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Doxorubicin (Dox), an effective anticancer agent, is known for its genotoxic effects on normal cells. Phenolic compounds, renowned for their antitumor, antioxidant, and antigenotoxic properties, have gained prominence in recent years. This study investigates the individual and combined protective effects of rosmarinic acid (RA) and epigallocatechin gallate (EGCG) against Dox-induced genotoxicity using various in vitro test systems. The synergistic/antagonistic interaction of these combinations on Dox's chemotherapeutic effect is explored in breast cancer cell lines. Both RA and EGCG significantly mitigate Dox-induced genotoxicity in comet, micronucleus, and Ames assays. While Dox exhibits higher selectivity against MCF-7 cells, EGCG and RA show greater selectivity against MDA-MB-231 cells. The coefficient of drug interaction reveals a synergistic effect when RA or EGCG is combined with Dox in breast cancer cells. In conclusion, both EGCG and RA effectively reduce Dox-induced genetic damage and enhance Dox's cell viability-reducing effect in breast cancer cells.

Natural non-psychoactive cannabinoids such as cannabigerol (CBG), cannabidiol (CBD), cannabichromene (CBC), cannabidivarin (CBDV), and cannabinol (CBN) are increasingly consumed as constituents of dietary products because of the health benefits claims. Cannabinoids may reduce certain types of pain, nausea, and anxiety. Anti-inflammatory and even anti-carcinogenic properties have been discussed. However, there are insufficient data available regarding their potential (geno-)toxic effects. Therefore, we tested CBG, CBD, CBC, CBDV, and CBN for their genotoxic potential and effects on mitosis and cell cycle in human lymphoblastoid TK6 cells. The selected cannabinoids (except CBDV) induced increased micronuclei formation, which was reduced with the addition of a metabolic activation system (S9 mix). CBDV induced micronuclei only after metabolic activation. Mitotic disturbances were observed with all tested cannabinoids, while G1 phase accumulation of cells was observed for CBG, CBD and CBDV. The genotoxic effects occurred at about 1000-fold higher concentrations than are reported as blood levels from human consumption. However, the results clearly indicate a need for further research into the genotoxic effects of cannabinoids. The mechanism of the mitotic disturbance, the shape of the dose-response curves and the possible effects of mixtures of cannabinoids are aspects which need clarification., (© 2024. The Author(s).)

