Adverse reproductive outcomes from exposure to environmental mutagens

Mutation Research 428 Ž1999. 203–215 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres

Adverse reproductive outcomes from exposure to environmental mutagens ˇ´ Radim J. Sram a

a,)

b , Blanka Binkova´ a , Pavel Rossner , Jirı ¨ ˇ´ Rubesˇ c , Jan Topinka a, a Jan Dejmek

Laboratory of Genetic Ecotoxicology, Regional Institute of Hygiene of Central Bohemia, c r o Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vıdenska ´ ´ 1083, 142 20 Prague 4, Czech Republic b National Institute of Public Health, Prague, Czech Republic c Veterinary Research Institute, Brno, Czech Republic Received 29 November 1998; accepted 20 February 1999

Abstract The effect of environmental pollution on reproductive outcomes has been studied in the research project ‘Teplice Program’ analyzing the impact of air pollution on human health. Genotoxicity of urban air particles - 10 mm ŽPM10. in in vitro system was determined by the analysis of DNA adducts. The highest DNA binding activity was observed in aromatic fraction, identifying DNA adducts of carcinogenic polycyclic aromatic hydrocarbons ŽPAHs. presumably diolepoxide-derived from: 9-hydroxybenzow axpyrene Ž9-OH-Bw axP., benzow axpyrene-r-7,-dihydrodiol-t-9,10-epoxidewqx Žanti-BPDE., benzow b xfluoranthene ŽBw b xF., chrysene ŽCHRY., benzw axantracene ŽBw axA., indenow1,2,3-cd xpyrene ŽIw cd xP.. Reproductive studies were conducted in both females and males. A study of the effects of PM10 exposure on pregnancy outcomes found the relationship between the intrauterine growth retardation ŽIUGR. and PM10 levels over 40 mgrm3 in the first gestational month ŽOdds Ratio for 40–50 mgrm3 s 1.6, ) 50 mgrm3 s 1.9.. Selected biomarkers were analyzed in venous blood, cord blood Žchromosomal aberrations, comet assay. and placenta ŽDNA adducts, genetic polymorphisms of GSTM1 and NAT2 genotypes. of women enrolled in a nested case-control study. DNA adduct levels were higher in polluted vs. control districts, in smoking vs. nonsmoking mothers, and in GSTM1 null genotype, which was more pronounced in polluted district. No effect of air pollution was observed by cytogenetic analysis of chromosomal aberrations or by comet assay. The

AbbreÕiations: AB.C., aberrant cells; Anti-BPDE, benzow axpyrene-r-7,t-8-dihydrodiol-t-9,10-epoxidew"x; Bw axA, benzw axanthracene; Bw ab xF, Bw aj xF, Bw k xF, benzow b,j,k xfluoranthenes; Bw ghi xP, benzow ghi xperylene; Bw axP, benzow axpyrene; CHEST, chick embryotoxicity screening test; DBw ah xA, dibenzw ah xanthracene; ED50, dose inducing in 50% of exposed embryos malformation andror death; FISH, fluorescence in situ hybridization; GC-MS, gas chromatography-mass spectrometry; GSTM1, glutathione-S-transferase M1; HiVol air sampler, high volume air sampler; HPLC, high pressure liquid chromatography; 9-OH-Bw axP, 9-hydroxybenzow axpyrene; IUGR, intrauterine growth retardation; Iw cd xP, indenow1,2,3-cd xpyrene; LBW, low birth weight; NAT2, N-acetyltransferase 2; NO x , nitrogen oxides; O.R., Odds Ratio; PAHs, polycyclic aromatic hydrocarbons; PCR, polymerase chain reaction; PM2.5, air particles - 2.5 mm; PM10, air particles - 10 mm; RFLP, restriction fragment length polymorphism; SO 2 , sulfur dioxide ) Corresponding author. Tel.: q42-02-475-2596; Fax: q42-02-475-2785; E-mail: [email protected] 0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 9 9 . 0 0 0 4 8 - 4

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reproductive development of young men was followed by measures of semen quality, adjusted for ambient SO 2 exposure. The analysis identified significant associations with air pollution for - 13% morphologically normal sperm, - 29% sperm with normal head shape, - 24% motile sperm. Analysis of aneuploidy in human sperm by FISH showed, aneuploidy YY8 was associated with season of heaviest air pollution. These findings are suggestive for an influence of air pollution on YY8 disomy. All these results indicate that air pollution may increase DNA damage in human population, which may be even higher for susceptible groups. Biomarkers of exposure ŽDNA adducts. and susceptibility ŽGSTM1 and NAT2. may indicate the risk of presumable low environmental exposure. Pregnancy outcome and semen studies imply that relatively low air pollution Žhigher than 40 mg PM10rm3 . can significantly increase the adverse reproductive outcomes affecting both genders. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Air pollution; Pregnancy outcome; Sperm morphology; PM10; Polycyclic aromatic hydrocarbon; DNA adduct; GSTM1 and NAT2 genotypes

