Micronucleus formation, DNA damage and repair in premenopausal women chronically exposed to high level of indoor air pollution from biomass fuel use in rural India

https://doi.org/10.1016/j.mrgentox.2010.02.006Get rights and content

Abstract

Genotoxicity of indoor air pollution from biomass fuel use has been examined in 132 biomass users (median age 34 years) and 85 age-matched control women from eastern India who used the cleaner fuel liquefied petroleum gas (LPG) to cook. Micronucleus (MN) frequency was evaluated in buccal (BEC) and airway epithelial cells (AEC); DNA damage was examined by comet assay in peripheral blood lymphocytes (PBL); and expressions of γ-H2AX, Mre11 and Ku70 proteins were localized in AEC and PBL by immunocytochemistry. Reactive oxygen species (ROS) generation in leukocytes was measured by flow cytometry, and the levels of superoxide dismutase (SOD) and total antioxidant status (TAS) in blood were measured by spectrophotometry. Real-time aerosol monitor was used to measure particulate pollutants in indoor air. Compared with controls, biomass users had increased frequencies of micronucleated cells in BEC (3.5 vs. 1.7, p < 0.001) and AEC (4.54 vs. 1.86, p < 0.001), and greater comet tail % DNA (18.6 vs. 11.7%, p < 0.01), tail length (45.5 vs. 31.4 μm, p < 0.01) and olive tail moment (4.0 vs. 1.4, p < 0.01) in PBL. Moreover, biomass users had more γ-H2AX-positive nuclei in PBL (49.5 vs. 8.5%, p < 0.01) and AEC (11.3 vs. 2.9%, p < 0.01) along with higher expression of DNA repair proteins Mre11 and Ku70 in these cells, suggesting stimulation of DNA repair mechanism. Biomass users showed rise in ROS generation and depletion of SOD and TAS. Biomass-using households had 2–4 times more particulate matter with diameter less than 10 and 2.5 μm in indoor air, and MN frequency and comet tail % DNA were positively associated with these pollutants after controlling potential confounders. Thus, chronic exposure to biomass smoke causes chromosomal and DNA damage and upregulation of DNA repair mechanism.

Introduction

Approximately half of the world's population and up to 90% of households in rural areas of developing countries still depend on unprocessed solid biomass fuel such as wood, dung, and crop residues for domestic cooking and room heating [1]. In the rural areas of India, women who bear the responsibility of daily household cooking with biomass for 2–5 h a day usually in poorly ventilated kitchen without chimney or smoke outlet are highly exposed to indoor air pollution (IAP) from biomass smoke. The severity of the problem can be gauged from the report that the concentration of PM10 (particulate matter having aerodynamic diameter of less than 10 μm) during cooking with biomass in a typical Indian household varies between 500 and 2000 μg/m3 [2] which is several times higher than the air quality standard recommended by the US Environmental Protection Agency [3].

Biomass smoke is essentially not different from cigarette smoke and it contains a large number of health-damaging chemicals including high level of fine and ultrafine particles, carbon monoxide, oxides of nitrogen, formaldehyde, acrolein, benzene, toluene, styrene, 1,3-butadiene, and polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene [4]. Many of these chemicals are mutagenic and carcinogenic, and international Agency for Research on Cancer has categorized indoor exposure to biomass combustion as probably carcinogenic to humans [5]. It is reasonable to assume therefore that chronic exposures to biomass smoke may cause genotoxic changes especially in cells at the direct line of exposure such as cells of the nasopharynx, oral cavity, airways and the lung. However, people have not paid attention to the cellular response of the airways to smoke emitted from burning biomass. In view of this, we have investigated the possibility of genetic damage and repair in a group of premenopausal women in eastern India who were chronically exposed to high level of IAP while cooking with unprocessed solid biomass such as dung cake, wood and agricultural wastes.

For assessment of genotoxicity, we have used micronucleus (MN) assay in exfoliated buccal (BEC) and airway epithelial cells (AEC) for the assessment of chromosomal damage, and single cell gel electrophoresis (Comet assay) in peripheral blood lymphocytes (PBL) for the detection of DNA damage. DNA damage may occur as single strand break (SSB) or double strand break (DSB). From the point of repair, DSB is more complex than SSB and comet assay cannot distinguish between SSB and DSB. In view of this, we have investigated the expression of phosphorylated (gamma) histone H2AX protein in AEC and PBL by immunocytochemistry (ICC) as phosphorylation of H2AX protein at serine 139 and resultant formation of γ-H2AX is associated with DSB [6].