Placental growth is most rapid during the first trimester (FT) of pregnancy, making it vulnerable to metabolic and endocrine influences. Obesity, with its inflammatory and oxidative stress, can cause cellular damage. We hypothesized that maternal obesity increases DNA damage in the FT placenta, affecting DNA damage response and trophoblast turnover. Examining placental tissue from lean and obese non-smoking women (4-12 gestational weeks), we observed higher overall DNA damage in obesity (COMET assay). Specifically, DNA double-strand breaks were found in villous cytotrophoblasts (vCTB; semi-quantitative γH2AX immunostaining), while oxidative DNA modifications (8-hydroxydeoxyguanosine; FPG-COMET assay) were absent. Increased DNA damage in obese FT placentas did not correlate with enhanced DNA damage sensing and repair. Indeed, obesity led to reduced expression of multiple DNA repair genes (mRNA array), which were further shown to be influenced by inflammation through in vitro experiments using tumor necrosis factor-α treatment on FT chorionic villous explants. Tissue changes included elevated vCTB apoptosis (TUNEL assay; caspase-cleaved cytokeratin 18), but unchanged senescence (p16) and reduced proliferation (Ki67) of vCTB, the main driver of FT placental growth. Overall, obesity is linked to heightened non-oxidative DNA damage in FT placentas, negatively affecting trophoblast growth and potentially leading to temporary reduction in early fetal growth., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Biomonitoring of human populations exposed to chemical substances that can act as potential mutagens or carcinogens, may enable the detection of damage and early disease prevention. In recent years, the comet assay has become an important tool for assessing DNA damage, both in environmental and occupational exposure contexts. To evidence the role of the comet assay in human biomonitoring, we have analysed original research studies of environmental or occupational exposure that used the comet assay in their assessments, following the PRISMA-ScR method (preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews). Groups of chemicals were designated according to a broad classification, and the results obtained from over 300 original studies (n = 123 on air pollutants, n = 14 on anaesthetics, n = 18 on antineoplastic drugs, n = 57 on heavy metals, n = 59 on pesticides, and n = 49 on solvents) showed overall higher values of DNA strand breaks in the exposed subjects in comparison with the unexposed. In summary, our systematic scoping review strengthens the relevance of the use of the comet assay in assessing DNA damage in human biomonitoring studies.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Smoking during pregnancy increases the risk of adverse pregnancy outcomes, such as stillbirth and fetal growth restriction. This suggests impaired placental function and restricted nutrient and oxygen supply. Studies investigating placental tissue at the end of pregnancy have revealed increased DNA damage as a potential underlying cause, which is driven by various toxic smoke ingredients and oxidative stress induced by reactive oxygen species (ROS). However, in the first trimester, the placenta develops and differentiates, and many pregnancy pathologies associated with reduced placental function originate here. Therefore, we determined DNA damage in a cohort of first-trimester placental samples of verified smokers and nonsmokers. In fact, we observed an 80% increase in DNA breaks (P < .001) and shortened telomeres by 5.8% (P = .04) in placentas exposed to maternal smoking. Surprisingly, there was a decrease in ROS-mediated DNA damage, ie, 8-oxo-guanidine modifications, in placentas of the smoking group (-41%; P = .021), which paralleled the reduced expression of base excision DNA repair machinery, which restores oxidative DNA damage. Moreover, we observed that the increase in placental oxidant defense machinery expression, which usually occurs at the end of the first trimester in a healthy pregnancy as a result of the full onset of uteroplacental blood flow, was absent in the smoking group. Therefore, in early pregnancy, maternal smoking causes placental DNA damage, contributing to placental malfunction and increased risk of stillbirth and fetal growth restriction in pregnant women. Additionally, reduced ROS-mediated DNA damage along with no increase in antioxidant enzymes suggests a delay in the establishment of physiological uteroplacental blood flow at the end of the first trimester, which may further add to a disturbed placental development and function as a result of smoking in pregnancy., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

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., (© 2023. Springer Nature Limited.)

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells., (© 2022. The Author(s).)

Objective: Leukemia represents a serious public health concern as the incidence is increasing worldwide. In this study we aimed to describe the epidemiological profile of acute lymphoblastic (ALL) and myeloid (AML) leukemia, identify disease clusters and find association with possible risk factors., Methods: Data on leukemia cases were provided by the National Institute of Health of the Republic of Armenia for the period of 2012-2018. Age-standardized incidence rate was calculated using Segi World Population. SaTScan purely spatial analysis was applied to find leukemia clusters. To find association between leukemia and agricultural and mining activities and demographic data Poisson regression model was used., Results: During the studied period 259 new cases of ALL and 478 AML were recorded. The age-standardized incidence rate was 1.5 and 1.9 per 100,000 inhabitants with male to female ratio of 0.97 and 1.1 for ALL and AML, respectively. No significant changes in ALL or AML incidence trends were found. For ALL significant cluster encompassing Shirak, Lori, Tavush and Armavir provinces of Armenia was identified, while Kotayk and Ararat was provinces with the highest incidence of AML. We found significant positive association of ALL with crop density, while no elevated risk estimates were found between AML and exposure variables., Conclusion: Altogether, our results suggested that acute leukemias incidence in Armenia follows the pattern described for developing countries.

