Cytogenetic effects in children and mothers exposed to air pollution assessed by the frequency of micronuclei and fluorescence in situ hybridization (FISH): A family pilot study in the Czech Republic

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Abstract

A family pilot study was conducted in the Czech Republic to test the hypothesis that exposure to air pollution with particulate matter (PM) in children results in detectable effects indicated by a number of biomarkers of exposure and early effects. The frequency of micronuclei (MN) in peripheral blood lymphocytes (PBLs) was analysed to assess the cytogenetic effects in children and mothers living in two different areas. From each area two groups of children from a total of 24 families (mean age: 6.0 ± 0.6 and 9.0 ± 1.2 years) in a total of 47 children and 19 mothers (mean age: 33.6 ± 3.9 years) participated. Chromosome aberrations determined with fluorescence in situ hybridization (FISH) painting for chromosomes #1 and #4 were analysed in 39 children and 20 parents. Teplice, a mining district, in Northern Bohemia was selected for the analyses of the effects in a population exposed to high levels of air pollution, especially during winter, and compared with a population from the rural area of Prachatice in Southern Bohemia. Significant higher frequencies of MN were found in the younger children living in the Teplice area as compared with those living in the Prachatice area (7.0 ± 2.3‰ versus 4.9 ± 2.0‰, p = 0.04). Higher levels of MN were also measured in the older children and the mothers from the Teplice area (9.2 ± 3.7‰ versus 6.6 ± 4.4‰) and (12.6 ± 3.4‰ versus 10.1 ± 4.0‰). The increased MN frequency may be associated with elevated carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) concentration of the PM2.5 measured in the ambient Teplice air, but other factors like genotoxic compounds from the diet or protective effect of micronutrients, which was not addressed in this pilot study, may also differ between the two areas. MN frequencies were found to increase with age in children. Lower MN frequency was found in boys as compared to girls. The result of the FISH analyses showed a low number of individuals with detectable levels of aberrations and no significant increases in genomic frequency of stable chromosome exchanges (FG/100) were found in children or parents from the Teplice area in comparison with those from the Prachatice area. The family pilot study indicates that MN is a valuable and sensitive biomarker for early biological effect in children and adults living in two different areas characterised with significant exposure differences in c-PAHs concentrations during winter.

Introduction

Exposure to ambient air pollution is a well-known environmental risk to adverse health effects. Several air pollutants have been evaluated for their carcinogenicity by the International Agency for Research on Cancer (IARC); for example benzo[a]pyrene (B[a]P) among other carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) and diesel engine exhaust emissions, are classified as group 2A whereas gasoline engine exhaust emissions, carbon black, and a number of PAHs are classified as group 2B [1], [2], [3]. Hazardous substances associated with particulate matter (PM) in the ambient air are of increasing concern. PAHs, volatile organic compounds (VOCs) and trace metals may absorb onto the surface of PM. In vitro studies have shown that extracts of PM collected from urban air are genotoxic to bacteria and mammalian cells [4]. Epidemiological studies have found an association between exposure to airborne PM and increased morbidity and mortality related to cardiovascular diseases [5], [6], [7]. Especially the fine fraction of particulate matter (PM2.5 and less) are retained in the alveolar regions of the lungs, and are able to diffuse into the blood circulation, and may subsequently induce inflammation, oxidative stress [8], and increased coagulation of the blood [9].

