Armen Nersesyan

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMNXL): The role of life-style, host factors, occupational exposures, health status, and assay protocol

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMN(XL) collaborative study. The HUMN(XL) project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p<0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40cig/day, FR=1.37; 95% CI 1.03-.82) and decreased with daily fruit consumption (FR=0.68; 95% CI 0.50-0.91). The results of the HUMN(XL) project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency.

Effect of Staining Procedures on the Results of Micronucleus Assays with Exfoliated Oral Mucosa Cells

Micronuclei in exfoliated epithelial cells are widely used as biomarkers of cancer risk in humans. To elucidate the effect of different staining procedures on the outcome of such investigation, we conducted a study in which the micronuclei frequencies in oral mucosa cells of heavy smokers (n = 20) and nonsmokers (n = 10) were evaluated with nonspecific (Giemsa, May-Grünwald-Giemsa) and DNA-specific (4′,6-diamidino-2-phenylindole, Feulgen, acridine orange) stains, whereas with Giemsa-based stains, the frequencies of micronuclei in smokers were significantly (4- to 5-fold) higher in the smokers group, no significant increase was observed with any of the DNA-specific stains. Furthermore, the evaluation of cells of the two study groups with Feulgen stain showed that oral mucosa cells from smokers had significantly increased levels of nuclear anomalies other than micronuclei. These anomalies are consequences of cell injury found in epithelial cells and are paralleled by formation of keratin bodies in the cytoplasm that resemble micronuclei. Correlation analyses showed that micronuclei frequencies scored in Giemsa-stained slides correlated significantly with karyorrhexis, karyolysis, condensed chromatin, and binucleates, whereas no such correlations were found with DNA-specific stains. These findings indicate that nuclear anomalies (and possibly keratin bodies) may be misinterpreted as micronuclei with nonspecific DNA stains and lead to false-positive results in studies with cells of epithelial origin. Furthermore, our results show that exposure of oral mucosa cells to genotoxic carcinogens contained in tobacco smoke does not lead to induction of micronuclei in these cells. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1835–40)

Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview.

This article describes the principles and limitations of methods used to investigate reactive oxygen species (ROS) protective properties of dietary constituents and is aimed at providing a better understanding of the requirements for science based health claims of antioxidant (AO) effects of foods. A number of currently used biochemical measurements aimed of determining the total antioxidant capacity and oxidised lipids and proteins are carried out under unphysiological conditions and are prone to artefact formation. Probably the most reliable approaches are measurements of isoprostanes as a parameter of lipid peroxidation and determination of oxidative DNA damage. Also the design of the experimental models has a strong impact on the reliability of AO studies: the common strategy is the identification of AO by in vitro screening with cell lines. This approach is based on the assumption that protection towards ROS is due to scavenging, but recent findings indicate that activation of transcription factors which regulate genes involved in antioxidant defence plays a key role in the mode of action of AO. These processes are not adequately represented in cell lines. Another shortcoming of in vitro experiments is that AO are metabolised in vivo and that most cell lines are lacking enzymes which catalyse these reactions. Compounds with large molecular configurations (chlorophylls, anthocyans and polyphenolics) are potent AO in vitro, but weak or no effects were observed in animal/human studies with realistic doses as they are poorly absorbed. The development of -omics approaches will improve the scientific basis for health claims. The evaluation of results from microarray and proteomics studies shows that it is not possible to establish a general signature of alterations of transcription and protein patterns by AO. However, it was shown that alterations of gene expression and protein levels caused by experimentally induced oxidative stress and ROS related diseases can be normalised by dietary AO.

The HUMNxl scoring criteria for different cell types and nuclear anomalies in the buccal micronucleus cytome assay - an update and expanded photogallery.

The buccal micronucleus cytome assay is a minimally invasive cytological and interphase cytogenetic technique for measuring DNA damage and cell death biomarkers in the oral epithelium. In this report we provide an updated and more comprehensive version of the cellular and nuclear scoring criteria used in the assay accompanied with a photogallery of the various cell types and nuclear anomalies. These detailed scoring criteria complement previous published protocols of this assay and form the basis for guiding intra- and inter-laboratory slide scoring comparisons. The scoring criteria update described in this paper is the outcome of ongoing efforts of the HUMN and HUMNxl projects (www.humn.org) to standardize the application of micronucleus assay for use in human biomonitoring and to update procedures as knowledge on mechanisms and technical capability improvements.

