Grażyna Motykiewicz

Modulation of DNA adduct levels in human mononuclear white blood cells and granulocytes by CYP1A1, CYP2D6 and GSTM1 genetic polymorphisms

The CYP1A1, CYP2D6 and GSTM1 genes encode biotransforming enzymes involved in activation and detoxification of xenobiotics. Metabolically activated chemical compounds may interact with DNA and form adducts. In this study, the effect of the GSTM1, CYP1A1 exon 7 and CYP2D6 polymorphisms on DNA adduct levels was studied in 170 healthy volunteers. DNA adducts levels were measured by 32P-postlabelling in mononuclear white blood cells (WBC, lymphocytes and monocytes) and granulocytes collected in summer and winter. The influence of the genotype on the level of DNA adducts in both types of WBCs was observed only in summer samples. Individuals with GSTM1 deficient (null) genotype had significantly elevated level of adducts in mononuclear WBCs (p = 0.045) and granulocytes (p = 0.031) compared to GSTM1 positives. Higher adduct levels in carriers of combined GSTM1(null)/CYP1A1-Ile/Val genotype were found in both types of WBCs when compared to GSTM1(+)/CYP1A1-Ile/Ile genotype carriers (p = 0.046 in granulocytes, p = 0.092 in mononuclear WBCs). CYP2D6 wild-type homozygotes (EMs) and heterozygotes (HEMs) were shown to have significantly higher mononuclear WBC DNA adduct levels than mutant homozygotes (PMs) (p = 0.037 and p = 0.014). When confounding factors associated with PAH exposure were taken into account a statistically significant effect of CYP1A1 exon 7 polymorphism on DNA adduct levels was found (p = 0.012 in mononuclear WBCs, p = 0.043 in granulocytes). In a subgroup of current smokers (n = 95) high DNA adduct levels in granulocytes were associated with GSTM1(null) genotype, and increased adduct levels in mononuclear WBCs correlated with CYP2D6 EM and HEM genotypes. In winter samples the association between the genotype and DNA adduct levels was not observed.

A molecular epidemiology study in women from Upper Silesia, Poland

The present report is a follow-up to our previous molecular epidemiology studies on DNA damage in residents of the industrial region of Upper Silesia. The study was designed to focus on environmental exposure to airborne pollutants; other exposures or confounding factors (e.g. smoking status and age) were eliminated. A Silesian population consisting of 67 donors was compared to 72 inhabitants of a less polluted but similarly urbanized area, surrounded by a rural part of Poland. In both regions the donors were non-smoking females with similar age range, and occupation. Eight biomarkers including urinary mutagenicity and 1-hydroxypyrene, polycylic aromatic hydrocarbon PAH-DNA adducts in oral mucosa, sister chromatid exchanges (SCE), high frequency cells (HFC), chromosomal aberrations (CA), and sensitivity to bleomycin in lymphocytes as well as glutathione s-transferase (GSTM1)/cytochrome P4501A1 (CYP1A1) genotypes were evaluated in samples collected in summer and winter seasons. All the biomarkers of internal and biological doses of mutagens and their early biologic effects indicated statistically significant increases in the Silesian group when compared to the controls. Immunohistochemical quantitation of PAH-DNA adducts additionally revealed significant seasonal changes in the levels of adducts. No influence of susceptibility genotypes (GSTM1 and CYP1A1) on biomarker levels was observed.

A cytogenetic study of men environmentally and occupationally exposed to airborne pollutants

The level of sister-chromatid exchanges (SCE), high-frequency cells (HFC), chromosomal aberrations (CA) as well as the proliferation rate index (PRI) were measured in peripheral blood lymphocytes from three groups of volunteers. The environmentally exposed donors were residents from the vicinity of a coke factory; the occupationally exposed persons were cokery workers, while rural region inhabitants served as a control group. Compared with the control group, statistically significant increases of SCE and HFC, as well as decreased cell kinetics (PRI) were observed for both occupationally and environmentally exposed groups. The effect was especially pronounced when only smokers were taken into account. A statistically significant increase of CA was observed in the environmentally exposed group when CA including gaps (CA + G) were evaluated. The proportion of HFC was found to be the most sensitive method to detect genetic effects on the tested human population. This study demonstrates the usefulness of all 4 biomarkers (SCE, HFC, CA and PRI) in monitoring populations exposed to ambient pollution and clearly indicates effects from residential as well as occupational exposure to industrial air pollutants.

