Milada Spatova

Automated scoring of lymphocyte micronuclei by the MetaSystems Metafer image cytometry system and its application in studies of human mutagen sensitivity and biodosimetry of genotoxin exposure

Automated image analysis scoring of micronuclei (MN) in cells can facilitate the objective and rapid measurement of genetic damage in mammalian and human cells. This approach was repeatedly developed and tested over the past two decades but none of the systems were sufficiently robust for routine analysis of MN until recently. New methodological, hardware and software developments have now allowed more advanced systems to become available. This mini-review presents the current stage of development and validation of the Metasystems Metafer MNScore system for automated image analysis scoring of MN in cytokinesis-blocked binucleated lymphocytes, which is the best-established method for studying MN formation in humans. The results and experience of users of this system from 2004 until today are reviewed in this paper. Significant achievements in the application of this method in research related to mutagen sensitivity phenotype in cancer risk, radiation biodosimetry and biomonitoring studies of air pollution (enriched by new data) are described. Advantages as well as limitations of automated image analysis in comparison with traditional visual analysis are discussed. The current increased use of the Metasystems Metafer MNScore system in various studies and the growing number of publications based on automated image analysis scoring of MN is promising for the ongoing and future application of this approach.

The impact of air pollution on the levels of micronuclei measured by automated image analysis.

The measurement of micronuclei (MN) in human peripheral blood lymphocytes is frequently used in molecular epidemiology as one of the preferred methods for assessing chromosomal damage resulting from environmental mutagen exposure. In the present study, we evaluated the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), volatile organic compounds (VOC) and smoking on the frequency of MN in a group of 56 city policemen living and working in Prague. The average age of the participants was 34+/-6 years. The study was conducted on the same subjects in February and May 2007. The concentrations of air pollutants were obtained from personal and stationary monitoring. A statistically significant decrease in the levels of pollutants was observed in May when compared with February, with the exception of toluene levels measured by stationary monitoring. The frequency of MN was determined by the automatic image scoring (MetaSystems Metafer 4, version 3.2.1) of DAPI-stained slides. The results of the image analysis indicated a significant difference in the frequency of MN (mean levels 7.32+/-3.42 and 4.67+/-2.92, for February and May, respectively). Our study suggests that automatic image analysis of MN is a highly sensitive method for evaluating the effect of c-PAHs and confirms that there are no differences between smokers and nonsmokers. These results demonstrate the ability of c-PAHs to increase MN frequency, even if the exposure to c-PAHs occurred up to 60 days before the collection of biological material. Our work is the first human biomonitoring study focused on the measurement of MN by automated image analysis for assessing chromosomal damage as a result of environmental mutagen exposure.

Factors affecting the frequency of micronuclei in asthmatic and healthy children from Ostrava

A higher incidence of asthma is one of the serious problems confronting urban populations worldwide. The aim of the present study was to analyze the effect of age, gender, smoking, vitamin intake, genetic polymorphisms in genes related to the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) and their detoxification and oxidative damage to DNA, lipids and proteins on the frequency of micronuclei (MN) in a group of 175 children (81 with bronchial asthma and 94 healthy controls) aged 6-15 years. The study group from the most polluted region of the Czech Republic, Ostrava, was followed in November 2008, when the mean concentration of benzo[a]pyrene (B[a]P) measured by stationary monitoring was 11.4±9.8ng/m(3). The results of cotinine analysis revealed active smoking in 15 children. The frequency of MN per 1000 binucleated cells (MN/1000 BNC), measured by automated image analysis, indicated a significant risk for smoking children with asthma in comparison with smoking control children (4.25±1.54 and 3.00±0.77, respectively, p<0.05). Girls in the control group had 16% higher levels of MN in comparison with boys. Markers of oxidative damage to DNA, proteins and lipids were not associated with asthma in this study. Higher levels of MN were associated with increased levels of protein carbonyl groups. We conclude that smoking asthmatic children are at higher risk of DNA damage measured as the frequency of micronuclei in peripheral blood lymphocytes.

Genetic, Biochemical, and Environmental Factors Associated with Pregnancy Outcomes in Newborns from the Czech Republic