The increase in the prevalence of obesity has led to an elevated risk for several associated diseases including cancer. Several studies have investigated the DNA damage in human blood samples and showed a clear trend towards increased DNA damage in obesity. Reduced genomic stability is thus one of the consequences of obesity, which may contribute to the related cancer risk. Whether this is influenced by compromised DNA repair has not been elucidated sufficiently yet. On the other hand, obesity has also been linked to reduced therapy survival and increased adverse effects during chemotherapy, although the available data are controversial. Despite some indications that obesity might alter hepatic metabolism, current literature in humans is insufficient, and results from animal studies are inconclusive. Here we have summarised published data on hepatic drug metabolism to understand the impact of obesity on cancer therapy better. Furthermore, we highlight knowledge gaps in the interrelationship between obesity and drug metabolism from a toxicological perspective.

A phytochemical investigation of the liana of Artabotrys thomsonii led to the isolation of a new oxoberberine alkaloid, 2,10-dihydroxy-3,9-dimethoxy-8-oxo-protoberberine ( 7 ), along with six known compounds. Their chemical structures were elucidated by 1 D and 2 D NMR spectroscopic methods and HRESI-MS n data analysis. Compounds 4 and 7 were selected for further in vitro investigations. In accordance with expectations from their chemical structures, compounds 7 and 4 showed a clear antioxidant activity in a cell-free assay, with compound 7 being 7-fold more active than 4 . Cytotoxicity, cytostatic and genotoxic effects only occurred at high micromolar concentrations of 50 µM or more. Compound 7 was slightly less effective than compound 4 . A low micromolar concentration of 10 µM did not cause any damaging cellular effects but showed potential for a protection against the micronucleus-inducing effect of reactive oxygen species hydrogen peroxide, although not to a significant extent.

The comet assay is widely used for quantification of genomic damage in humans. Peripheral blood derived mononuclear cells (PBMCs) are the most often used cell type for this purpose. Since the comet assay can be performed in an enhanced throughput format, it can be applied to large sample collections such as biobanks. The European Prospective Investigation into Cancer and Nutrition (EPIC) study is one of the largest existing prospective cohort studies, and the German Cancer Research Institute (DKFZ) in Heidelberg is a participating center with 25.000 frozen blood samples stored from around 25 years ago, enabling retrospective assessment of disease risk factors. However, experience with decades long frozen samples in the comet assay is so far missing. In Heidelberg, 800 study participants were re-invited twice between 2010 and 2012 to donate further blood samples. Here, we analyzed 299 Heidelberg-EPIC samples, compiled from frozen PBMC and buffy coat preparations selected from the different sampling time points. In addition, 47 frozen PBMC samples from morbidly obese individuals were included. For buffy coat samples, we observed a poor correlation between DNA damage in the same donors assessed at two sampling time points. Additionally, no correlation between DNA damage in buffy coat samples and PBMCs was found. For PBMCs, a good correlation was observed between samples of the same donors at the two time points. DNA damage was not affected by age and smoking status, but high BMI (>30; obesity) was associated with increased DNA damage in PBMCs. There was no indication for a threshold of a certain BMI for increased DNA damage. In conclusion, while 25 year-long stored buffy coat preparations may require adaptation of certain experimental parameters such as cell density and electrophoresis conditions, frozen PBMC biobank samples can be analyzed in the comet assay even after a decade of storage., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H 2 O 2 ) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H 2 O 2 or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided., (© 2021. The Author(s).)

