Increased oxidative DNA damage and decreased expression of base excision repair proteins in airway epithelial cells of women who cook with biomass fuels

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 341–352

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Increased oxidative DNA damage and decreased expression of base excision repair proteins in airway epithelial cells of women who cook with biomass fuels Bidisha Mukherjee, Banani Bindhani, Hirak Saha, Manas Ranjan Ray ∗ Department of Experimental Hematology, Chittaranjan National Cancer Institute, Kolkata 700 026, India

a r t i c l e

i n f o

a b s t r a c t

Article history:

To investigate whether biomass burning causes oxidative DNA damage and alters the

Received 30 October 2013

expression of DNA base excision repair (BER) proteins in airway cells, sputum samples

Received in revised form 8 May 2014

were collected from 80 premenopausal rural biomass-users and 70 age-matched control

Accepted 25 June 2014

women who cooked with liquefied petroleum gas. Compared with control the airway cells

Available online 3 July 2014

of biomass-users showed increased DNA damage in alkaline comet assay. Biomass-users showed higher percentage of cells expressing oxidative DNA damage marker 8-oxoguanine

Keywords:

and lower percentages of BER proteins OGG1 and APE1 by immunocytochemical staining.

Biomass fuel

Reactive oxygen species (ROS) generation was doubled and level of superoxide dismutase

Particulate matter

was depleted significantly among biomass-users. The concentrations of particulate matters

Oxidative stress

were higher in biomass-using households which positively correlated with ROS generation

Oxidative DNA damage

and negatively with BER proteins expressions. ROS generation was positively correlated with

Base excision repair

8-oxoguanine and negatively with BER proteins suggesting cooking with biomass is a risk

Airway cells

for genotoxicity among rural women in their child-bearing age. © 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Unprocessed, solid biomass fuel (BMF) such as firewood, cow dung cake and crop residues accounts for more than one-half of the domestic energy needs in the developing nations of the world (Smith et al., 2004). Burning of BMF emits smoke that significantly increases the level of indoor air pollution (IAP; Naeher et al., 2007; Torres-Dosal et al., 2008). In India, about 74% of the population especially in the rural areas is still dependent on BMF for cooking and room heating (WHO, 2006),

and the estimated per capita use of biomass is 1.2–2.1 kg/day (Smith, 1987). Women who cook with BMF in traditional stoves for 2–5 h a day usually in poorly ventilated kitchen without chimney or smoke outlet became highly exposed to IAP (Dutta et al., 2011; Mondal et al., 2010). BMF smoke contains a wide spectrum of combustion products in the form of gases and suspended particulates. Hundreds of organic chemicals and several transitional metals get adsorbed on the surface of these particulates, and are inhaled during respiration. The International Agency for Research on Cancer has categorized biomass smoke as a probable human carcinogen (Group

∗ Corresponding author at: Department of Experimental Hematology, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700 026, India. Tel.: +91 033 2476 5101x322; fax: +91 033 2475 7606. E-mail address: [email protected] (M.R. Ray). http://dx.doi.org/10.1016/j.etap.2014.06.010 1382-6689/© 2014 Elsevier B.V. All rights reserved.

