Use of micronucleus cytome assays with buccal cells for the detection of genotoxic effects: a systematic review and meta-analysis of occupational exposures to metals.

Micronucleus (MN) assays with buccal cells are at present widely used to investigate occupational exposures to genotoxic carcinogens. This article describes their use for the monitoring of metal exposed workers. We found in total 73 relevant articles, in the majority (97%) increased MN and/or other nuclear anomalies were reported. Most studies were realized in South East Asia and South America. A variety of different occupations was studied including welders, electroplaters, painters, workers in battery recycling and production, tannery workers, dental technicians, miners, workers in foundries and smelters, and also subjects working in waste recycling, glass, aluminum and steel production. In many investigations the effects increased with the duration of the working period. The quality of individual studies was evaluated with a quality score tool. The number of cells was in most studies sufficient and DNA-specific stains were used. However, many studies have shortcomings, e.g. they focused solely on MN formation and did not evaluate anomalies, which provide additional information about the stability of the genetic material and acute cytotoxic effects. Only 35% of the investigations contain quantitative information about exposures to metals and other toxicants. In 6 of these studies, correlations were observed between the concentrations of specific metals (As, Pb, Cr, Cd) in body fluids and MN frequencies. Taken together, the available data indicate that the MN assay can be used to detect chromosomal damage in metal exposed groups; furthermore, it enables also comparisons between subgroups differing in regard to their exposure and allows an estimation of the efficiency of protective measures. The exposure of workers to metals is currently controlled with chemical analytical measurements only, MN assays with buccal cells could contribute to further improve the safety at workplaces as they reflect the biological consequences including synergistic and antagonistic interactions between toxicants.

Impact of high (1950 MHz) and extremely low (50 Hz) frequency electromagnetic fields on DNA damage caused by occupationally relevant exposures in human derived cell lines.

Epidemiological studies indicate that electromagnetic fields are associated with cancer in humans. Exposure to mobile phone specific high frequency fields (HF-EMF) may lead to increased glioma risks, while low frequency radiation (LF-EMF) is associated with childhood leukemia. We studied the impact of HF-EMF (1950 MHz, UMTS signal) on DNA stability in an astrocytoma cell line (1321N1), and the effect of LF-EMF (50 Hz) in human derived lymphoma (Jurkat) cells. To find out if these fields affect chemically induced DNA damage, co-exposure experiments were performed. The cells were exposed to HF-EMF or LF-EMF and treated simultaneously and sequentially with mutagens. The compounds cause DNA damage via different molecular mechanisms, i.e. pyrimidine dimers which are characteristic for UV light (4-nitroquinoline 1-oxide, 4NQO), bulky base adducts (benzo[a]pyrene diolepoxide, BPDE), DNA-DNA and DNA-protein cross links and oxidative damage (NiCl2, CrO3). DNA damage was measured in single cell gel electrophoresis (comet) assays. We found a moderate reduction of basal and 4NQO-induced DNA damage in the astrocytoma line, but no significant alterations of chemically induced DNA migration by the HF and LF fields under all other experimental series. The biological consequences of the moderate reduction remain unclear, but our findings indicate that acute mobile phone and power line specific EMF exposures do not enhance genotoxic effects caused by occupationally relevant chemical exposures.

Entinostat Enhances the Efficacy of Chemotherapy in Small Cell Lung Cancer Through S-phase Arrest and Decreased Base Excision Repair.

PURPOSE: Acquired chemoresistance is a frequent event in small cell lung cancer (SCLC), one of the deadliest human malignancies. Histone deacetylase inhibitors (HDACi) have been shown to synergize with different chemotherapeutic agents including cisplatin. Accordingly, we aimed to investigate the dual targeting of HDAC inhibition and chemotherapy in SCLC. EXPERIMENTAL DESIGN: The efficacy of HDACi and chemotherapy in SCLC was investigated both in vitro and in vivo. Synergistic drug interactions were calculated based on the HSA model (Combenefit software). Results from the proteomic analysis were confirmed via ICP-MS, cell-cycle analysis, and comet assays. RESULTS: Single entinostat- or chemotherapy significantly reduced cell viability in human neuroendocrine SCLC cells. The combination of entinostat with either cisplatin, carboplatin, irinotecan, epirubicin, or etoposide led to strong synergy in a subset of resistant SCLC cells. Combination treatment with entinostat and cisplatin significantly decreased tumor growth in vivo. Proteomic analysis comparing the groups of SCLC cell lines with synergistic and additive response patterns indicated alterations in cell-cycle regulation and DNA damage repair. Cell-cycle analysis revealed that cells exhibiting synergistic drug responses displayed a shift from G1 to S-phase compared with cells showing additive features upon dual treatment. Comet assays demonstrated more DNA damage and decreased base excision repair in SCLC cells more responsive to combination therapy. CONCLUSIONS: In this study, we decipher the molecular processes behind synergistic interactions between chemotherapy and HDAC inhibition. Moreover, we report novel mechanisms to overcome drug resistance in SCLC, which may be relevant to increasing therapeutic success.