Use of single cell gel electrophoresis assays for the detection of DNA-protective effects of dietary factors in humans: Recent results and trends

This article summarises the results of human dietary intervention trials employing the comet assay (single cell gel electrophoresis, SCGE), which have been published in the last few years (i.e., between 2005 and 2008) and describes new trends and developments as well as current problems concerning the design of intervention trials and the interpretation of the results. Most new studies were carried out with complex plant derived foods and juices; only a few were conducted with individual food constituents. With specific vegetables, for example with water cress and Brussels sprouts, potent antioxidant effects were observed; also coffee caused a protective effect and it is notable that it was more effective than consumption of a diet containing increased levels of fruits and vegetables. Interesting recent developments include the development of protocols which enable us to monitor protection towards genotoxic chemicals contained in the human diet, and it was shown in preliminary studies that alterations of the activities of drug metabolising enzymes by dietary factors lead to altered sensitivity of lymphocytes against DNA damage caused by certain dietary carcinogens. Another novel approach is the development of methods to monitor the effects of dietary factors on DNA repair. The development of protocols for experiments with exfoliated buccal cells is another potentially valuable innovation. The adequate experimental design of SCGE trials is still a matter of debate and the evaluation of the available data shows that there is an urgent need to develop guidelines concerning the number of participants, sampling periods, duration of trials, use of placebos, and definition of adequate run-in and wash-out phases. Recent studies showed that the results of dietary studies could be biased by factors such as age, sex, body mass index and life style habits and by seasonal effects. Another still unsolved problem is the interpretation of the results of SCGE trials in regard to potential beneficial health effects. The use of -omics techniques may contribute to provide mechanistic explanations in addition to conventional approaches (such as enzyme measurements). Information on health effects of dietary factors and on prevention of diseases related to DNA damage can also be obtained in experiments with animals, using SCGE to detect decreases in DNA damage in inner organs.

Inhalative exposure to vanadium pentoxide causes DNA damage in workers: results of a multiple end point study.

Background Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents. Objective The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory. Methods We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method. Results Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 μg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance. Conclusions V2O5 causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.

Consumption of Brussels sprouts protects peripheral human lymphocytes against 2-amino-1- methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and oxidative DNA-damage: results of a controlled human intervention trial

To find out if the cancer protective effects of Brussels sprouts seen in epidemiological studies are due to protection against DNA-damage, an intervention trial was conducted in which the impact of vegetable consumption on DNA-stability was monitored in lymphocytes with the comet assay. After consumption of the sprouts (300 g/p/d, n = 8), a reduction of DNA-migration (97%) induced by the heterocyclic aromatic amine 2-amino-1-methyl-6-phenyl-imidazo-[4,5-b]pyridine (PhIP) was observed whereas no effect was seen with 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2). This effect protection may be due to inhibition of sulfotransferase 1A1, which plays a key role in the activation of PhIP. In addition, a decrease of the endogenous formation of oxidized bases was observed and DNA-damage caused by hydrogen peroxide was significantly (39%) lower after the intervention. These effects could not be explained by induction of antioxidant enzymes glutathione peroxidase and superoxide dismutase, but in vitro experiments indicate that sprouts contain compounds, which act as direct scavengers of reactive oxygen species. Serum vitamin C levels were increased by 37% after sprout consumption but no correlations were seen between prevention of DNA-damage and individual alterations of the vitamin levels. Our study shows for the first time that sprout consumption leads to inhibition of sulfotransferases in humans and to protection against PhIP and oxidative DNA-damage.

DNA-protective effects of sumach (Rhus coriaria L.), a common spice : Results of human and animal studies

