The Department of Genetic Ecotoxicology (DGE) is a joint venture of the Health Institute of Central Bohemia and the Institute of Experimental Medicine.
      The research of the DGE is concerned with the genetic damage caused by genotoxic and carcinogenic compounds such as PAHs, alkenes, etc. The effects of genotoxic xenobiotics are studied in vitro using acelullar assays and cell cultures, such as human diploid lung fibroblasts, as well as in vivo by carrying out human transitional molecular epidemiologic studies and observational epidemiologic studies. The research is based on the application of advanced scientific technologies such as the 32P-postlabelling assay for DNA adduct analysis in WBC, lymphocytes and placentas, HPLC analysis of 32P-labeled DNA adducts, the expression of p53 and p21WAF1 proteins in plasma and lymphocytes, the comet assay to assess DNA damage in lymphocytes, cytogenetic analysis of chromosomal aberrations by conventional method as well as by fluorescence in situ hybridization (FISH), radioimmunological methods, flow cytometry for the phenotyping of lymphocyte subpopulations and methods for the analysis of genetic polymorphisms CYP1A1, GSTM1, GSTT1, GSTP1, NAT2, EPHX, XPD, XRCC1, hOGG1, p53, MS, MTHFR and APOE (PCR, RLFP techniques).
      The DGE has been responsible for the scientific management of the Program "AIR POLLUTION AND HEALTH", which consists of several projects studying the impact of environmental pollution on the health of the population in North Bohemia and in the capital, Prague.
      Current research at the Department focuses on the following main topics:
- Molecular epidemiology studies: biomarkers of exposure to mutagens and carcinogens
- Pregnancy outcome
- Immunity and childhood morbidity
- In vitro studies
- Embryotoxicity of genotoxic compounds
- Apoptosis in developing systems

      The objectives of these studies are to simultaneously evaluate personal exposure, internal measure of exposure, biologically effective dose of exposure, early biological effect and metabolic susceptibility using different biomarkers as mentioned above. Cotinine in urine, as determined by an RIA assay, as a marker of exposure to tobacco smoke and the levels of vitamin A, C, E and folic acid in plasma are measured as possible confounding factors.

Fig. 1 Evaluation of chromosomal aberrations by fluorescent in situ hybridization (FISH) method (reciprocal translocation of chromosome 1).

      In the project "Exposure of model population" (Program AIR POLLUTION AND HEALTH) the impact of air pollution on city policemen working outdoors in downtown Prague for more than 8 h/day (EXP) was studied.Volunteers from a suburban area working indoors were selected as controls (CON). The results of multivariate regression analysis indicate that smoking, vitamin C, polymorphisms of GSTM1, XPD repair gene in exon 23, and epoxide hydrolase gene (EPHX) in exon 4 were significant predictors for total DNA adduct levels. Smoking, exposure to ambient air pollution and polymorphism of XPD repair gene in exon 6 were significant predictors for "like" B[a]P-DNA adduct. Using FISH analysis a significant increase in all cytogenetic endpoints was observed in the EXP group, due to a significant increase in the genomic frequency of translocations in EXP vs. CON groups. The frequencies of chromosomal aberrations determined by FISH were affected by GSTP1, hOGG1 and XPD polymorphisms. Ambient air exposure to PAHs significantly increased FISH cytogenetic endpoints in nonsmoking policemen working in downtown Prague. The project "Atherosclerotic changes and DNA adducts" (IGA MZ - grant NM-10-3) was based on the idea that atherosclerosis and carcinogenesis may share some common mechanisms of the genotoxic action of exogenous compounds such as polycyclic aromatic hydrocarbons (PAHs). The main objective of this study was to test the hypothesis that "bulky" aromatic DNA adducts in the smooth muscle cells (SMC) of thoracic aortas taken at autopsy from sudden and accidental death male subjects are associated with the stage of atherosclerosis. We found significantly higher DNA adduct levels in "Cases" as compared with "Controls". "Cases" also had significantly greater age, elevated heart weight and plasma cholesterol levels, and a higher frequency of overweight subjects as compared to "Controls". Multivariate logistic regression revealed that the "bulky" aromatic DNA adducts, which are most likely related to environmental exposure to genotoxic chemicals, remain a statistically and biologically significant predictor of the stage of atherosclerosis even after adjustment for all other significant predictors in the model. Our results showed that the risk of atherosclerosis is also associated with some of genetic susceptibility markers (GSTT1 and CYP1A1-MspI polymorphisms). The fact that "bulky" aromatic DNA adduct levels predict the progression of atherosclerosis independently of smoking indicates that the formation of atherosclerotic plaques may also be initiated by environmental exposures other than tobacco smoke.
      The DGE has also participated in several EC projects: "Air pollution distribution of adult population in Europe", "Biomarkers of genotoxicity of urban air pollution: A dose-response study", "Biomonitoring of occupationally exposed population to acrylonitrile" and "Effects of PAHs in environmental pollution on exogeneous and endogeneous DNA damage" as well as the HEI project "Transitional epidemiology study for 1,3-butadiene biomarkers".