Thebaine is an alkaloid and can be found in poppy seeds in relatively high concentrations. Acute toxicity of thebaine is fairly high, but not much is known about chronic toxicity. To investigate the genotoxicity of thebaine, cytokinesis-block micronucleus test and comet assay were conducted in TK6 cells. In addition, effects of putative thebaine metabolites were analysed using metabolically active HepG2 cells and TK6 cells with S9 mix. FDA test and trypan blue test were used together with the frequency of mitotic and apoptotic cells to assess potential cytotoxicity of thebaine treatment. Micronucleus induction was observed after high doses (150 and 500 μM) of thebaine without metabolic activation in the presence of slight to moderate cytotoxicity. No effects were observed in the comet assay or after metabolic activation up to the highest dose of 500 μM. A potential protective effect on micronucleus induction after thebaine treatment was investigated via co-treatment with MMC and BaP in TK6 cells. Only after co-treatment with MMC, a reduction of micronucleus frequency was found. Overall, this study shows a potential of thebaine to induce genotoxic effects at high concentrations. The observation of cytotoxicity at these concentrations supports the hypothesis that genotoxicity may be caused by cytotoxic effects. Further studies will need to elucidate whether these effects are directly genotoxic or indeed result from cytotoxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples., (© The Author(s) 2021. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Delphinidin (DEL) is a plant-derived antioxidant with clinical potential to treat inflammatory pain but suffers from poor solubility and low bioavailability. The aim of the study was to develop a well-tolerated cyclodextrin (CD)-DEL complex with enhanced bioavailability and to investigate the mechanisms behind its antinociceptive effects in a preclinical model of inflammatory pain. Results: CD-DEL was highly soluble and stable in aqueous solution, and was nontoxic. Systemic administration of CD-DEL reversed mechanical and heat hyperalgesia, while its local application into the complete Freund's adjuvant (CFA)-induced inflamed paw dose-dependently reduced mechanical hyperalgesia, paw volume, formation of the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE), and tissue migration of CD68 + macrophages. CD-DEL also directly prevented 4-HNE-induced mechanical hyperalgesia, cold allodynia, and an increase in the intracellular calcium concentration into transient receptor potential ankyrin 1 expressing cells. Both 4-HNE- and CFA-induced reactive oxygen species (ROS) levels were sensitive to CD-DEL, while its capacity to scavenge superoxide anion radicals (inhibitory concentration 50 [IC 50 ]: 70 ± 5 μ M ) was higher than that observed for hydroxyl radicals (IC 50 : 600 ± 50 μ M ). Finally, CD-DEL upregulated heme oxygenase 1 that was prevented by HMOX -1 siRNA in vitro . Innovation: In vivo application of DEL to treat inflammatory pain is facilitated by complexation with CD. Apart from its antioxidant effects, the CD-DEL has a unique second antioxidative mechanism involving capturing of 4-HNE into the CD cavity followed by displacement and release of the ROS scavenger DEL. Conclusion: CD-DEL has antinociceptive, antioxidative, and anti-inflammatory effects making it a promising formulation for the local treatment of inflammatory pain.

The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.