1. Introduction The Northern Bohemia brown coal basin was perceived as one of the worst environmentally polluted regions in Europe. Conifers in Krusne ˇ ´ Hory ŽOre Mountains. forming the northern border of this region have been essentially destroyed. This process started more than 20 years ago. As first consequences of environmental pollution on the health were remarkable increase of allergies, immunodeficiencies and respiratory diseases in children. Unfavorable effect of environment on pregnant women was understood as the reason for the increase of birth

defects and higher number of children with low birth weight. Exploratory analysis of data collected prior to 1999 suggested a higher incidence of cancer and reproductive and behavioral effects in this region w1,2x. The Northern Bohemia brown coal basin comprises several mining districts. The coal in this region is of low quality with high sulfur content, and is from surface-mines with open pits. It is primarily used to produce steam and power for this heavy industrialized region. Coal-fire power plants produced 50% of the electricity used in the Czech Republic. After major political changes in the Czech

Fig. 1. Czech Republic, Districts of Teplice and Prachatice.

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Republic in November 1989, a new research program, the Teplice Program, was developed to evaluate the short-term and long-term health impact of air pollution on the population. Teplice, one of the mining districts in Northern Bohemia, was designated as a model district for investigation of the health effects of air pollution. The district of Prachatice, an agricultural area without heavy industry, was selected as a control district ŽFig. 1. w3x. The Teplice Program was initiated by the Czech Ministry of Environment in 1990 to provide scientifically valid information needed to assess environmental health problems in the Northern Bohemia basin area. In collaboration with the U.S. Environmental Protection Agency ŽUS EPA., the program started in 1991. Simultaneously, this program was incorporated into PHARE II, as ECrHEAr18-CZ project ‘Impact of Environmental Pollution on the Health of Population ŽTeplice Program.’. This program has succeeded in bringing together many different research organization and government laboratories in the Czech Republic, United States and EC countries to accomplish the multidisciplinary program. The hypothesis in the Teplice Program has been that the air pollution in the Teplice district adversely affects the health of population. The principal objective of this program was to assess human exposure to toxic air pollutants, to relate ambient concentrations of pollutants to health risks. First studies indicated higher level of carcinogenic PAHs, responsible for an increased mutagenicity of ambient air w4x as well as DNA adducts in women working outdoor w5,6x. The Teplice Program was originally composed from 25 different projects. We review here the results of adverse reproductive outcomes from exposure to environmental mutagens.

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aromatic hydrocarbons ŽPAHs., toxic metals, sulfates and crustal elements w7x. These data were used to characterize the ambient exposures of population to air pollutants for the health studies. The ambient monitoring and source characterization data were used in receptor modeling calculations, the results of which indicate that residential space heating and power plant emissions accounted for most of respirable particle mass concentrations. The source of pollution from residential heating was two to three times more significant than from power plants. Mobile sources account for the total mass of PM2.5 more in summer than in winter. Stevens et al. w8x concluded: ‘‘ . . . The extremely high concentrations of sulfur dioxide and particulate matter Žespecially respirable particles. measured in the Czech Republic in these studies may pose a health risk to people living in the region.’’ During the last eight years, the concentrations of SO 2 were substantially decreased, and they are now approximately 20% of those seen in the winter 1989. Surprisingly, the concentrations of PM 10 did not

2. Air pollution concentrations and source contributions Air pollution monitoring was carried out continuously during this study at both regions, in Teplice and Prachatice cities. The daily concentrations of the following pollutants were collected: sulfur dioxide ŽSO 2 ., nitrogen oxides ŽNO x ., particles - 10 mm ŽPM10., particles - 2.5 mm ŽPM2.5., polycyclic

Fig. 2. Seasonal concentrations of PM10 in the districts of Teplice and Prachatice, period 1993–1998.

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substantially change in the period 1994–1998 ŽFig. 2..