Influence of airborne suspended matter on mitotic cell division.

The effect of organic extracts of airborne suspended matter collected in the highly polluted industrial region of Silesia (Poland) on mitotic cell division was evaluated in the Chinese hamster V79 cell line. Crude benzene extracts as well as sequential elution solvent chromatography (SESC) fractions were investigated for their ability to affect the mitotic index, the proportion of anaphases-telophases to metaphases (AT/M ratio), the cloning efficiency and to produce aneuploid cells. The incidence of cell division disturbances in V79 cells exposed to extracts increased in a concentration-dependent manner. Mitotic arrest, manifested as a highly increased mitotic index and a concomitant decrease in the AT/M ratio, was found for the crude extract at a dose corresponding to 0.75 m3 of air. Comparable effects were noticed for SESC fraction 4, probably containing monophenol compounds. A strong dose-dependent reduction of cloning efficiency of V79 cells demonstrated cytotoxic activity of both the crude extract and fraction 4.

Sister chromatid exchanges and high-frequency cells in men environmentally and occupationally exposed to ambient air pollutants: an intergroup comparison with respect to seasonal changes and smoking habit

Sister chromatid exchanges (SCE) and high-frequency cells (HFC) were measured in peripheral blood lymphocytes from men environmentally and occupationally exposed to a mixture of ambient air pollutants. The environmentally exposed individuals were inhabitants of the industrial region of Upper Silesia; those occupationally exposed were Silesian cokery or steel plant workers, while the control group consisted of rural region residents. A total of 147 males were enrolled in the study. Blood samples were collected in winter (February) and summer (September) seasons. Three major areas were investigated during the study: exposure-based dose dependency, seasonal changes, and influence of smoking habits on the SCE frequencies. The latter is frequently reported as a confounding factor in SCE analyses. In both winter and summer samples, statistically significant increases of SCE were observed in the environmentally and occupationally exposed groups compared to the controls (p < 0.001). The difference between both exposed groups was also significant (p < 0.001). An intergroup comparison was based on ANOVA after adjustment for smoking status. In all three groups of interest, a seasonal variation was found with higher levels in winter. However, in a part of the study in which each donor served as his own control, statistical differences were only found within the exposed groups. Control region inhabitants did not have significantly higher frequencies of SCE in winter, compared to summer samples. The impact of two major confounders, age of the donor and smoking habit, was investigated by multiple regression analysis. Smoking was a major factor influencing the level of SCE. Nevertheless, the effect was seen in winter samples only, which suggests an additive response and adds new information to this known effect.

The effect of the genetic polymorphisms of CYP1A1,CYP2D6, GSTM1 and GSTP1 on aromatic DNA adduct levels in the population of healthy women

Aromatic DNA adduct levels and polymorphisms of two phase I enzymes - CYP1A1 and CYP2D6 and two phase II enzymes - GSTM1 and GSTP1 were analyzed in a group of 133 nonsmoking healthy women 35-45 years old and holding jobs not connected with the exposure to the combustion products of organic matter. They were office workers from the south and north-eastern parts of Poland. Blood samples were collected in winter and in summer. Aromatic DNA adduct levels were measured in all winter and summer samples. The frequencies of CYP1A1, CYP2D6, GSTM1 and GSTP1 polymorphisms in samples from the studied women did not show any differences when compared with other Caucasian populations and the Polish male population studied previously. The differences in the levels of DNA adducts among the carriers of different genotypes were statistically non-significant. Analysis of combined genotypes selected the groups of volunteers with the highest and the lowest DNA adduct levels. The highest levels of DNA adducts were observed in the carriers of GSTM1(null)/CYP1A1Ile/Val (8.00+/-13.00 adducts/10(8) nucleotides in summer samples) and GSTP1-AA/CYP1A1Ile/Val genotypes (7.00+/-4.32 in winter and 7.30+/-7. 27/10(8) nucleotides in summer). The lowest levels of DNA adducts (3. 00+/-2.30 in winter and 2.00+/-3.16/10(8) nucleotides in summer) were found in the carriers of the genotype GSTP1-AG+GG/CYP1A1Ile/Val. The levels of DNA adducts in these groups were determined by the polymorphisms of GSTM1 and GSTP1 phase II detoxifying enzymes.