Exposure to air pollution in children is different from that for adults. In general, children have higher levels of physical activity and spend more time outside and for that reason they are more exposed to outdoor air pollution. The air intake of a resting infant of an age less than 1 year is twice that for an adult. Children also exhibit a higher intake of food and water per kilogram body weighs [10]. The differences in behaviour between adults and infants are obvious; e.g., ability to remove themselves from a noxious environment, hand-to-mouth behaviour and playing on the floor [11]. Children are considered more vulnerable to the adverse effects of air pollution than adults due to physiological differences related to their body size, growth, development and immaturity of organs and body functions. Eighty percent of alveoli are formed during postnatal development. Hence, changes in the lungs continue through out adolescence, and exposure to harmful air pollutants during childhood has an important impact on lung development [12]. The immaturity of the child immune system, metabolic pathways and differences in sensitivity of tissue and organs, and cell repair mechanism are factors contributing to increased susceptibility of children [13], [14]. Available data suggest that genetic damage early in life and an exceptional susceptibility of children exposed to genotoxins may influence risk of carcinogenesis [15]. Humans are widely exposed to genotoxins suspended in the outdoor air as well as in the indoor air. Also, individual variation such as lifestyle, diet, occupation, age, health status, and genetic susceptibility influence the personal exposure, in addition to the environmental factors.

A family pilot study was conducted in order to assess the relationship between exposure to air pollution and cytogenetic effects in 48 children; i.e., two siblings, and their mothers from two different areas in the Czech Republic. In family studies useful information on interactions between environmental exposure and genetic background may be gained. Biomarker measures in children can be compared with their siblings and parents in response to genotoxic exposure and age related factors of variation can be investigated. Family related variation can also be evaluated by comparison of the variation between families. The results of the analyses of micronuclei (MN) frequencies and the fluorescence in situ hybridization (FISH) whole chromosome painting of chromosomes #1 and #4 in peripheral blood lymphocytes (PBLs) are reported in this paper. Microarray analysis of differential gene expression and exposure is reported in details, separately in this issue [16].

MN formation in PBLs is assumed to be biologically relevant for carcinogenesis, as MN are expressed in different dividing cells following genotoxic exposure as a result of either chromosome breakage or chromosome loss [17]. The family pilot study was part of in the European ChildrenGenoNetwork which had the aim to collect, validate and review the available studies on children's exposure and susceptibility to, especially, airborne genotoxins.

Section snippets

Study area and study populations

A total of 24 study families were identified and the families were selected with the aim of obtaining equal groups of children from each area in terms of age and gender. From each of the selected families, the mother and her two children aged 5–11 years were asked to participate.

Families were drawn from the Teplice area, a mining region. Teplice is located in a valley surrounded by mountains and during winter periods of cold temperature and absence of wind, air pollution may be trapped and

Exposure assessment

Mean airborne ultrafine particles (UFP) number concentrations, measured at the front doors of the homes of the selected families during five different days in the early spring 2004 resulted in no significant differences in total number of UFP neither between the Teplice and the Prachatice areas (15,000 ± 1900 UFP ml−1 versus 13,000 ± 1900 UFP ml−1), nor the various sampling sites within the same area, the different days or times of the day. The highest concentrations of fine particles during the period

Discussion

The air quality of Northern Bohemia is significantly improved since the time where the area was described as the Black triangle area [30]. Levels of air pollution are now reaching a level comparable to other European Union member states, as the annual mean concentrations of common pollutants did not exceed limits and recommended values for health and ecosystems protection defined in the current European Commission Directives [31]. A difference in air pollution levels between the two areas was

Conclusions

The result of the CBMN assay in children, as well as in mothers, reflected the difference in exposure between the two areas of residence. Pilot sampling of UFPs and PM2.5 in the residential areas did not result in major differences, however significant differences in c-PAH concentrations were measured at the stationary ambient air quality stations. It was hypothesised that differences in MN frequencies between the areas could be associated with differences in genotoxic exposure from airborne

Acknowledgements

The authors would like to thank the study persons; the Czech paediatricians for their administrations of questionnaires and blood sampling Dr. D. Horakova (Teplice), Dr. J. Ruzickova (Krupka), and Dr. M. Weigartova (Vimperk); Laboratory of Genetic Toxicology, National Institute of Public Health, Prague, for the processing of MN cultures. This study was part of the concerted action (QLK4-CT-2002-02198), ChildrenGenoNetwork.

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