3. Genotoxicity and embryotoxicity of urban air particulate matter In order to assess a possible health risk associated with a complex mixture of hundreds of organic compounds adsorbed to air particles we used the biomarker-directed fractionated procedure to evaluate biological activities of different chemical compound classes w9x. Urban air particles PM10 were collected using an Anderson Hi-Vol air sampler in both Teplice and Prachatice during the winter ŽOctober–March. and summer ŽApril–September. seasons in the years 1993–1994. Particulate organic matters were extracted by dichloromethane and crude extracts were fractionated according to the procedure of Lewtas et al. w10x by acid-base partitioning into organic bases, acids and neutral fractions. The neutral organic fractions were subsequently fractionated into five subfractions according to increasing polarity by silica gel column chromatography. The principal aim of this study was to compare the DNA binding activities of the different classes of compounds using an in vitro acellular assay coupled with 32 P-postlabeling and an embryotoxicity assay using Chick Embryotoxicity Screening Test ŽCHEST. w11x. DNA adducts were determined by 32 P-postlabeling analysis using nuclease P1 and butanol extraction procedures. Qualitative measurement of 32 P-postlabeled DNA adducts was determined by high pressure liquid chromatography ŽHPLC. to identify some of the major DNA adducts originated from the in vitro assay. Using CHEST, embryotoxicity was defined as the sum of dead and malformed embryos and expressed Žcalculated. as ED 50 Ža dose inducing in 50% of exposed embryos malformation andror death. w11x. Both the assays evaluating DNA binding activities and embryotoxicity showed that the different organic compound classes behaved qualitatively similarly. For both assays, the highest toxicity was observed for the neutral fractions from which the aromatic subfractions were the most toxic for both the localities and seasons. These subfractions contained mainly PAHs and their methyl-derivatives as was confirmed

by GC-MS analysis. These results are in agreement with the other studies showing that PAHs account for most of the mutagenic activity of neutral fraction of urban air w12,13x. A good correlation between DNA adduct levels formed in the presence of the S9 metabolic activation system and the ED 50 was observed for all of the different complex mixtures of organic compounds tested Ž r s 0.773, p - 0.001.. The major adduct spots were analyzed by HPLC analysis of 32 P-labeled DNA adducts. DNA adduct maps and HPLC profiles were similar for the samples from both the districts and seasons. The major DNA adducts resulting from the crude extracts were identical to those derived from the aromatic fractions. We tentatively identified presumably diolepoxide-derived adducts from: 9-OH-benzow axpyrene Ž9OH-Bw a xP., benzow a xpyrene-r-7,t-8-dihydrodiol-t9,10-epoxidewqx Žanti-BPDE., benzow b xfluoranthene ŽBw b xF., benzow j xfluoranthene ŽBw j xF., benzow k xfluoranthene ŽBw k xF., chrysene ŽCHRY., benzow axanthracene ŽBw axA., and indenow1,2,3-cd xpyrene ŽIw cd xP.. The results confirmed the similarities of the major ubiquitous emission sources of organic compounds in both districts, which are presumably residential home heating and motor vehicles in winter seasons and motor vehicles in the summer period.

4. Pregnancy outcome The main aim of this study was to evaluate the impact of air pollution and lifestyle variables on all hospitalized pregnancies in two districts ŽTeplice and Prachatice. using biomarkers as a measure of exposure. The hypothesis was that the pregnancy outcomes would be generally worse in the district with higher pollution level ŽTeplice.. Exposure was estimated by air pollution monitoring and modeling, questionnaire information and selected biomarkers. Based on the power analysis, the main reproductive effects were chosen, namely, low birth weight Žbelow 2500 g., premature births Žbelow 37 weeks., and intrauterine growth retardation Žsmall for gestational age. w14x. Two basic approaches were combined: Ža. In the prospective cohort study, all hospitalized pregnancies terminated in Teplice and Prachatice districts during a 2-year period Ž1994–1996.

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were involved. Personal and lifestyle data were obtained via self-administered questionnaires, together with information about reproductive history, work and environmental exposures, health status, diseases, and medications. Žb. Selected biomarkers were analyzed in venous blood, cord blood, and placentas of the women enrolled in a nested case-control study. Women with pregnancies less than 37 weeks or with babies weighing less than 2500 g were classified as cases. An equal number of women giving birth to babies with normal parameters were selected systematically from the whole cohort as controls Žeach fifth noncase birth was selected.. Biomarkers such as DNA adducts, chromosomal aberrations, comet assay, cell and humoral immunity markers, and vitamins A, E, and C were analyzed in blood and placenta obtained from cases and controls. Metabolic genotypes ŽGSTM1, and NAT2. were determined. Preliminary analysis of the data collected during the first 18 months of the study resulted in two general observations: Ž1. Air pollution level in the polluted region Žnamely SO 2 , PM10, PAHs. was highest during winter and lower during summer, as measured by continuous air monitoring. Ž2. The prevalence of low birth weight ŽLBW. and intrauterine growth retardation ŽIUGR. was