Sumach (Rhus coriaria L.) is widely used as a spice. The aim of this study was the investigation of its DNA-protective effects in humans and animals. Prevention of the formation of strand breaks and oxidized DNA bases as well as the protection against H(2)O(2)- and (+/-)-anti-benzo[a]pyrene-7,8-dihydro-diol-9,10-epoxide (BPDE)-induced DNA-damage were monitored in human lymphocytes in a placebo controlled trial (N=8/group) with ethanolic extract of sumach (3.0g/day, 3 days) in single cell gel electrophoresis assays. Furthermore, DNA-protective effects of sumach were monitored in different inner organs of rats under identical conditions. No alteration of DNA-migration was detectable in human lymphocytes under standard conditions, but a decrease of the tail-lengths due to formation of oxidized purines and pyrimidines (52% and 36%) was found with lesion-specific enzymes. Also damage caused by H(2)O(2) and BPDE was significantly reduced by 30% and 69%, respectively. The later effect may be due to induction of glutathione S-transferase (GST). After the intervention, the overall GST (CDNB) activity in plasma was increased by 40%, GST-alpha by 52% and GST-pi by 26% (ELISA). The antioxidant effects of extract are probably due to scavenging which was observed in in vitro experiments, which also indicated that gallic acid is the active principle of sumach. The animal experiments showed that sumach also causes protection in inner organs. Supplementation of the drinking water (0.02g/kg per animal) decreased the formation of oxidized DNA bases in colon, liver, lung and lymphocytes; also after gamma-irradiation pronounced effects were seen.

Impact of smoking on the frequencies of micronuclei and other nuclear abnormalities in exfoliated oral cells: a comparative study with different cigarette types

The primary aim of the study was to investigate the impact of tar and nicotine contents of cigarettes on chromosomal damage in oral mucosa cells of smokers. We monitored the effect of smoking different cigarette types (i.e., of ultralight filter, light filter, medium filter and unfiltered cigarettes) on induction of nuclear anomalies including micronuclei (MN), broken eggs (BE), binucleates (BN), condensed chromatin (CC), karyorrhexis (KR), karyolysis (KL) and pyknosis (P) in exfoliated buccal cells. The cells were collected from 83 healthy heavy smokers (n=15-25/group) consuming a similar number of cigarettes (26-33) per day and from never smokers as controls (n=20). The frequencies of KR, CC, KL, BE and BN were increased significantly only in smokers of medium (MF) and non-filtered (NF) types of cigarettes while MN levels were only elevated (p < 0.0001) in the group that smoked NF cigarettes. Since BN and BE were increased (p < 00001) as a consequence of exposure to lower levels of toxic constituents in tobacco, it suggests that these endpoints, which both reflect DNA damage, are more sensitive than MN, which is the only parameter scored in most earlier studies. The induction of MN, BN, KR and KL increased significantly with daily tar exposure and decreased simultaneously with daily nicotine uptake (in all cases, P was < 0.0001). These findings also suggest that nicotine potentially protects cells against DNA reactive carcinogens contained in tobacco smoke although earlier in vitro and animal studies showed that the alkaloid induces DNA damage per se. A significant inverse correlation between the frequencies of endpoints such as cells with MN (- 1.56), MN (-1.69), BN (-1.36), KR (-1.10) and KL (-1.87) with the nicotine levels in cigarettes was found. However, this observation requires further verification by a controlled intervention study. In case it can be substantiated it will have an impact on the ongoing discussion of the health risks associated with nicotine replacement therapy.

Impact of paper filtered coffee on oxidative DNA-damage: results of a clinical trial.

Coffee is among the most frequently consumed beverages worldwide and epidemiological studies indicate that its consumption is inversely related to the incidence of diseases in which reactive oxygen species (ROS) are involved (liver cirrhosis, certain forms of cancer and neurodegenerative disorders). It has been postulated that antioxidant properties of coffee may account for this phenomenon. To find out if consumption of paper filtered coffee which is the most widely consumed form in Central Europe and the US protects humans against oxidative DNA-damage, a controlled intervention trial with a cross-over design was conducted in which the participants (n=38) consumed 800ml coffee or water daily over 5 days. DNA-damage was measured in peripheral lymphocytes in single cell gel electrophoresis assays. The extent of DNA-migration attributable to formation of oxidised purines (formamidopyrimidine glycosylase sensitive sites) was decreased after coffee intake by 12.3% (p=0.006). Biochemical parameters of the redox status (malondialdehyde, 3-nitrotyrosine and the total antioxidant levels in plasma, glutathione concentrations in blood, intracellular ROS levels and the activities of superoxide dismutase and glutathione peroxidase in lymphocytes) were not markedly altered at the end of the trial, also the urinary 8-isoprostaglandine F2α concentrations were not affected. Overall, the results indicate that coffee consumption prevents endogenous formation of oxidative DNA-damage in human, this observation may be causally related to beneficial health effects of coffee seen in earlier studies.