      Several recent studies suggested that the increased risk of adverse human reproductive effects in the District of Teplice could be related to the high level of air pollution in this region. A population-based project "Pregnancy outcome" was carried out to test this presumption. A long-term project, the major aim of which is to evaluate the impact of urban air pollution on reproduction quality in the capital city of Prague (with heavy traffic pollution), has been recently initiated. The major objective of this project is a comparison of the reproductive effects of air pollution in different regions.
      Exposure is estimated from air pollution monitoring, questionnaire information and the assessment of selected biomarkers. Based on statistical power analysis, low birthweight, premature birth and intrauterine growth retardation (IUGR) were chosen as the main reproductive outcomes. Two basic approaches were combined: In a prospective cohort study of all pregnancies in Teplice and Prachatice, the effect of the simultaneous action of air pollution and many other factors on pregnancy outcomes was analyzed using methods of multivariate logistic regression analysis. The statistical models accounted for possible critical exposure windows for different outcomes. In a nested case control study, a sample of cases (adverse outcomes) and suitable controls were studied. Selected biomarkers were determined in venous blood, cord blood and placenta (DNA adducts assayed by 32P-postlabelling, comet assay, cell immunity markers and levels of vitamins A, E, C and folic acid) with the aim of evaluating the exposure, response and state of mother or child. Metabolic genotypes (GSTM1, GSTT1, NAT2, CYP1A1 and EPHX) were also determined to investigate the effect of genetically determined susceptibility on the level of internal exposure.
      A considerably higher prevalence of all adverse outcomes was found in the highly polluted district of Teplice compared with Prachatice. A significant and dose-related relationship between the risk of fetal growth retardation and the levels of fine particles (PM10, PM2.5) was observed in the Teplice region. No similar association was observed in Prachatice - a region with considerably lower levels of particles. On the other hand, a strong dose-related association between the risk of IUGR and the concentration of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) was found in both Teplice and Prachatice.

Fig. 2: Intrauterine growth retardation (IUGR) by carcinogenic PAHs in Teplice. Adjusted Odds Ratios for IUGR were significantly increased for medium and high concentrations of c-PAHs during the first month of gestation. Concentration intervals for c-PAHs were:
Low = < 15 ng/m3 (reference level), Med = 15 to 30ng/m3 and
High = 30 ng/m3 or higher.

      PAHs are mostly adsorbed on the surfaces of fine particles. Therefore, based on the above results, it was hypothesized that c-PAHs, rather than particles, adversely affect fetal growth. Carcinogenic PAHs are known to inhibit epidermal growth factor receptors and to alter early trophoblast proliferation. In this way the fetoplacental exchange of oxygen and nutrients may be reduced to a sufficient extent to affect fetal growth.
      The results concerning the impact of air pollution on pregnancy outcome for the first time revealed that air pollution may influence human reproductive processes, such as fetal growth or fecundability. The induced changes may include not only morphologic birth defects but also some subtle functional changes affecting their carriers throughout their lives. Children are thought to be very sensitive to xenobiotics from conception. Their development is complex and is influenced by various environmental factors as well as by their mother´s deleterious lifestyle such as cigarette smoking or poor diet.

      A study of the morbidity of children aged between 0-3 years has been carried out in the families enrolled in the Pregnancy Outcome Study. Immune biomarkers in cord and maternal blood were determined in study participants at delivery. Comparisons were made between children living in the district Teplice, known for its high air pollution, and children living in Prachatice, a district with consistently lower particulate and SO2 exposures. Children born in Teplice experienced a significantly higher rate of otitis media (and otalgia), gastrointestinal infections, upper respiratory infections, and pneumonia, but did not differ in their risk of bronchitis or viral infections such as varicella. These findings remained significant even when multiple linear regression models accounted for education, maternal age, maternal smoking, and other smokers in the household, breastfeeding, and attendance at day care. The results suggest that air pollution may alter early childhood susceptibility to infections. This stage of the study was supported by the Ministry of Environment of the Czech Republic in the period 1997-1999. The study now continues (2001-2004) under the sponsorship of the Health Effects Institute (Boston, MA) to follow-up children born in 1997-1999.

      These studies are concerned with the investigation of the DNA binding activities of single carcinogenic chemicals, their environmental mixtures, and different chemical compounds associated with PM10 air particles. DNA adduct formation is studied using an acelullar system, human diploid lung fibroblasts, NCI-H322 and Hep G2 cell lines with the aim to better understand the mechanism of action of these complex mixtures. Western blotting and ELISA are used to examine the induction of p53 and p21WAF1 protein levels following treatment of these cells with PAH compounds and environmental mixtures. Both these proteins are involved in cell cycle control; cell cycle alteration has been studied as well.

      The significance of the genotoxic effects of xenobiotics during the embryonic period of life is analyzed using a chick embryo model. DNA and chromosome damage is related to (i) the kinetics of the tested compound in embryonic and extraembryonic tissues , (ii) cell proliferation and cell death and (iii) final morphological and functional development of particular morphogenetic systems and the whole embryo. Special attention is devoted to hematopoietic cells and the embryonic kidney. Image analysis is used to quantify the differences between control and exposed individuals.

Fig.3 Erythroid cells of the chick embryo exhibiting a different degree of nuclear DNA fragmentation.
(a) healthy cell - all DNA in the nucleus ("head of comet"),
(b) cell with the low level of DNA damage (DNA fragments in the "tail of comet"),
(c) cell with the high degree of DNA damage (minimum of DNA in the "head of comet").

      Apoptosis represents a mechanism to eliminate mutant cells from an organism. Using single cell gel electrophoresis, the extent of apoptotic DNA fragmentation is measured in bone marrow aspirates of patients with myelodysplastic syndromes with the aims of (i) precise diagnosis, (ii) a comparison of the impairment of particular cell lineages, (iii) a follow-up of disease progression and the efficiency of therapy. Besides DNA fragmentation, membrane markers of cell differentiation and cell death, involving mapping the levels of sialylation of membrane glycoproteins, are investigated.
      The role of apoptosis in the development of genetically determined male sterility is studied in rats with the Hd mutation. The levels of apoptosis in particular stages of spermatogenesis in control and mutant rats are correlated with histological findings.