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greater for infants conceived during winter months than for those conceived in summer ŽFig. 3.. These preliminary observations were consistent with recent hypotheses for the etiology of IUGR, that initial changes leading to fetal growth retardation may be triggered in very early pregnancy, around the time of implantation w15,16x. The study group included all singleton full-term births of European origin in Teplice district. Information on reproductive history, health and lifestyle was obtained from maternal questionnaire. The mean concentration of PM10 for each month of gestation was calculated using continuous monitoring data. Each mother’s PM10 level was estimated using averages for each of nine consecutive 30-day periods after the estimated date of conception ŽEDC.; these periods correspond roughly to the nine months of gestation. The PM10 data were divided into three categories for analysis: low ŽL s- 40 mgrm3 ., medium ŽM s 40 to - 50 mgrm3 . and high ŽH s) 50 mgrm3 .. Each month of pregnancy was analyzed separately, allowing some factors to vary over time Že.g., particulate matter, maternal smoking and alcohol consumption, and season.. Thirty-day averages of PM10, calculated for nine time periods for each pregnancy, varied between 29 and 86 mgrm3. The values were highest in winter due to inversions and

Fig. 3. Prevalence of adverse outcomes in pregnancies conceived during summer and winter periods in the district of Teplice. LBWs low birth weight, IUGR s intrauterine growth retardation.

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the use of coal for heating, and lowest in summer months. The correlation between season and particulate matter levels was significant, ranging between 0.49–0.60 Ž p - 0.01. for monthly average PM10. The study included 1943 women who gave birth to infants between the 37th and 43rd gestational weeks in the period 1994–1996. A total of 9.8% infants in the study were below the 10th percentile of birth weight for gestational age. Many potentially confounding factors were included into transitional logistic models. The final model contains maternal height and pre-pregnancy weight, parity, marital status, education and maternal smoking, season and the year of the study. Adjusted Odds Ratios ŽOR. and their 95% confidence intervals were estimated using Proc logistic w17x. Evaluating crude ORs, we observed the effect of air pollution on IUGR at medium and high PM10 levels in the first month of pregnancy ŽFig. 4.. After adjustment, both the medium and high exposure were statistically significant for PM10: the medium level OR was 1.62 ŽCI: 1.07–2.46. and the OR for

the high level was 2.64 ŽCI: 1.48–4.71.. Though PM10 levels were lower in Prachatice than in Teplice, a similar significant association between IUGR and PM10 level in the first month of pregnancy was observed. These data suggest that exposure to particulate matter Žor an associated air pollutants. early in pregnancy may adversely affect fetal growth. Regardless of which particular toxicant associated with particulate matter could affect fetal growth, the biological mechanisms remain to be explained. The effective components would need to be inhaled and absorbed into the maternal bloodstream. Highly biologically active compounds Že.g., PAHs. might interfere with some processes affecting development or nutrition of the fetus. Effects during the first month of gestation were observed for monthly average concentrations of PM10 above 40 mgrm3. These levels are also observed in other industrialized parts of the Czech Republic w18x and other countries w19x. Therefore, potential adverse effects of pollutants may be relevant for a significant number of births anywhere.

Fig. 4. Adjusted OR of IUGR by PM10 levels during pregnancy.

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Fig. 5. Prevalence of IUGR for PM10 levels in the first month of gestation by maternal smoking and season.

Data on indoorroutdoor exposures from the Teplice region suggest that PM10 levels in nonsmokers’ homes are approximately 50–80% of outdoor PM10 levels. Concentrations of PM10 in smokers’ homes are substantially higher and therefore less affected by outdoor levels. Therefore, it is important to control for active and passive smoking in this type of studies. Active smoking of the mother and her exposure to the environmental tobacco smoke substantially increases the IUGR among their children ŽFig. 5..

