[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 07/2013; 8(7):e69197. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Ostrava Region in the Czech Republic is a heavily polluted industrial area. Concentrations of PM10, PM2.5, and benzo[a]pyrene (B[a]P) significantly exceed limit values. To investigate the impact of these levels on human health, epidemiological, molecular epidemiology, and in vitro studies were done in 2008–2011. Morbidity of children was followed in 10 pediatric districts. In the most polluted district, children suffered higher incidence of acute respiratory diseases in the first year of life, and higher prevalence of asthma bronchiale. Gene expression was studied in children from Ostrava and from a control rural area. Genes specific to asthma bronchiale differed, suggesting a different molecular phenotype in children in the polluted region compared to children in the control area. A molecular epidemiology study showed adverse effect of the Ostrava exposures, but also an increased expression of XRCC5, which probably protects these exposed subjects against the degree of genetic damage that would otherwise be expected. In vitro studies clearly related concentration of B[a]P from PM2.5 extracts to induced PAH-DNA adducts. These studies clearly demonstrate that under the present local environmental conditions, the health of the population is severely impaired and will likely remain so for a significant period of time.
[show abstract][hide abstract] ABSTRACT: The use of micronucleus (MN) assays in in vitro genetic toxicology testing, radiation biodosimetry and population biomonitoring to study the genotoxic impacts of environment gene-interactions has steadily increased over the past two decades. As a consequence there has been a strong interest in developing automated systems to score micronuclei, a biomarker of chromosome breakage or loss, in mammalian and human cells. This paper summarises the outcomes of a workshop on this topic, organised by the HUMN project, at the 6th International Conference on Environmental Mutagenesis in Human Populations at Doha, Qatar, 2012. The aim of this paper is to summarise the outcomes of the workshop with respect to the set objectives which were: (i) Review current developments in automation of micronucleus assays by image cytometry; (ii) define the performance characteristics of automated MN scoring using image cytometry and methods of assessment for instrument validation and quality control and (iii) discuss the design of inter-laboratory comparisons and standardisation of micronucleus assays using automated image cytometry systems. It is evident that automated scoring of micronuclei by automated image cytometry using different commercially available platforms [e.g. Metafer (MetaSystems), Pathfinder™ (IMSTAR), iCyte(®) (Compucyte)], particularly for lymphocytes, is at a mature stage of development with good agreement between visual and automated scoring across systems (correlation factors ranging from 0.58 to 0.99). However, a standardised system of validation and calibration is required to enable more reliable comparison of data across laboratories and across platforms. This review identifies recent progress, important limitations and steps that need to be taken into account to enable the successful universal implementation of automated micronucleus assays by image cytometry.
International journal of hygiene and environmental health 03/2013; · 2.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gene expression levels are significantly regulated by DNA methylation. Differences in gene expression profiles in the populations from various locations with different environmental conditions were repeatedly observed. In this study we compare the methylation profiles in 200 blood samples of children (aged 7-15 years) with and without bronchial asthma from two regions in the Czech Republic with different levels of air pollution (a highly polluted Ostrava region and a control Prachatice region). Samples were collected in March 2010 when the mean concentrations of benzo[a]pyrene (B[a]P) measured by stationary monitoring were 10.1±2.4 ng/m3 in Ostrava Bartovice (5.6 times higher than in the control region). Significantly higher concentrations of other pollutants (benzene, NO2, respirable air particles and metals) were also found in Ostrava. We applied the Infinium Methylation Assay, using the Human Methylation 27K BeadChip with 27,578 CpG loci for identification of the DNA methylation pattern in studied groups. Results demonstrate a significant impact of different environmental conditions on the DNA methylation patterns of children from the two regions. We found 9,916 CpG sites with significantly different methylation (beta value) between children from Ostrava vs. Prachatice from which 58 CpG sites had differences > 10%. The methylation of all these 58 CpG sites was lower in children from polluted Ostrava, which indicates a higher gene expression in comparison with the control Prachatice region. We did not find a difference in DNA methylation patterns between children with and without bronchial asthma in individual locations, but patterns in both asthmatics and healthy children differed between Ostrava and Prachatice. Further, we show differences in DNA methylation pattern depending on gender and urinary cotinine levels. Other factors including length of gestation, birth weight and length of full breastfeeding are suggested as possible factors that can impact the DNA methylation pattern in future life.