Impact of mobile phone-specific electromagnetic fields on DNA damage caused by occupationally relevant exposures: results of ex vivo experiments with peripheral blood mononuclear cells from different demographic groups.

The aim of this study was to investigate if age and body mass of humans have an impact on the DNA-damaging properties of high-frequency mobile phone-specific electromagnetic fields (HF-EMF, 1950 MHz, universal mobile telecommunications system, UMTS signal) and if this form of radiation has an impact on the genotoxic effects of occupationally relevant exposures. Pooled peripheral blood mononuclear cells (PBMC) from three groups [young normal weight, young obese (YO), and older age normal weight individuals] were exposed to different doses of HF-EMF (0.25, 0.5, and 1.0 W/kg specific absorption rate-SAR) and simultaneously or sequentially to different chemicals which cause DNA damage (CrO3, NiCl2, benzo[a]pyrene diol epoxide-BPDE, and 4-nitroquinoline 1-oxide-4NQO) via different molecular mechanisms. We found no difference in regard to the background values in the three groups but a significant increase of DNA damage (81% without and 36% with serum) in cells from old participants after radiation with 1.0 W/kg SAR 16 h. In combined treatment experiments we found no impact of the UMTS signal on chemically induced DNA damage in the different groups in general. However, a moderate decrease of DNA damage was seen in simultaneous treatment experiments with BPDE and 1.0 W/kg SAR in the YO group (decline 18%). Taken together our findings indicate that HF-EMF cause DNA damage in PBMC from older subjects (69.1 years). Furthermore, they show that the radiation does not increase induction of DNA damage by occupationally relevant chemicals.

Use of the single cell gel electrophoresis assay for the detection of DNA-protective dietary factors: Results of human intervention studies.

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.

Impact of Bariatric Surgery on the Stability of the Genetic Material, Oxidation, and Repair of DNA and Telomere Lengths.

Obesity causes genetic instability, which plays a key-role in the etiology of cancer and aging. We investigated the impact of bariatric surgery (BS) on DNA repair, oxidative DNA damage, telomere lengths, alterations of antioxidant enzymes and, selected proteins which reflect inflammation. The study was realized with BS patients (n = 35). DNA damage, base oxidation, BER, and NER were measured before and 1 month and 6 months after surgery with the single-cell gel electrophoresis technique. SOD and GPx were quantified spectrophotometrically, malondealdehyde (MDA) was quantified by HPLC. Telomere lengths were determined with qPCR, and plasma proteome profiling was performed with high-resolution mass spectrophotometry. Six months after the operations, reduction of body weight by 27.5% was observed. DNA damage decreased after this period, this effect was paralleled by reduced formation of oxidized DNA bases, a decline in the MDA levels and of BER and NER, and an increase in the telomere lengths. The activities of antioxidant enzymes were not altered. Clear downregulation of certain proteins (CRP, SAA1) which reflect inflammation and cancer risks was observed. Our findings show that BS causes reduced oxidative damage of DNA bases, possibly as a consequence of reduction of inflammation and lipid peroxidation, and indicate that the surgery has beneficial long-term health effects.

Micronucleus assays with the human derived liver cell line (Huh6): A promising approach to reduce the use of laboratory animals in genetic toxicology.