5. Biomarkers and pregnancy The impact of air pollution on pregnancy outcome was analyzed by several biomarkers in venous blood of mothers, cord blood and placenta of the women enrolled in a nested case-control study.

the course of pregnancy, mothers in the course of delivery and in newborns cord blood. The increase of chromosomal aberrations in the Teplice district compared to Prachatice district in pregnant mothers was observed ŽTable 1.. No differences between the groups of mothers Žsampled during delivery. as well as groups of newborn children were found. The frequency of aberrant cells in newborns from both districts corresponds to spontaneous level of chromosomal aberrations in peripheral blood lymphocytes of newborns in the Czech Republic. The level of chromosomal aberrations in mothers in both the regions were significantly lower than the frequency of aberrant cells for the same age group, which was 1.59% AB.C. w20x. It seems that exposure to air pollutants may have more effect on chromosomes of mothers during pregnancy than at the end of pregnancy and in the newborn.

5.1. Chromosomal aberrations

5.2. Comet assay

In the period 1992–1994, the level of chromosomal aberrations was analyzed in pregnant mothers in

Comet assay Žthe single cell gel electrophoresis assay. was selected as a biomarker of exposure to

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Table 1 Cytogenetic analysis of peripheral lymphocytes in pregnant women and cord blood, period 1992–1994 District

Pregnant a N

Teplice Prachatice

131 48

Mothersb % AB.C. U

1.54 1.04

c

BrC

Cord blood

d

N

% AB.C.

BrC

N

% AB.C.

BrC

U

54 20

1.22 1.05

0.016 0.012

86 29

0.89 1.00

0.010 0.011

0.017 0.012

U

p - 0.05. Pregnant—samples during pregnancy. b Mothers—samples in the course of delivery. c AB.C.—aberrant cells. d BrC—breaks per cell. Cultivation 48 h, analyzed 100 metaphasesrsubject. a

evaluate the impact of air pollution and lifestyle variables on hospitalized pregnancies in both the districts. We supposed that the DNA damage detected as single strand breaks would be generally higher in the district with higher pollution. A total of 322 pregnancies in Teplice and 220 in Prachatice were enrolled w21x. Venous and cord blood were analyzed using the alkaline Comet assay procedure with lysis for 60 min, unwinding for 40 min and electrophoresis for 24 min. The modified procedure with unwinding prolonged to 60 min and electrophoresis to 40 min was used later. Peripheral white blood cells were analyzed using an image analyzer system. No differences for Comet parameters in the blood of mothers and children between polluted and control districts were found. The prolongation of alkali unwinding and electrophoresis did not increase sensitivity of the assay. No effects of pre-maturity, low birth weight, ethnicity, smoking or GSTM1 polymorphism were observed for any of the Comet parameters. Multiple regression analyses were performed for the European population Ž n s 285.. A statistical model was fitted to determine the relationship between the Comet parameters of mothers and their children, the only ones, which were statistically and biologically significant. The Comet assay was not a particularly sensitive technique in determining the effects of environmental pollution at the DNA level if peripheral white blood cells were used w21x. 5.3. DNA adducts The placenta bulky DNA adducts have been studied in relation to metabolic genotypes for glu-

tathione-S-transferase M1ŽGSTM1. and N-acetyl transferase 2 ŽNAT2., cotinine and vitamins A, C and E in 158 mothers Ž113 nonsmokers and 45 smokers.. DNA adduct levels were determined using the butanol extraction enrichment procedure of 32 Ppostlabeling. GSTM1 and NAT2 genotypes were studied using polymerase chain reaction ŽPCR. and RFLP techniques. All sets of data were analyzed by multiple regression analysis. Using this approach more complete relationship could be observed as compared to descriptive statistics in the original paper w22x. Total DNA adduct levels were significantly higher in the polluted region and in smoking mothers. Higher DNA adduct levels were detected in the group of mothers carrying the GSTM1-null genotype. This finding seems more relevant to subjects living in polluted industrial regions w23x. The biomarker data has been employed for the following reasons: Ža. to test the biomarkers for the prediction of environmental exposure, Žb. to evaluate the effect of metabolic andror nutritional status on DNA adduct levels, and Žc. to test the association between the biomarkers of exposure, susceptibility and pregnancy outcome. For sound valid results, many social, demographic and lifestyle characteristics were controlled Že.g., maternal age, parity, pre-pregnancy weight, education, marital status, active and passive smoking.. Environmental exposures of the mothers during the last three months of gestation were estimated using data of continuously monitoring of PM10, PAHs, and SO 2 . Multiple regression models using stepwise procedure for evaluation of interaction of all possible