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2013; · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Health impact of air pollution to children was studied over the last twenty years in heavily polluted parts of the Czech Republic during. The research program (Teplice Program) analyzed these effects in the polluted district Teplice (North Bohemia) and control district Prachatice (Southern Bohemia). Study of pregnancy outcomes for newborns delivered between 1994 and 1998 demonstrated that increase in intrauterine growth retardation (IUGR) was associated with PM10 and c-PAHs exposure (carcinogenic polycyclic aromatic hydrocarbons) in the first month of gestation. Morbidity was followed in the cohort of newborns (N=1492) up to the age of 10years. Coal combustion in homes was associated with increased incidence of lower respiratory track illness and impaired early childhood skeletal growth up to the age of 3years. In preschool children, we observed the effect of increased concentrations of PM2.5 and PAHs on development of bronchitis. The Northern Moravia Region (Silesia) is characterized by high concentrations of c-PAHs due to industrial air pollution. Exposure to B[a]P (benzo[a]pyrene) in Ostrava-Radvanice is the highest in the EU. Children from this part of the city of Ostrava suffered higher incidence of acute respiratory diseases in the first year of life. Gene expression profiles in leukocytes of asthmatic children compared to children without asthma were evaluated in groups from Ostrava-Radvanice and Prachatice. The results suggest the distinct molecular phenotype of asthma bronchiale in children living in polluted Ostrava region compared to children living in Prachatice. The effect of exposure to air pollution to biomarkers in newborns was analyzed in Prague vs. Ceske Budejovice, two locations with different levels of pollution in winter season. B[a]P concentrations were higher in Ceske Budejovice. DNA adducts and micronuclei were also elevated in cord blood in Ceske Budejovice in comparison to Prague. Study of gene expression profiles in the cord blood showed differential expression of 104 genes. Specifically, biological processes related to immune and defense response were down-regulated in Ceske Budejovice. Our studies demonstrate that air pollution significantly affect child health. Especially noticeable is the increase of respiratory morbidity. With the development of molecular epidemiology, we can further evaluate the health risk of air pollution using biomarkers.
International journal of hygiene and environmental health 01/2013; · 2.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Populations living in industrialised regions are at higher risk of a number of diseases and shortened life span. These negative effects are primarily brought about by damage to cells and macromolecules caused by environmental pollutants. In this study, we analysed the effect of exposure to benzo[a]pyrene, a particulate matter of aerodynamic diameter < 2.5 µm (PM2.5), and benzene on oxidative stress markers [including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 15-F(2t)-isoprostane (15-F2t-IsoP) and protein carbonyls] and cytogenetic parameters (stable and unstable chromosomal aberrations). The samples were collected from subjects living in the Ostrava region characterised by very high levels of air pollution and in Prague with comparatively lower concentrations of pollutants in three seasons (winter 2009, summer 2009 and winter 2010). Despite several-fold higher concentrations of air pollutants in the Ostrava region, the levels of stable aberrations (genomic frequency of translocations per 100 cells, percentage of aberrant cells and frequency of acentric fragments) were mostly comparable in both locations. The frequency of unstable aberrations measured as the number of micronuclei was unexpectedly significantly lower in the Ostrava region subjects in both seasons of 2009. Urinary excretion of 8-oxodG did not differ between locations in either season. Lipid peroxidation measured as levels of 15-F2t-IsoP in blood plasma was elevated in the Ostrava subjects sampled in 2009. Protein oxidation was higher in Prague samples collected in summer 2009. Multivariate analyses conducted separately in subjects from Prague and Ostrava showed a negative association between the frequency of micronuclei and concentrations of benzo[a]pyrene and PM2.5 in both regions. A positive relationship was observed between lipid peroxidation and air pollution; protein oxidation seems to be positively affected by PM2.5 in both regions.