Introduction: Pyrrolizidine alkaloids (PAs) are found in many plant species as secondary metabolites which affect humans via contaminated food sources, herbal medicines and dietary supplements. Hundreds of compounds belonging to PAs have been identified. PAs undergo hepatic metabolism, after which they can induce hepatotoxicity and carcinogenicity. Many aspects of their mechanism of carcinogenicity are still unclear and it is important for human risk assessment to investigate this class of compounds further., Material and Methods: Human hepatoma cells HepG2 were used to investigate the genotoxicity of different chemical structural classes of PAs, namely europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine and lasiocarpine, in the cytokinesis-block micronucleus (CBMN) assay. The different ester type PAs europine, seneciphylline, and lasiocarpine were also tested in human hepatoma Huh6 cells. Six different PAs were investigated in a crosslink comet assay in HepG2 cells., Results: The maximal increase of micronucleus formation was for all PAs in the range of 1.64-2.0 fold. The lowest concentrations at which significant induction of micronuclei were found were 3.2 μM for lasiocarpine and riddelliine, 32 μM for retrorsine and echimidine, and 100 μM for seneciphylline, europine and lycopsamine. Significant induction of micronuclei by lasiocarpine, seneciphylline, and europine were achieved in Huh6 cells at similar concentrations. Reduced tail formation after hydrogen peroxide treatment was found in the crosslink comet assay for all diester type PAs, while an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration., Conclusion: The widely available human hepatoma cell lines HepG2 and Huh6 were suitable for the assessment of PA-induced genotoxicity. Selected PAs confirmed previously published potency rankings in the micronucleus assay. In HepG2 cells, the crosslinking activity was related to the ester type, which is a first report of PA mediated effects in the comet assay., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Regional changes to the intestinal microenvironment brought about by Roux-en-Y gastric bypass (RYGB) surgery may contribute to some of its potent systemic metabolic benefits through favorably regulating various local cellular processes. Here, we show that the intestinal contents of RYGB-operated compared with sham-operated rats region-dependently confer superior glycemic control to recipient germ-free mice in association with suppression of endotoxemia. Correspondingly, they had direct barrier-stabilizing effects on an intestinal epithelial cell line which, bile-exposed intestinal contents, were partly farnesoid X receptor (FXR)-dependent. Further, circulating fibroblast growth factor 19 levels, a readout of intestinal FXR activation, negatively correlated with endotoxemia severity in longitudinal cohort of RYGB patients. These findings suggest that various host- and/or microbiota-derived luminal factors region-specifically and synergistically stabilize the intestinal epithelial barrier following RYGB through FXR signaling, which could potentially be leveraged to better treat endotoxemia-induced insulin resistance in obesity in a non-invasive and more targeted manner., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Background: Chronic kidney disease (CKD) is defined as a gradual loss of renal function progressing from very mild damage, with no obvious symptoms in stage one, to complete kidney failure in stage five, which ultimately requires kidney replacement therapy by organ transplantation or dialysis. Cancer incidence and other health problems, mainly diabetes and hypertension, are elevated in CKD, ultimately leading to elevated mortality., Methods: A literature search on the induction of micronuclei (MN) as endpoint for genomic damage in white blood cells and buccal mucosa cells of CKD patients was conducted. Possible associations with disease stage, treatment modalities, and vitamin or antioxidant supplementations were analyzed., Results: In total, 26 studies were enclosed in the data analysis. Patient groups in the predialysis or hemodialysis state of the disease exhibit higher levels of genomic damage, measured as micronucleus frequency in peripheral blood lymphocytes and buccal mucosa cells, than healthy control groups. Genomic damage seems to increase with the disease stage during the predialysis phase. The association with dialysis regimens or with years on dialysis is less clear, but there are indications that efficient removal of uremic toxins is beneficial. Patients with CKD receive a variety of medications, some of which could modulate genomic damage levels and thus contribute to the observed heterogeneity. In addition, supplementation with vitamins or antioxidants may in some cases lower the genomic damage. Meta-Analysis confirmed the high and significant levels of genomic damage present in CKD patients compared to matched healthy controls., Conclusion: Genomic damage, as measured by the MN frequency, is elevated in CKD patients. Different strategies, including supplementation with antioxidants and optimizing dialysis processes, can reduce the levels of genomic damage and the different associated pathologies. Whether MN frequency can in the future also be used to assist in certain therapeutic decisions in CKD will have to be investigated further in larger studies., Competing Interests: Declaration of Competing Interest There are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control., (© The Author(s) 2020. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.