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variables was used. DNA adducts as outcomes response variables were utilized. As predictors were applied air pollutants, smoking status, genotypes, antioxidant vitamin levels, and IUGR. Using the stepwise approach the final models contained only covariates which contribute significantly to the model predictivity. Analyzing the effect of carcinogenic PAHs concentrations and vitamin C levels in nonsmoking women, an inverse relationship between vitamin C levels and DNA adduct levels was found Ž b s y0.649, p - 0.05.. The higher DNA adduct levels was observed in nonsmoking mothers delivering children with IUGR Ž b s y0.741, p s 0.01.. The DNA adduct levels was significantly affected by GSTM1 resulting in higher DNA adduct levels in placentas of the GSTM1-null nonsmoking mothers Ž b s y0.513, p - 0.05.. We also at first observed the effect of passive smoking on DNA adducts Ž b s 0.031, p - 0.01.. DNA adducts were also increased by the exposure to carcinogenic PAHs in the last month of pregnancy Ž b s 0.043, p - 0.05.. Summing up these results ŽTable 2., multiple regression analysis indicates that DNA adduct levels in placentas of all women are affected by smoking, NAT2 genotype, vitamin C level, IUGR and education. In the group of smoking women the DNA adduct levels are affected by NAT2 genotype, carcinogenic PAHs in the last month of pregnancy, IUGR, the length of gestation and education. The effect of the education on DNA adduct levels may indicate the role of better knowledge of good diet and healthy lifestyle. The results of multiple regres-

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sion analysis showed that DNA adducts together with analysis of genotypes are sensitive biomarkers of exposure. These biomarkers may be used to find various factors which may affect the development of pregnancy. It is probably relevant to expect Žbelieve. that the DNA adduct levels correspond to the exposure during the last month or, in the best case, during the last three months of pregnancy. A surprising association was observed between DNA adduct levels and IUGR which are anticipated to be induced by PM10 already in the first month of pregnancy w14x. DNA adduct data in placenta are complementary with in vitro DNA binding activity and embryotoxicity studies w11x which proved genotoxic and embryotoxic potential of the organic extracts from the air in the Teplice and Prachatice districts. The complementarity of both results may be used to put forward a hypothesis about the risk factors after modelling genotoxicity of complex mixtures in human cells in vitro. We can propose: - to use the analysis of DNA adducts and identification of specific DNA adducts, - to determine mutations using the analysis of HPRT and p53 with the identification of specific mutation spectra, - to use cytogenetic endpoints as chromosomal aberrations, SCE or micronuclei and determine the sensitivity of cytogenetic endpoints, - to determine genotypes to be studied Žmetabolic polymorphisms..

Table 2 Multiple regression analysis of factors affecting DNA adducts in placenta Group

All

Nonsmokers

Smokers

N R

104 0.5084

77 0.2842

27 0.9530

B

P-level

B

P-level

B

P-level

IUGR Length of gestation Education Smoking Vitamin C NAT2 Carcinogenic PAHs Žin ninth month of pregnancy.

y0.971

0.0004

y1.115

0.0079

y0.153 0.704 y0.753 y0.453

0.0374 0.0006 0.0047 0.0026

y0.889 0.198 y1.103

0.0015 0.0001 0.0001

y0.323 y0.240

0.0019 0.0202

y0.560 y0.371

0.0021 0.0012

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The results of in vitro studies might help us to formulate hypotheses about risk factors in selecting the population to study as an exposed and control groups and help us in the selection of validated and sensitive biomarkers. The availability of extensive exposure measurements is critical to molecular epidemiology studies. However, it is very difficult to establish monitoring of all relevant pollutants on a routine basis as it is expensive and very laborious. Still for the future, it should be the basic requirement for molecular epidemiology studies. It seems that to realize proper molecular epidemiology studies with a broad spectrum of the biomarkers of exposure, effects and susceptibility, only the international collaboration of many laboratories could be successful.

6. Semen quality This study examined whether exposure to high levels of air pollution over the entire process of spermatogenesis Žabout 90 days. is associated with abnormal semen parameters w24x. Certain components of air pollution, e.g., PAHs, have altered male reproductive function in test species providing additional rationale for the undertaking w25,26x. In the overall study, 325 young men from both communities, Teplice and Prachatice were examined. Surveys in the fall and late winter included interviews, physi-