The inadequate representation of enzymes which catalyze the activation/detoxification of xenobiotics in cells that are currently used in genotoxicity testing of chemicals leads to a high number of false positive results and the number of follow up studies with rodents could be reduced by use of more reliable in vitro models. We found earlier that several xenobiotic drug metabolizing enzymes are represented in the human derived liver cell line Huh6 and developed a protocol for micronucleus (MN) experiments which is in agreement with the current OECD guideline. This protocol was used to test 23 genotoxic and non-genotoxic reference chemicals; based on these results and of earlier findings (with 9 chemicals) we calculated the predictive value of the assay for the detection of genotoxic carcinogens. We found a sensitivity of 80% and a specificity of 94% for a total number of 32 chemicals; comparisons with results obtained with other in vitro assays show that the validity of MN tests with Huh6 is higher as that of other experimental models. These results are promising and indicate that the use of Huh6 cells in genetic toxicology may contribute to the reduction of the use of laboratory rodents; further experimental work to confirm this assumption is warranted.

The Single-Cell Gel Electrophoresis Genotoxin Sensitivity Assay.

The single-cell gel electrophoresis-based genotoxin sensitivity assay (GSA) is an ex vivo approach which enables to study the impact of a variety of dietary factors, occupational exposures, and diseases on the sensitivity of humans towards genotoxic chemicals which cause adverse health effects such as cancer, accelerated aging, and infertility.

p53 Loss Mediates Hypersensitivity to ETS Transcription Factor Inhibition Based on PARylation-Mediated Cell Death Induction.

The small-molecule E26 transformation-specific (ETS) factor inhibitor YK-4-279 was developed for therapy of ETS/EWS fusion-driven Ewing's sarcoma. Here we aimed to identify molecular factors underlying YK-4-279 responsiveness in ETS fusion-negative cancers. Cell viability screenings that deletion of P53 induced hypersensitization against YK-4-279 especially in the BRAFV600E-mutated colon cancer model RKO. This effect was comparably minor in the BRAF wild-type HCT116 colon cancer model. Out of all ETS transcription factor family members, especially ETS1 overexpression at mRNA and protein level was induced by deletion of P53 specifically under BRAF-mutated conditions. Exposure to YK-4-279 reverted ETS1 upregulation induced by P53 knock-out in RKO cells. Despite upregulation of p53 by YK-4-279 itself in RKOp53 wild-type cells, YK-4-279-mediated hyperphosphorylation of histone histone H2A.x was distinctly more pronounced in the P53 knock-out background. YK-4-279-induced cell death in RKOp53-knock-out cells involved hyperPARylation of PARP1, translocation of the apoptosis-inducible factor AIF into nuclei, and induction of mitochondrial membrane depolarization, all hallmarks of parthanatos. Accordingly, pharmacological PARP as well as BRAFV600E inhibition showed antagonistic activity with YK-4-279 especially in the P53 knock-out background. Taken together, we identified ETS factor inhibition as a promising strategy for the treatment of notoriously therapy-resistant p53-null solid tumours with activating MAPK mutations.

Genotoxic activities of wastewater after ozonation and activated carbon filtration: Different effects in liver-derived cells and bacterial indicators.

Aim of this study was to investigate the impact of advanced wastewater treatment techniques (combining ozonation with activated carbon filtration) on acute and genotoxic activities of tertiary treated wastewater. Concentrated samples were tested in Salmonella/microsome assays. Furthermore, induction of DNA damage was measured in liver-derived cells (human hepatoma and primary rat hepatocytes) in single cell gel electrophoresis experiments, which are based on the measurement of DNA migration in an electric field. These cell types possess phase I and phase II enzymes, which catalyze the activation/detoxification of mutagens. Acute toxicity was determined with the trypan blue exclusion technique. We found no evidence for mutagenic effects of non-ozonated samples in several bacterial tester strains (TA98, TA100, YG7108, YG7104, YG7112 and YG7113) but clear induction of His+ mutants after O3 treatment in two strains with defective genes encoding for DNA repair, which are highly sensitive towards alkylating agents (YG7108 and YG7104). These effects were reduced after activated carbon filtration. Furthermore, we detected a slight increase of mutagenic activity in strain YG1024 with increased acetyltransferase activity, which is sensitive towards aromatic amines and nitro compounds in untreated water, which was not reduced by O3 treatment. A completely different pattern of mutagenic activity was seen in liver-derived cells; non ozonated samples caused in both cell types pronounced DNA damage, which was reduced (by ca. 25%) after ozonation. Activated carbon treatment did not cause a substantial further reduction of DNA damage. Additional experiments with liver homogenate indicate that the compounds which cause the effects in the human cells are promutagens which require enzymatic activation. None of the waters caused acute toxicity in the liver-derived cells and in the bacterial indicators. Assuming that hepatic mammalian cells reflect the genotoxic properties of the waters in vertebrates (including humans) more adequately as genetically modified bacterial indicators, we conclude that ozonation has beneficial effects in regard to the reduction of genotoxic properties of treated wastewaters.