Seasonal variations in mutagenic activity of air pollutants at an industrial district of Silesia

Organic material from airborne particulate pollutants collected over a 7-month period at a highly industrialized region in Silesia (Poland) was tested for mutagenicity using the Ames test. Sequential elution solvent chromatography (SESC) was used for the separation of crude benzene extracts. Five out of 8 fractions showed mutagenic activity with differential direct and indirect responses. The mutagenicity of each active fraction was tested during the whole sampling period (from August to February 1984/1985) and seasonal variations were observed. All of the fractions, except fraction 3, showed only quantitative distinctions in mutagenic potential, expressed as a number of revertants per m3 of air. Over a period of 7 months, a steady increase of activity of fractions 2 and 4 was observed but the type of mutagenic response, indirect and direct respectively, remained unchanged in the summer and winter months. Fraction 3 (the most abundant component, probably containing polar derivatives of PAHs and heterocyclics) differed quantitatively and qualitatively between summer and winter time. From August to December samples showed enhanced mutagenic potency upon addition of rat liver microsomal enzymes, whereas in January a 4-5-fold increase in direct response was noted. This significant increase in direct mutagenic activity was accompanied by a considerable decrease in mean air temperature and resulted most probably from the intensive use of coal for domestic heating.

Measurement of cytogenetic endpoints in women environmentally exposed to air pollution.

The levels of sister chromatid exchanges (SCE), high-frequency cells (HFC) and chromosomal aberrations (CA) were studied in lymphocytes of Silesian women environmentally exposed to ambient air pollutants. Inhabitants of a less polluted but similarly urbanized area, in a rural region of Poland, served as controls. The study population was selected to minimize the major confounding factors influencing SCE and CA. These factors include age, gender, smoking status, and occupation. All donors were 35-46 years old non-smoking City Hall clerks. The levels of all three biomarkers were significantly higher in the exposed group than in controls as analyzed by the Mann-Whitney U-test. No correlation was found between levels of CA and SCE. Additional possible confounders, such as passive smoking, ex-smoking and X-ray chest examination did not influence the levels of biomarkers. This study builds upon our previous research in a male population but better controls for confounders. Thus, the results reveal genetic damage resulting from low-dose but chronic environmental exposure.

Bleomycin sensitivity test in the exposed and reference human populations.

Sensitivity to bleomycin was investigated in lymphocytes collected from three groups of males: 30 occupationally exposed cokery workers, 38 environmentally exposed Silesian citizen and 35 rural inhabitants. The data were analyzed at both the individual and group levels. The first analysis has revealed a substantial interindividual variability in the level of generated breaks (breaks per cell, b/c). This variability was independent of the age of the donor, smoking habit and X-ray exposure as tested in the multiple regression model. The means per group for the occupationally and environmentally exposed persons were almost the same with the values of 0.674 and 0.639, respectively. These two groups differed significantly from the rural population (b/c=0.448, p<0.001 by MANOVA). The reproducibility of the assay was satisfying (p>0.49 by the Wilcoxon matched paired test) after omitting 7 out of 49 repeatedly sampled donors. Those persons exhibited extremely high b/c rates in the first sampling.

Assessment of cancer hazard from environmental pollution in Silesia

New concepts of cancer risk estimation have been developed during the past decade. Short-term bioassays dealing with mutagenicity and carcinogenicity of environmental samples are being replaced by more relevant molecular epidemiology studies. The general idea of using a battery of bioassays remains unchanged while the origin of tested samples is different. Instead of testing samples collected from the environment, body fluids or human cells from exposed populations are under investigation. This paper reviews the collaborative study on cancer risk assessment in highly polluted industrial region of Silesia in which both approaches had been employed during the 1985-1995 period. A potent carcinogenic activity of airborne pollutants was indicated in a battery of in vitro and in vivo short-term assays. These studies were followed by the molecular epidemiology study performed on human populations inhabiting the region of Silesia. An elevated damage of genetic material on the chromosome and/or DNA levels was observed in the Silesian populations as compared with proper rural controls.