Endogenous hormones systemically regulate the growth and metabolism and some prior studies have shown that their imbalance can have a potential to induce genomic damage in in vitro and animal models. Some conditions that are associated with elevated levels of endogenous hormones are hyperinsulinemia and intense exercise-induced stress causing increased adrenaline. In this study we test whether these two hormones, could cause an additive increase in genomic damage and whether they have an overlapping mechanism of action. For this, we use the human promyelocytic HL60 cells, as they express the receptors for both hormones. At doses taken from the saturation level of the individual dose response curves, no additivity in genomic damage was detected through micronucleus induction. This hints towards a common step in the pathway, which is under these conditions fully activated by each of the individual hormone. To investigate this further, individual and common parts in insulin and adrenaline signalling such as their respective hormone receptors, the downstream protein AKT and the involvement of mitochondria and NADPH oxidase (NOX) enzymes were studied. The results indicate no additive effect of high hormone concentrations in genomic damage in the in vitro model, which may be due to exhaustion of the NOX 2-mediated reactive oxygen production. It remains to be determined whether a similar situation may occur in in vivo situations., Competing Interests: Declaration of Competing Interest The author declares no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Arsenolipids, especially arsenic-containing hydrocarbons (AsHC), are an emerging class of seafood originating contaminants. Here we toxicologically characterize a recently identified oxo-AsHC 332 metabolite, thioxo-AsHC 348 in cultured human liver (HepG2) cells. Compared to results of previous studies of the parent compound oxo-AsHC 332, thioxo-AsHC 348 substantially affected cell viability in the same concentration range but exerted about 10-fold lower cellular bioavailability. Similar to oxo-AsHC 332, thioxo-AsHC 348 did not substantially induce oxidative stress nor DNA damage. Moreover, in contrast to oxo-AsHC 332 mitochondria seem not to be a primary subcellular toxicity target for thioxo-AsHC 348. This study indicates that thioxo-AsHC 348 is at least as toxic as its parent compound oxo-AsHC 332 but very likely acts via a different mode of toxic action, which still needs to be identified., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Obesity is associated with elevated cancer risk, which may be represented by elevated genomic damage. Oxidative stress plays a key role in obesity related detrimental health consequences including DNA oxidation damage. The elevated cancer risk in obesity may be a consequence. Weight loss has been shown to reduce genomic damage, but the role of oxidative stress in that has not been clarified. The aim of this study is therefore to investigate the influence of bariatric surgery induced weight loss on DNA oxidation damage in morbidly obese subjects. For this aim, we used cryopreserved peripheral blood mononuclear cells in the FPG comet assay. Advanced protein oxidation products and 3-nitrotyrosine were measured as oxidative and nitrative protein stress markers. Furthermore, expression of oxidative stress related proteins HSP70 and Nrf2 as well as mitochondrial enzyme citrate synthase and NADPH oxidase subunit p22 phox were analysed. Our findings revealed significantly reduced DNA strand breaks, but DNA base oxidation was not reduced. We observed significant reduction in plasma AOPPs and 3-nitrotyrosine, which indicated an improvement in oxidative/nitrative stress. However, expression of HSP70 and Nrf2 were not altered after weight loss. In addition, expression of citrate synthase and p22 phox were also unaltered. Overall, bariatric surgery induced significant reduction in excess body weight and improved the patients' health status, including reduced DNA strand breaks and slightly improved antioxidant status in some of the investigated endpoints, while cellular ROS formation and DNA oxidation damage stayed unaltered. This complex situation may be due to combined beneficial effects of weight loss and burdening of the body with fat breakdown products. In the future, collecting samples two years after surgery, when patients have been in a weight plateau for some time, might be a promising approach., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This review describes the use of the comet assay for assessment of DNA damage in human colon cells. We screened 98 papers, which employed human colon -derived cells to analyse DNA damage induced by different insults with the comet assay. In most cases tumour cell lines were used, and only a few studies were performed with primary colon cells. The comet assay was mostly applied to test chemotherapeutics and natural products. We could not find a clear difference between the susceptibility of cell lines to genotoxic insults and they were all suitable for comet assay experiments. Further comparisons between cell lines, and with primary cells and stem cells would be desirable to understand the relevance of the established cell lines as model for the human target tissue better., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation. A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status. Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Diabetes represents one of the major health concerns, especially in developed countries. Some hormones such as the stress hormone adrenaline can induce reactive oxygen species (ROS) and may worsen the diabetes. Therefore, the main aim of the investigation was to find out whether peripheral blood mononuclear cells (PBMCs) from normal persons have less DNA damage induced by adrenaline (0.1, 1 and 10 μM) in comparison to PBMCs from obese, prediabetic and diabetic patients. Also, the biochemical parameters of oxidative stress (TBARS, catalase) and lactate dehydrogenase were monitored. It was observed that higher concentrations of adrenaline (1 and 10 μM) induced DNA damage in the obese, prediabetic and diabetic groups. In healthy individuals only the highest concentration of adrenaline caused significant increase in the DNA damage. In summary, total comet score (TCS) comparison has shown significant differences between groups, and DNA damaging effects of adrenaline were most evident in diabetic patients. The results of the biochemical analysis also demonstrate that adrenaline exerts most obvious effects in diabetic individuals which is manifested as significant change of parameters of oxidative stress. In summary, the obtained results demonstrated that diabetics are more sensitive to genotoxic effects of adrenaline and this effect probably resulted from decreased antioxidative defence mechanisms in various stages of progression through diabetes. Therefore, these results could contribute to a better understanding of a role of endocrine factors to damage of cellular biomolecules which could be useful in finding novel therapeutic approaches and lifestyle changes with an aim to lower the possibility of diabetes complications., (Copyright © 2019. Published by Elsevier B.V.)

