DHN-MA). Twofold elevation of fasting insulin level and HOMA index in H. pylori-positive subjects (p H. pylori-triggered metabolic derangements in sedentary subjects include development of insulin resistance in H. pylori-positive subjects; however, there is no evidence of systemic inflammatory and OS-related changes.

This study aimed to compare the frequencies of nuclear anomalies in buccal cells between diabetic and non-diabetic individuals and to assess the impact of a 'healthy diet'-a cornerstone in the treatment of diabetes. Seventy-six diabetic and 21 non-diabetic individuals participated in this parallel, randomised, intervention trial. All participants received information about the importance of a healthy diet, while participants randomly assigned to the intervention group received additionally 300g of vegetables and 25ml of plant oil rich in polyunsaturated fatty acids (PUFA) per day for 8 weeks. Cytogenetic damage in buccal cells was assessed at baseline and after 8 weeks using the buccal micronucleus cytome assay. Micronucleus (MN) frequency at baseline was significantly higher in participants with diabetes (0.58±0.30‰) compared with non-diabetic individuals (0.28±0.29‰). Further analysis of baseline data revealed significantly higher MN levels in participants of the highest tertile of waist circumference (+40%), fasting plasma glucose (+55%), glycated haemoglobin (+41%) and cardiovascular disease risk (+39%) relative to participants of the lowest tertile. The dietary intervention had no effect on MN frequencies. Glycated haemoglobin and biomarkers reflecting cytokinetic defect or acute cell death were reduced in both the intervention and 'information only' groups. The results of this study suggest a strong impact of abdominal obesity and glucose metabolism on genomic stability. Similar effects on nuclear anomalies were observed in the 'information only' group and the intervention group receiving vegetables and PUFA-rich plant oil.

cope Aim of the study was to investigate the protective properties of coffee towards aflatoxin B1 (AFB1) induced formation of pre-neoplastic hepatic foci and the identification of the constituents and molecular mechanisms that account for these effects. Materials and methods Rats consumed three different brews and were subsequently treated with AFB1 (0.75 mg/kg b.w. intraperitoneally). Ten weeks later, the numbers and areas of hepatic foci were determined. Furthermore, the impact of the brews on AFB1-induced DNA damage was quantified in single cell gel electrophoresis assays and the activities of drug metabolising enzymes and glutathione-related parameters were monitored. Additionally, single cell gel electrophoresis assay experiments were conducted with pure caffeine. Conclusion All brews reduced the frequencies of the hepatic foci. The most pronounced protection (reduction 82%) was seen with the caffeine containing metal and paper filtered brews. DNA migration was reduced between 65 and 75% with the caffeine containing brews. In additional experiments, clear protective effects were found with caffeine at dose levels that corresponded to those contained in the coffee. This observation indicates that the alkaloid accounts partly for the protective effects of coffee. Furthermore, our findings indicate that induction of UDP-glucuronosyltransferase contributes to the chemopreventive effects of coffee since all brews increased the activity of this detoxifying enzyme.