The Comet Assay as a Tool in Human Biomonitoring Studies of Environmental and Occupational Exposure to Chemicals-A Systematic Scoping Review.

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 ameliorative effect of rosmarinic acid and epigallocatechin gallate against doxorubicin-induced genotoxicity.

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.

Rosmarinic acid (RA) showed protective effect against doxorubicin-induced genotoxicity.Epigallocatechin gallate (EGCG) demonstrated pro-oxidant properties at high concentrations.EGCG and RA selectively targeted MDA-MB-231 cells.Synergistic effect was observed when EGCG or RA was administered together with Dox on breast cancer cells.

Differential Modulation of Markers of Oxidative Stress and DNA Damage in Arterial Hypertension.

Patients with arterial hypertension have an increased risk of developing tumors, particularly renal cell carcinoma. Arterial hypertension is linked to DNA damage via the generation of oxidative stress, in which an upregulated renin-angiotensin-aldosterone system plays a crucial role. The current study investigated surrogates of oxidative stress and DNA damage in a group of hypertensive patients (HypAll, n = 64) and subgroups of well (HypWell, n = 36) and poorly (HypPoor, n = 28) controlled hypertensive patients compared to healthy controls (n = 8). In addition, a longitudinal analysis was performed with some of the hypertensive patients. Markers for oxidative stress in plasma (SHp, D-ROM, and 3-nitrotyrosine) and urine (8-oxodG, 15-F2t-isoprostane, and malondialdehyde) and markers for DNA damage in lymphocytes (γ-H2AX and micronuclei) were measured. In HypAll, all markers of oxidative stress except malondialdehyde were increased compared to the controls. After adjustment for age, this association was maintained for the protein stress markers SHp and 3-nitrotyrosine. With regard to the markers for DNA damage, there was no difference between HypAll and the controls. Further, no significant differences became apparent in the levels of both oxidative stress and DNA damage between HypWell and HypPoor. Finally, a positive correlation between the development of blood pressure and oxidative stress was observed in the longitudinal study based on the changes in D-ROM and systolic blood pressure. In conclusion, we found increased oxidative stress in extensively treated hypertensive patients correlating with the level of blood-pressure control but no association with DNA damage.

Maternal Smoking in the First Trimester and its Consequence on the Early Placenta.

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.

Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells.

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.

Incidence and Risk Factors of Acute Leukemias in Armenia: A Population-Based Study.

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.

Obesity-related genomic instability and altered xenobiotic metabolism: possible consequences for cancer risk and chemotherapy.

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.

Short- and long-term reproducibility of the COMET assay for measuring DNA damage biomarkers in frozen blood samples of the EPIC-Heidelberg cohort.

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.

Cytotoxic and genotoxic properties of artathomsonine, a new oxoberberine alkaloid from Artabotrys thomsonii (annonaceae).

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-MSn 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.

"Micronuclei and Disease" special issue: Aims, scope, and synthesis of outcomes.

The purpose of the "Micronuclei and Disease" special issue (SI) is to: (i) Determine the level of evidence for association of micronuclei (MN), a biomarker of numerical and structural chromosomal aberrations, with risk of specific diseases in humans; (ii) Define plausible mechanisms that explain association of MN with each disease; (iii) Identify knowledge gaps and research needed to translate MN assays into clinical practice. The "MN and Disease" SI includes 14 papers. The first is a review of mechanisms of MN formation and their consequences in humans. 11 papers are systematic reviews and/or meta-analyses of the association of MN with reproduction, child health, inflammation, auto-immune disease, glycation, metabolic diseases, chronic kidney disease, cardiovascular disease, eleven common cancers, ageing and frailty. The penultimate paper focuses on effect of interventions on MN frequency in the elderly. A road map for translation of MN data into clinical practice is the topic of the final paper. The majority of reviewed studies were case-control studies in which the ratio of mean MN frequency in disease cases relative to controls, i.e. the mean ratio (MR), was calculated. The mean of these MR values, estimated by meta-analyses, for lymphocyte and buccal cell MN in non-cancer diseases were 2.3 and 3.6 respectively, and for cancers they were 1.7 and 2.6 respectively. The highest MR values were observed in studies of cancer cases in which MN were measured in the same tissue as the tumour (MR = 4.9-10.8). This special issue is an important milestone in the evidence supporting MN as a reliable genomic biomarker of developmental and degenerative disease risk. These advances, together with results from prospective cohort studies, are helping to identify diseases in which MN assays can be practically employed in the clinical setting to better identify high risk patients and to prioritise them for preventive therapy.

Cell survival after DNA damage in the comet assay.

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 (H2O2) 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 H2O2 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.

Genotoxicity of selected pyrrolizidine alkaloids in human hepatoma cell lines HepG2 and Huh6.

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.

Cytokinesis-block micronucleus assay of celecoxib and celecoxib derivatives.

Celecoxib is used widely for the acute treatment of pain and for pain relief in various diseases. Furthermore, it shows potential in chemoprevention, although chronic treatment with celecoxib could lead to adverse effects like cardiovascular events. New derivatives of celecoxib were synthesised that may be suitable as chemopreventive agent without inducing adverse effects. Critical endpoint for a safe use of pharmaceuticals is genotoxicity after application. A standard test for the assessment of genotoxicity is the cytokinesis-block micronucleus assay, that evaluates the number micronuclei after treatment of cells with a test compound as biomarker for DNA damage. Various promising derivatives of celecoxib have been assessed with the cytokinesis-block micronucleus assay in HeLa-H2B-GFP cells. It could be demonstrated, that neither celecoxib nor its derivatives were genotoxic in this assay and therefore celecoxib derivatives could be developed further for a safe use as chemopreventive agent.

Micronucleus frequency in buccal mucosa cells of patients with neurodegenerative diseases.

Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer's disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome.

Micronucleus frequency in chronic kidney disease patients: A review.

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.

A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice: Impact of Sex and Age.

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.

Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems.

Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.

Use of micronucleus assays for the prediction and detection of cervical cancer: a meta-analysis.

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.

Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells.

Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.

Influence of bariatric surgery induced weight loss on oxidative DNA damage.

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.