cal examinations and collection of semen samples. Semen quality measures included concentration, volume, motility and morphology, computer-aided motion analysis and sperm chromatin structure assay. Men were classified into exposure groups using relative levels of ambient sulfur dioxide. Semen was collected on site, measures included sperm concentrations and volume, percentage of motile sperm, percentage of sperm with normal morphology and percentage with normal head morphology. For the morphology evaluation 200–300 sperms per sample were assessed from air dried preparations and classified into WHO categories w27x. To estimate the relevant exposure scenarios SO 2 data were grouped by district, sampling time, early fall or late winter, year and exposure window, as shown in Table 3. An examination of these exposure windows showed that mean SO 2 values were similar regardless of the exposure window used. There were no significant relationship observed between measures of sperm production Žsemen volumes, sperm concentrations or total sperm count. and air pollution in the logistic regression model. The most notable findings of this report are the significant relationships observed between air pollution and sperm morphology ŽTable 3. with the lower air pollution levels in the late winter in Prachatice with ORs of 0.2 Ž0.1–0.7. and 0.5 Ž0.2–2.0. and increasing with medium levels to an OR of 4.1 Ž1.2–13.9. and high levels to an OR of 10.1 Ž2.8– 36.0. in Teplice. The results were consistent for the

Table 3 Semen outcomes by level of exposure late winter surveys versus all fall surveysa Prachatice

Total sperm count - 40 millionrsample Percent motile - 24% Sperm morphology - 13% normal Sperm head shape - 29% normal a

Teplice

Very low

Low

Medium

High

Late winter 94 Ž n s 48, mean SO 2 s 17.5.

Late winter 93 Ž n s 28, mean SO 2 s 41.5.

Late winter 94 Ž n s 63, mean SO 2 s 79.4.

Late winter 93 Ž n s 47, mean SO 2 s 164.0.

0.6 Ž0.2–1.7. 2.0 Ž0.7–5.5. 0.2 Ž0.1–0.7. 0.5 Ž0.2–1.6.

1.5 Ž0.5–4.5. 2.0 Ž0.6–6.8. 0.5 Ž0.2–2.0. 0.2 Ž0.0–1.2.

1.1 Ž0.5–2.5. 9.8 Ž3.6–27.2. 4.1 Ž1.2–13.9. 6.1 Ž2.0–18.4.

0.7 Ž0.3–1.9. 3.5 Ž1.1–11.5. 10.1 Ž2.8–36.0. 1.4 Ž0.4–5.0.

For all fall: Teplice: Fall, 1992, mean SO 2 s 23.8, n s 36; Fall, 1993, mean SO 2 s 29, n s 44; Prachatice: Fall, 1992, mean SO 2 s 8.9, n s 17; Fall, 1993, mean SO 2 s 6, n s 42. Odds Ratios Ž95% confidence intervals..

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linear regression: sperm morphology, very low b s 3.31 Ž0.12, 6.52., low b s 5.90 Ž1.88, 9.93., medium b s 5.80 Žy8.84, y2.75., p s 0.0002 and high b s y7.25 Žy10.62, y3.89., p s 0.0001. These analyses suggest a strong relationship between sperm morphology and air pollution with a possible seasonal effect in the opposite direction. An increase in abnormal sperm head morphology was also noted with medium wOR s 6.1 Ž2.0–18.4.x but not high exposure wOR s 1.4 Ž0.4–5.0.x, respectively ŽTable 3.. The linear regression was consistent with these findings: medium exposure b s 9.58 Žy13.29, y5.86., p s 0.0001; high b s y3.84 Žy7.95, y0.26., p s 0.07. Thus alteration of sperm head shape may be a significant component of the sperm morphology effect, but does not account for all of it. The main effects associated with air pollution appear to be postmeiotic effects on sperm motility and morphology. Effects on sperm morphology suggest effects during spermiogenesis when the normal spermatids are transformed into differentiated sperm cells. Severe alterations in motility and morphology can be associated with infertility. Accumulating evidence suggests that exposure to environmental pollution may contribute to a decline in sperm counts worldwide w28,29x. In general, these data suggest that exposure to air pollution for one spermatogenic cycle may increase the risk of altered semen quality. This appears to be reversible since the young men evaluated six months after high pollution episodes have improved semen quality. In the cohort of 25 subjects from the Teplice district aneuploidy human sperm by FISH were analyzed w30x. Semen samples were provided by 10 men who reported smoking 20 cigarettesrday, and 15 nonsmokers who reported no more than minimal exposure to passive smoking. Sperm FISH aneuploidy was determined with the use of three probes directly labeled for chromosome X, Y and 8 ŽTable 4.. There were no statistically significant differences between smokers and nonsmokers with regard to occupational exposure, health status, passive smoking, age at first seminal emission and other activities. Smokers produced significantly elevated aggregate frequencies of sperm, aneuploidy for chromosomes X, Y and 8 Ž p - 0.01.. The frequencies of sperm with Y disomy were significantly elevated among

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Table 4 Aneuploidy in human sperm by FISH, effect of smoking w30x Sperm aneuploidy type

Group Nonsmokers Ž ns15.