Methamphetamine ("crystal meth") causes induction of DNA damage and chromosomal aberrations in human derived cells.

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.

Low doses of widely consumed cannabinoids (cannabidiol and cannabidivarin) cause DNA damage and chromosomal aberrations in human-derived cells.

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.

Cytome micronucleus assays with a metabolically competent human derived liver cell line (Huh6): A promising approach for routine testing of chemicals?

One of the main problems of in vitro genotoxicity tests is the inadequate representation of drug metabolizing enzymes in most indicator cell lines which are currently used. We identified recently a human derived liver cell line (Huh6) which detected induction of DNA damage by representatives of different groups of promutagens without enzyme mix and showed that these cells are more suitable in terms of reproducibility and sensitivity as other currently used liver derived lines. We developed a protocol for micronucleus (MN) cytome assays with these cells and validated the procedure in experiments with representatives of different groups of directly and indirectly acting genotoxic carcinogens (MMS, cisplatin, PhIP, IQ, NDMA, B(a)P, AFB1, etoposide, and H2 O2 ). The optimal cytochalasin B concentration in combination with 48 hr treatment was found to be 1.5 µg/mL and leads to a cytokinesis block proliferation index in the range between 1.7 and 2.0. The morphological characteristics of different nuclear anomalies which reflect DNA damage (MN, nuclear bridges, and buds) and their baseline frequencies in untreated cells were characterized, and the rates which are required to cause significant effects were calculated. All compounds caused dose dependent induction of MN when the cells were treated for 24 hr, longer and shorter exposure times were less effective. Experiments with different serum levels (fetal bovine serum [FBS]) showed that 10% FBS in the medium (instead of 4%) causes a substantial increase of the sensitivity of the cells. Our results indicate that the new protocol is a promising approach for routine testing of chemicals. Environ. Mol. Mutagen. 60: 134-144, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Impact of obesity and overweight on DNA stability: Few facts and many hypotheses.

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.

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells.

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.

Overt Increase of Oxidative Stress and DNA Damage in Murine and Human Colitis and Colitis-Associated Neoplasia.

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.

Gallic Acid Improves Health-Associated Biochemical Parameters and Prevents Oxidative Damage of DNA in Type 2 Diabetes Patients: Results of a Placebo-Controlled Pilot Study.

SCOPE: Oxidative imbalance plays a key role in cancer induction and cardiovascular diseases (CVD) in patients with type 2 diabetes mellitus (T2DM). The aim of this study is to find out if gallic acid (GA) prevents oxidative stress in diabetic patients. Therefore, we investigate its impact on oxidation of DNA bases and on other health-related macromolecules. METHODS AND RESULTS: We perform an intervention study (n = 19) with GA and monitored alterations of the DNA stability in single cell gel electrophoresis (SCGE) assays in lymphocytes. Furthermore, a panel of health-related biomarkers is measured before and after consumption of GA (15 mg p-1  d-1 ) for 7 d. Significant reduction of oxidized purines (by 31%, p < 0.001, effect size 0.404) and pyrimidines (by 2%, p < 0.022, effect size 0.089) is observed in SCGE assays. Furthermore, the plasma concentrations of oxidized-LDL and C-reactive protein are reduced after the intervention by 24% (p = 0.014, effect size 0.384) and 39% (p < 0.001, effect size 0.686), respectively. No alterations of other biomarkers are found. CONCLUSIONS: A small amount of GA (in the range of daily consumption in Central Europe) prevents oxidative DNA damage and reduces markers which reflect inflammation and increased risks of cancer and CVD.

Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins: a new hope for laboratory animals?

Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1 (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.

Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer.

Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factor-α (TNF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.

Vitamin E Modifies High-Fat Diet-Induced Increase of DNA Strand Breaks, and Changes in Expression and DNA Methylation of Dnmt1 and MLH1 in C57BL/6J Male Mice.

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.