The potential protective effect of tricetinidin as novel antioxidant is investigated and compared with selected known antioxidant substances in vitro. Dihydroethidium staining was performed to detect intracellular ROS formation and the protective effect of the antioxidant substances in combination with the superoxide-inducer antimycin a (AMA). Glutathione level, mitochondrial membrane potential and HO-1 expression were analysed for further characterization of the cellular response. The cytokinesis block micronucleus test was applied to investigate the anti-genotoxic effect of the substances against insulin induced genomic damage. AMA treatment caused a significant increase in intracellular ROS formation and insulin treatment induced a significant micronucleus induction in NRK cells. Combination of the antioxidant substances with AMA or insulin protected from the oxidative stress and the micronucleus-induction. All analysed antioxidants showed comparable effects on GSH production and mitochondrial membrane potential. Only delphinidin and tricetinidin caused an increase in HO-1 expression. Tricetinidin and delphinidin might be good candidates for development as an antioxidant supplement. Further research is necessary to show possible therapeutic and preventive effects of tricetinidin and delphinidin in vivo., (Copyright © 2018. Published by Elsevier Ltd.)

The number of bariatric surgeries being performed worldwide has markedly risen. While the improvement in obesity-associated comorbidities after bariatric surgery is well-established, very little is known about its impact on cancer risk. The peripheral lymphocyte micronucleus test is a widely used method for the monitoring of chromosomal damage levels in vivo, and micronucleus frequency positively correlates with cancer risk. Therefore, the aim of this study was to compare the micronucleus frequency before and after bariatric surgery in obese subjects. Peripheral blood mononuclear cells were collected from 45 obese subjects before and at two time-points after bariatric surgery (6 and 12 months) to assess spontaneous micronucleus frequency. Consistent with the increased cancer risk previously shown, bariatric surgery-induced weight loss led to a significant reduction in lymphocyte micronucleus frequency after 12 months. Interestingly, comorbidities such as type 2 diabetes mellitus and metabolic syndrome further seemed to have an impact on the lymphocyte micronucleus frequency. Our findings may indicate a successful reduction of cancer risk in patients following weight loss caused by bariatric surgery.

Obesity is associated with several detrimental health consequences, among them an increased risk for development of cancer, and an overall elevated mortality. Multiple factors like hyperinsulinemia, chronic microinflammation and oxidative stress may be involved. The comet assay has been proven to be very sensitive for detection of DNA damage and has been used to explore the relationship between overweight/obesity and DNA damage, but results are controversial. Very few investigations have been performed to correlate weight loss of obese individuals and possible reduction of DNA damage and these studies have not provided clear results. As currently, only surgical interventions (metabolic/bariatric surgery) enable substantial and sustained weight loss in the vast majority of morbidly obese patients, we analyzed whole blood samples of 56 subsequent patients prior, 6 and 12 months after bariatric surgery. No reduction of DNA damage was observed in comet assay analysis after 6 months despite efficient weight loss, but a significant reduction was observed 12 months after surgery. Concurrently, the ferric-reducing antioxidant power assay showed a significant reduction after 6 and 12 months. The level of oxidised glutathione and lipid peroxidation products were increased at 6 months but normalised at 12 months after surgery. As conclusion, a significant weight reduction in obese patients may help to diminish existing DNA damage besides improving many other health aspects in these patients.