Background Oxidative damage to placental DNA can result in negative pregnancy outcomes, including intrauterine growth restriction (IUGR) and low birth weight (LBW). Objective We investigated associations between the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage, in placental DNA, exposure to air pollutants during pregnancy, genetic polymorphisms in 94 selected genes, and pregnancy outcomes. Methods We studied 891 newborns who were IUGR- or LBW-affected or normal weight and were born between 1994 and 1999 in the Czech Republic in two districts with different levels of air pollution. Results We found nonsignificantly elevated 8-oxodG levels in the IUGR-affected group compared with the non-IUGR group (p = 0.055). Similarly, slightly elevated 8-oxodG levels were found in the LBW-affected group compared with the non-LBW group (p < 0.050). In univariate analyses, we identified single nucleotide polymorphisms associated with 8-oxodG levels, IUGR, and LBW. Exposure to particulate matter < 2.5 μm was associated with increased 8-oxodG levels in placental DNA and LBW. However, multivariate-adjusted logistic regression revealed that above-median 8-oxodG levels were the only factor significantly associated with IUGR [OR = 1.56; 95% confidence interval (CI), 1.07–2.37; p = 0.022]. Above-median levels of 8-oxodG were associated with LBW (OR = 1.88; 95% CI, 1.15–3.06; p = 0.011). Other variables associated with LBW included sex and gestational age of the newborn, maternal smoking, and haplotypes in the promoter region of the gene encoding mannose-binding lectin 2 (MBL2). The role of air pollutants in the risk of adverse pregnancy outcomes seemed to be less important. Conclusions Levels of 8-oxodG in placental DNA were associated with the risk of IUGR as well as LBW. Newborn’s sex, gestational age, maternal smoking, and genetic polymorphisms in the promoter region of the MBL2 gene were associated with LBW incidence.

Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.

Abstract 4648: Biomarkers of exposure and effecft short-term vs. chronic environmental exposure to carcinogenic polycyclic aromatic hydrocarbons

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background. Ostrava Region in the Northern Moravia (Silesia) is the most polluted region in the Czech Republic by particulate matter (PM10 and PM2.5) and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as benzo[a]pyrene (B[a]P). Sources of this pollution are industry (steel production, coke oven), traffic and local heating. In the most polluted district of Ostrava City Bartovice in the year 2009 was PM10 47.6±42.1 μg/m3, B[a]P was 9.2±4.2 ng/ m3. Objectives. The aim of the study is to gain new knowledge on the mechanisms of the effects of complex mixtures bound on dust particles (c-PAHs) in the ambient air on biomarkers of exposure and effect during the period of inversion in January-February 2010. Molecular epidemiology study. In the Ostrava City were selected 78 volunteers- working in the office, 31 city policemen from the near town Karvina, 12 city policemen from the near town Havirov, and 28 volunteers from Ostrava-Bartovice. As controls were city policemen from Prague (N=65). All volunteers were nonsmokers. The personal monitoring to c-PAHs was carried on in January-February 2010. The determined concentration of B[a]P was in the Ostrava region vs. Prague 14.8±13.3 vs. 2.80±1.87 ng/ m3. The impact of these concentration to biomarkers as DNA adducts, chromosomal aberrations, 8-oxodG, lipid peroxidation, genetic polymorphisms (metabolic and DNA repair genes) and gene expression profiles are followed. DNA adducts were analysed in lymphocytes by 32P-postlabeling assay, chromosomal aberrations by fluorescent in situ hybridization (FISH) by whole chromosome painting #1 and #4, and by the determination of micronuclei, oxidative damage as 8-oxodG and 15-F2t-IsoP. Obtained results indicate no differences between the level of biomarkers determined in the Ostrava region and Prague. But different results were observed in four subjects from Prague, who spent 3 weeks in Ostrava during this inversion (exposed), compared to controls from Prague. Analyzing 6000 cells/subject, the frequency of micronuclei in exposed group increased from 7.96±4.92 to 12.91±6.49 MN/1000 vs. 8.32±1.63 and 8.47±1.55 MN/1000 in controls (p<0.05). Similar tendency was observed using FISH and analysing 5000 cells/subject, genomic frequency of translocations (FG/100) in exposed group increased from 1.44±0.66 to 2.44±1.82 FG/100 vs. 1.51±0.61 and 1.45±0.0.52 FG/100. Conclusion: We may speculate, that high environmental exposure to B[a]P does not correspond to DNA damage according to dose effect relationship in a population affected by these concentrations for years, probably due to a possible adaptive response. Acknowledgement: Supported by the Czech Ministry of Environment (SP/1b3/8/08) and the Czech Ministry Education (2B08005). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4648. doi:10.1158/1538-7445.AM2011-4648

Abstract 4392: Oxidative damage to placental DNA, air pollution, genetic polymorphisms and pregnancy outcomes

Oxidative stress to placenta DNA may result in negative pregnancy outcomes, including intrauterine growth retardation (IUGR) and low birth weight (LBW). The aim of our study was to investigate the association between the levels of 8-oxodeoxuanosine (8-oxodG), a marker of oxidative DNA damage, in placenta DNA, exposure to particulate matter 1.80 8-oxodG/105 dG) had 64% higher probability of IUGR (OR (95% CI): 1.64 (1.12-2.42), p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4392.