The genome of eukaryotic cells is under constant attack from environmental agents and from products of normal cellular metabolism such as reactive oxygen species (ROS) derived from oxidative respiration, and products of lipid metabolism. A variety of changes can be seen in the DNA including SSBs, DSBs, mismatches and chemical adducts, and to repair these lesions multiple repair pathways have been evolved [7]. There are five main DNA repair pathways in human body, viz., homologous recombination (HR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). Each pathway consists of several proteins and about 130 genes have been identified so far that are associated with DNA repair [8]. DSBs can be repaired either by NHEJ or by HR. At the onset of these pathways, a DNA repair protein meiotic recombination 11 (Mre11) forms a complex with two other proteins Rad50 and Nsb1. The MRN complex thus formed senses DNA damage and initiates DNA repair mechanism through activation of ataxia telangiectasia mutated (ATM) kinase protein [9]. Considering the important role of Mre11 in initiating DNA repair process, we have examined the expression of this protein by ICC in epithelial cells and PBL of biomass-using women and have compared the findings with that of LPG-using women. In addition, we have examined the expression of Ku70 which forms a heterodimer with Ku80 that binds with the broken ends of the damaged DNA and activates DNA-dependent protein kinase leading to initiation of the NHEJ pathway of DSB repair [10].

Section snippets

Subjects

A total of 217 premenopausal women aged between 27 and 42 years from rural areas of West Bengal, a state in eastern India were enrolled for this study. They attended health check-up camps organized in different villages with the active cooperation of the local administrative bodies and non-government organizations. Among the participants, 132 women (age 28–42 years, median 34 years) used to cook daily for 2.5–5.5 h exclusively with wood, cow dung and agricultural wastes, such as bamboo, jute

Demographic and socio-economic characteristics

Demographic and socio-economic characteristics of study population are summarized in Table 1. LPG users and biomass users were comparable with respect to age, body mass index, cooking time, smoking and chewing habit and environmental tobacco smoke due to the presence of smokers in family. However, the income and education levels in biomass users were significantly lower than those in LPG users (p < 0.05). The rates without separate kitchen in biomass-using households and LPG-using households were

Discussion

Compared with LPG users, biomass-using women of this study had significantly higher MCFs in BEC and AEC that are at the direct route of smoke exposure. Chronic exposure of oral mucosa to toxic substances such as tobacco smoke leads to keratinization with synthesis of keratohyalin bodies that appear as granule in the cytoplasm [20]. Although these round cytoplasmic bodies do not contain DNA, they could be erroneously classified as MN when using a non-DNA-specific stain such as Pap. We frequently

Conflicts of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors gratefully acknowledge the financial support received from Central Pollution Control Board, Delhi in carrying out this study.

References (43)

  • P.H. Danielsen et al.

    Oxidative damage to DNA and repair induced by Norwegian wood smoke particles in human A549 and THP-1 cell lines

    Mutat. Res.

    (2009)
  • M. Ishihama et al.

    Generation of phosphorylated histone H2AX by benzene metabolites

    Toxicol. In Vitro

    (2008)
  • N. Bruce et al.

    Indoor air pollution in developing countries: a major environmental and public health challenge for the new millennium

    Bull. World Health Org.

    (2000)
  • K. Balakrishnan et al.

    Daily average exposures to respirable particulate matter from combustion of biomass fuels in rural households of southern India

    Environ. Health Perspect.

    (2002)
  • US EPA. US Environmental Protection Agency National Ambient Air Quality Standards (NAAQS), Retrieved from...
  • J. Zhang et al.

    Hydrocarbon emissions and health risks from cook stoves in developing countries

    J. Exposure Anal. Environ. Epidemiol.

    (1996)
  • D.R. Pilch et al.

    Characteristics of γ-H2AX foci at DNA double-strand breaks sites

    Biochem. Cell Biol.

    (2003)
  • J.H. Houtgraff et al.

    A concise review of DNA damage checkpoints and repair in mammalian cells

    Cardiovasc. Revasc. Med.

    (2006)
  • P. Baumann et al.

    DNA end joining catalyzed by human cell-free extracts

    Proc. Natl. Acad. Sci. U. S. A.

    (1998)
  • H.E. Hughes et al.

    Handbook of Diagnostic Cytology

    (1968)
  • P. Tolbert et al.

    Micronucleus and other nuclear anomalies in buccal smears: methods development

    Mutat. Res.

    (1991)
  • Cited by (54)

    View all citing articles on Scopus
    View full text