Type 2 diabetes (T2DM) and obesity are frequently associated with non-alcoholic fatty liver disease (NAFLD) and with an elevated cancer incidence. The molecular mechanisms of carcinogenesis in this context are only partially understood. High blood insulin levels are typical in early T2DM and excessive insulin can cause elevated reactive oxygen species (ROS) production and genomic instability. ROS are important for various cellular functions in signaling and host defense. However, elevated ROS formation is thought to be involved in cancer induction. In the molecular events from insulin receptor binding to genomic damage, some signaling steps have been identified, pointing at the PI3K/AKT pathway. For further elucidation Phosphatase and Tensin homolog (Pten), a tumour suppressor phosphatase that plays a role in insulin signaling by negative regulation of PI3K/AKT and its downstream targets, was investigated here. Dihydroethidium (DHE) staining was used to detect ROS formation in immortalized human hepatocytes. Comet assay and micronucleus test were performed to investigate genomic damage in vitro. In liver samples, DHE staining and western blot detection of HSP70 and HO-1 were performed to evaluate oxidative stress response. DNA double strand breaks (DSBs) were detected by immunohistostaining. Inhibition of PTEN with the pharmacologic inhibitor VO-OHpic resulted in increased ROS production and genomic damage in a liver cell line. Knockdown of Pten in a mouse model yielded increased oxidative stress levels, detected by ROS levels and expression of the two stress-proteins HSP70 and HO-1 and elevated genomic damage in the liver, which was significant in mice fed with a high fat diet. We conclude that PTEN is involved in oxidative stress and genomic damage induction in vitro and that this may also explain the in vivo observations. This further supports the hypothesis that the PI3K/AKT pathway is responsible for damaging effects of high levels of insulin., Competing Interests: The authors have declared that no competing interests exist.

Background: Evidence on bariatric surgery induced weight loss and its possible impact on cancer risk is limited, but also controversial. We used obese Zucker(fa/fa) and lean Zucker(fa/+) to investigate the association between obesity, oxidative stress and genomic damage after weight loss induced either by Roux-en-Y gastric bypass surgery (RYGB) or caloric restriction., Methods: Male Zucker(fa/fa) rats underwent RYGB (n=15) or sham surgery (n=17). Five shams were food restricted and body weight matched (BWM) to RYGB. Twelve Zucker(fa/+) rats served as lean controls. Body weight and food intake were measured daily. An oral glucose tolerance test was performed on day 27. DHE staining and western blots of HSP70 and HO-1 were used to evaluate oxidative stress and anti-3-nitrotyrosine antibody staining for nitrative stress detection in colon and kidney. Lipid peroxidation products in urine were quantified by TBARS assay. LC/MS/MS was applied to measure urinary excretion of 8-oxoGua (oxidized DNA derived base), 8-oxodG (oxidized DNA derived nucleoside) and 8-oxoGuo (oxidized RNA derived nucleoside). DNA double strand breaks (DSBs) and cell proliferation (PCNA) were detected by immunohistochemistry., Results: Sham-operated rats showed impaired glucose tolerance, elevated plasma insulin levels as well as elevated oxidative stress and nitrative stress markers, which were less severe after weight loss by RYGB or caloric restriction. Cell proliferation showed similar trends but no significant alteration. DNA DSBs were more frequent in sham-operated compared to all other groups. DNA damage in Zucker(fa/fa) rats positively correlated with basal plasma insulin values (Spearman's correlation coefficient for colon, 0.634 and for kidney, 0.525)., Conclusions: RYGB and caloric restriction were sufficient to significantly reduce elevated oxidative/nitrative stress and genomic damage in obese Zucker(fa/fa) rats. Further investigations are needed to elucidate the underlying mechanism of these genome protective effects., (Copyright © 2016 Elsevier Inc. All rights reserved.)