Smokers Ž ns10.

Disomy X Y X–Y 8

5.8"0.6 2.2"0.3 14.0"1.4 4.7"0.6

5.9"0.8 4.5"0.6U 20.4"4.4 6.5"1.0

Diploidy X–X–8–8 Y–Y–8–8 X–Y–8–8

2.3"0.6 2.6"0.6 19.0"5.3

4.7"1.4 2.5"1.1 23.9"5.7

U

p- 0.001. Note: Values are frequency per 10 4 sperm Žmean"SE.; all nuclei are confirmed as sperm heads by the presence of flagella.

smokers in comparison with nonsmokers Ž p 0.001.. There have been no previous report relating an exogenous exposure to increased frequencies of Y disomy in sperm. The finding of increased frequencies of Y disomy in sperm may have implications for the risk of having an aneuploid child. The proportion of affected children may be higher in men who smoke more. Relationship between air pollution in the district of Teplice, other covariates of interest and sperm aneuploidy was described using Poisson and linear regression modeling w31x. YY8 aneuploidy was significantly associated with the season of heaviest air pollution ŽOR s 5.25, 95% CI 2.5–11.0; linear modeling of normally transformed YY8, coefficient 1.44, p - 0.0001.. No other cytogenetic endpoints were significantly associated with seasonal air pollution. These findings are suggestive of an influence of seasonal air pollution on YY8 disomy. Rubesˇ put forward an idea for further study if disomy of chromosome Y in sperm could be used as a marker of sperm injury by chronic exposure to environmental pollutants. The sperm disomy findings for the Teplice nonsmokers compared closely with those of a group of Californian non-smokers ŽTable 5.. The slides for both cohorts were prepared and scored in the same laboratory w32,33x using the same probes and scoring criteria, but different scorers. Overall, there were no differences in the levels of sperm diploidies or Y

ˇ ´ et al.r Mutation Research 428 (1999) 203–215 R.J. Sram

214

Table 5 Comparison of sperm aneuploidy levels between Teplice and California nonsmokers w32,33x Sperm aneuploidy type

Group Teplice Ž ns15.

Disomy X Y X–Y 8

5.8"0.6 2.2"0.3 14.0"1.4 4.7"0.6

Diploidy X–X–8–8 Y–Y–8–8 X–Y–8–8

2.3"0.6 2.6"0.6 19.0"5.3

California Ž ns14. 3.1"0.5U 3.1"0.7 9.5"0.7U 6.6"0.7U

2.2"0.4 1.7"0.4 10.6"1.4

U

p- 0.001. Note: Values are frequency per 10 4 sperm Žmean"SE.; all nuclei are confirmed as sperm heads by the presence of flagella.

disomy. The Czech non-smokers, however, were elevated in the levels of X, X–Y and 8 disomies Ž p s 0.02.. These differences may be related to the geographic and ethnic differences, pollution differences, the slight age difference, or cultural differences between the two cohorts.

7. Conclusion Data generated from the Teplice Program indicate that air pollution in Central Europe may represent a very specific problem. We should expect health impact on adverse reproductive outcomes, both in females and males. Probably the most prominent environmental pollutants are organic compounds, especially PAHs. Another risk factor is lifestyle. We can speculate that in fact smoking and mutagens from diet could be a more significant risk factor than air pollution. All these ideas indicate that this region seems to be suitable for systematic molecular epidemiology studies. It seems that pregnant women, newborn children and young men are groups most susceptible to the air pollution expressed as PM10 or PAHs. Precise determination of their health risk for disease prevention is an important task for molecular epidemiology.

Acknowledgements The authors would like to thank Prof. R.J. Albertini, University of Vermont, USA, and Prof. W.W. Au, University of Texas Medical Branch at Galveston, USA, for their critical comments in the preparation of this manuscript. The authors are grateful to Dr. Sally Perreaut-Darney and Dr. Sherry G. Selevan for their support in those studies. The authors acknowledge the technical support of the staff of the District Institutes of Hygiene in Teplice and Prachatice for providing of the air monitoring under the superiority by Dr. I. Benesˇ and Dr. J. Nozicka. ˇ ˇ This study was supported by the Czech Ministry of Environment ŽTeplice Program II—VaV 340r1r1997., U.S. EPArU.S. AID and CEC ŽPHARE II, ECr HEAr18-CZ..

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