[show abstract][hide abstract] ABSTRACT: The effect of exposure to carcinogenic polycyclic aromatic hydrocarbons adsorbed onto respirable air particles (PM2.5, diameter < 2.5 μm) on DNA adducts and chromosomal aberrations was repeatedly studied in Prague, Czech Republic, in groups of policemen working in the downtown area and in bus drivers. Personal exposure was evaluated using personal samplers during working shifts. DNA adducts were analyzed in lymphocytes by the (32)P-postlabeling assay and chromosomal aberrations were analyzed by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH). The impact of environmental pollution on DNA adducts and chromosomal aberrations was studied in a total of 950 subjects. Our results suggest that the environmental exposure of nonsmokers to concentrations higher than 1 ng benzo[a]pyrene/m(3) represents a risk of DNA damage, as indicated by an increase in DNA adducts and the genomic frequency of translocations determined by FISH.
Air Quality Atmosphere & Health 12/2011; 4(3-4):161-167. · 1.98 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to analyze genetic damage in human lymphocytes measured using automated image analysis of micronuclei (MN) in a group of 178 mothers and their newborns from two locations in the Czech Republic. The concentrations of benzo[a]pyrene (B[a]P), particulate matter of aerodynamic diameter <2.5 μm (PM2.5) and benzene were measured by stationary monitoring in the winter season of 2008/2009 in the capital city of Prague and in Ceske Budejovice, a regional city in a rural area. The 3-month mean concentration of B[a]P before delivery was lower in Prague in comparison with Ceske Budejovice: 1.9 ± 0.5ng/m³ vs. 3.2 ± 0.2ng/m³ (p<0.001). The opposite trend was found for PM2.5 and benzene: 27.0 ± 2.5μg/m³ and 2.5 ± 0.5μg/m³ vs. 24.5 ± 0.7μg/m³ and 2.1 ± 0.8μg/m³ (p<0.001) for Prague vs. Ceske Budejovice, respectively. The average age of the mothers was 31 years (range, 18-49 years). The frequencies of MN per 1000 binucleated cells were 8.35 ± 3.06 vs. 6.47 ± 2.35 (p<0.001) for mothers from Prague and Ceske Budejovice, respectively, and 2.17 ± 1.32 vs. 3.82 ± 2.43 (p<0.001) for newborns from Prague and Ceske Budejovice, respectively. Other factors, including vitamin intake, exposure to tobacco smoke, body mass index (BMI) before pregnancy, the education level of the mothers and the impact of the mothers' and fathers' ages were analyzed in our study. The results suggest that the different sensitivity of the study groups to various mixtures of carcinogenic pollutants could be affected by significant differences in lifestyle factors. Possible higher genetic damage was analyzed in newborns of smoking mothers, and the birth weight of this group was 7.4% lower (p<0.05) in comparison with the newborns of nonsmoking mothers. No impact of the age of the mothers or fathers on MN frequency in the newborns was observed.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2011; 715(1-2):72-8. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Air pollution causes oxidative damage to macromolecules, chromosomal aberrations and changes in gene expression. We investigated the levels of oxidative stress markers [8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 15-F(2t)-isoprostane (15-F2t-IsoP), protein carbonyls] and cytogenetic parameters [genomic frequency of translocations (F(G)/100), percentage of aberrant cells (%AB.C.) and acentric fragments (ace)] in subjects living in Prague and in the heavily polluted Ostrava region. We also compared the expression of genes participating in base excision repair (BER) and non-homologous end-joining (NHEJ). We analyzed 64 subjects from Prague and 75 subjects from Ostrava. We measured oxidative stress markers by ELISA, cytogenetic parameters by fluorescence in situ hybridization and gene expression by quantitative PCR. The levels of air pollutants (benzo[a]pyrene, B[a]P; carcinogenic polycyclic aromatic hydrocarbons, c-PAHs; benzene) measured by personal monitors were significantly elevated in Ostrava compared to Prague (p<0.001). Despite this fact, we observed no differences in biomarkers of oxidative stress between the two locations. Moreover, subjects from Ostrava were less likely to have above-median levels of %AB.C. (OR; 95% CI: 0.18; 0.05-0.67; p=0.010). Multivariate analyses revealed that subjects living in Ostrava had increased odds of having above-median levels of XRCC5 expression (OR; 95% CI: 3.33; 1.03-10.8; q=0.046). Above-median levels of 8-oxodG were associated with decreased levels of vitamins C (OR; 95% CI: 0.37; 0.16-0.83; p=0.016) and E (OR; 95% CI: 0.25; 0.08-0.75; p=0.013), which were elevated in subjects from Ostrava. We suggest that air pollution by c-PAHs affects XRCC5 gene expression, which probably protects subjects from Ostrava against the induction of a higher frequency of translocations; elevated vitamin C and E levels in the Ostrava subjects decrease the levels of 8-oxodG.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2011; 713(1-2):76-82. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2011; 708(1-2):44-9. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: We investigated the effect of the seasonal variability of environmental air pollutants on oxidative stress and cytogenetic biomarkers in a group of 59 city policemen working in Prague, Czech Republic. The studied group was monitored in February and May 2007. The exposure to environmental pollutants (carcinogenic polycyclic aromatic hydrocarbons, c-PAHs, including benzo[a]pyrene, B[a]P, and particulate matter of aerodynamic diameter<2.5μm, PM2.5) was measured by personal and/or stationary monitors. Levels of c-PAHs were significantly higher in winter than spring, while exposure to PM2.5 was higher in May than in February 2007. We did not observe any significant difference between the two seasons for any biomarker of oxidative stress (8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG, 15-F(2t)-isoprostane, 15-F(2t)-IsoP, protein carbonyl levels) or any cytogenetic parameter, including the genomic frequency of translocations (F(G)/100), the percentage of aberrant cells (%AB.C.) or the number of acentric fragments (ace). Analyses of associations between oxidative stress biomarkers and cytogenetic parameters showed a negative relationship between protein oxidation and F(G)/100, as well as protein oxidation and ace. We further analyzed the effect of air pollution on all subjects regardless of the season. Data from stationary monitors showed that 8-oxodG levels were significantly increased by exposure to PM2.5 over a 2-day period before sampling and by exposure to B[a]P over a 28-day period, days 57-84 before sampling. 15-F(2t)-IsoP levels were increased after exposure to B[a]P over both 2-day and 3-day periods preceding sample collection and after exposure to c-PAHs over a 2-day period before sampling. %AB.C. was significantly affected by exposure to B[a]P over a 14-day period, days 57-70 before sampling. In summary, our results indicate that the exposure to environmental pollutants affects urinary excretion of 8-oxodG, lipid peroxidation and the frequency of chromosomal aberrations.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2011; 707(1-2):34-41. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The capital city of Prague is one of the most polluted areas of the Czech Republic. The impact of air pollution on the level of chromosomal aberrations was systematically studied: analyses were performed using fluorescence in situ hybridization (FISH) with whole-chromosome painting for chromosomes #1 and #4. In the present study, we analyzed the levels of stable (one-way and two-way translocations) and unstable (acentric fragments) chromosomal aberrations in 42 mothers living in Prague and in their newborns. The average age of the mothers was 29 years (range, 20-40 years). Blood samples were collected from October 2007 to February 2008. The average levels of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) and benzo[a]pyrene (B[a]P) in respirable particles (PM2.5), as determined by stationary monitoring, were 21.0+/-12.3 ng/m(3) and 2.9+/-1.8 ng/m(3), respectively. We did not observe any effect of either c-PAH or B[a]P exposure on the genomic frequency of translocations (per 100 cells, F(G)/100) in either group due to their similar exposure during the winter months. The mean values of F(G)/100 representing stable aberrations were 0.09+/-0.13 vs 0.80+/-0.79 (p<0.001) for newborns vs mothers, indicating a significant increase of F(G)/100 with age. On the other hand, the frequency of unstable aberrations did not differ between the two groups. Our results demonstrate how the patterns of different types of aberration differed between newborns and mothers: we observed 64.3% unstable aberrations and 35.7% stable aberrations in newborns vs 19.7% and 80.3% in mothers, respectively. Our results indicate that after birth the frequencies of aberrations are very low and that the aberrations are represented mainly by acentric fragments. The changes observed in mothers show a shift to stable aberrations represented mainly by two-way translocations. The mother's age affected the level of aberrations in newborns: the group of children born to older mothers (31-40 years) had significantly increased F(G)/100 levels.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2010; 699(1-2):29-34. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2009; 669(1-2):42-7. · 3.90 Impact Factor