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2a), while the smoke constituents benzene, 1,3-butadine and benzo(a)pyrene as confirmed (Group 1) human carcinogens (Danielsen et al., 2009; Straif et al., 2006). Mammalian genomic DNA contains various moieties that can react with environmental pollutants. As a result, the integrity of the DNA can be affected. Oxidation and methylation of the DNA bases, for example, are particularly common. Oxidation can be mediated by reactive oxygen species (ROS) that accumulate in cells due to environmental stress (Prasad et al., 2010) or by the cell’s own metabolism (Fraga et al., 1990). Amongst the four bases, guanine is most susceptible to oxidation because of its low redox potential caused by the unsaturated N7 C8 bond (Neeley and Essigmann, 2006; Steenken and Jovanovic, 1997). The one-electron oxidation product of guanine 7,8-dihydro-8-oxoguanine (8-oxoG) is the most prevalent base lesion found in the DNA following oxidative stress (Faucher et al., 2012; Kasai et al., 1986) with an estimated frequency of ∼0.3–4 lesions/106 bases (Gedik and Collins, 2005). The presence of 8-oxoG is considered a major mutagenic DNA base lesion (Radak and Boldogh, 2010) that contributes to genomic instability and tumorigenesis because of replication-associated mispairing. 8-oxoG pairs with adenine during replication, promoting G:C to T:A transversion (Epe, 2002) and consequent transcriptional alterations (Saxowsky et al., 2008). However, the cells possess an efficient repair mechanism for oxidative DNA damage through the base excision repair (BER) pathway (Faucher et al., 2012; Fishel et al., 2007). The BER is accomplished in four steps involving: (i) recognition and removal of the damaged base such as 8-oxoG by 8-oxoguanine-DNA glycosylase (OGG1) leaving an apurinic/apyrimidinic (AP) site; (ii) incision of the AP site 5 to phosphodeoxyribose creating free 3 -hydroxyl termini by apurinic/apyrimidinic endonuclease/redox effector factor-1, (APE/Ref-1) (Cho et al., 2010); (iii) supplementation of a nucleotide onto a pre-existing 3 -OH group and synthesis of a new strand by DNA polymerase; and finally, (iv) ligation of the new and the existing strand by X-ray repair crosscomplementing protein 1 (XRCC1) and the enzyme DNA ligase (Martin, 2008). Dysregulation of any one of the four BER steps and repair proteins could result in unrepaired DNA damage that can promote oncogenesis (Chevillard et al., 1998; Fortini et al., 2003; Kelley et al., 2001; Kumar et al., 2012; Raffoul et al., 2007; Souza et al., 2011; Xie et al., 2010; Zaky et al., 2008). Several investigators have documented a higher risk of developing lung cancer among BMF users compared with users of cleaner fuels (Behera and Balamugesh, 2005; Delgado et al., 2005). Consistent with this, we have documented an excess of chromosomal breakage and DNA damage in peripheral blood lymphocytes, buccal epithelium, and airway cells of BMF-using rural women compared with age-matched control women from similar neighborhood who cooked with cleaner fuel liquefied petroleum gas (LPG) (Mondal et al., 2011, 2010; Mukherjee et al., 2013). However, it is still unknown whether chronic inhalation of biomass smoke causes oxidative DNA damage and whether it affects the BER pathway like its adverse effect on the non-homologous end-joining type of DNA double strand breakage repair that we reported earlier (Mondal et al., 2010). It is worth mentioning that polymorphism of the BER genes has been found in lung cancer (Li et al., 2011). In view of this, an attempt has been made in the present

study to examine whether chronic exposure to biomass smoke inflicts oxidative DNA damage and alters the expression of proteins involved in BER pathway. For this, we have evaluated by immunocytochemistry (ICC) the expressions of some proteins involved in the BER pathway in the airway cells exfoliated in spontaneously expectorated sputum of a group of pre-menopausal BMF-using housewives from rural India, and have compared the findings with that of age-matched control women from similar neighborhood who cooked with cleaner fuel LPG.

2.

Materials and methods

2.1.

Participants

A total number of 150 pre-menopausal women aged between 22 and 42 years, were enrolled in this study through health check-up camps organized in 5 villages in Hooghly and Burdwan districts of West Bengal, a state in eastern India, and ambient particulate matters with an aerodynamic diameter of <10 ␮m (PM10 ) in these areas for the past few years were within national ambient air quality standards for residential areas (60 ␮g/m3 annual average). The villages were at least 5 km away from the national or state highways so that vehicular pollution was low, there was no air-polluting industry such as thermal power plant, brick kiln, sponge iron factory and rice mill within 5 km radius. Among the participants, 80 women (age 22–42 years, median 34 years) cooked daily with wood, cow dung cake and agricultural refuse, such as bamboo, jute stick, paddy husk, hay and dried leaves for the past 5 years or more. Accordingly they were grouped as BMF users. The remaining 70 women aged 23–40 years, median age 33 years were residents of the same locality but they cooked with cleaner fuel LPG. They were considered as reference or control group. All the women worked and stayed indoors or within their villages all the time. The participants were all engaged in domestic chores like cooking family meals twice a day, care giving to children and elders in the family, attention to pets and cattle, washing the clothes of the entire family and collecting drinking water from community tube wells. Biomass users had the added responsibility of collecting wood and agricultural wastes, and making cow dung cake with their bare hands and sun-drying of these cakes after pasting them on mud walls. None of the participants had a regular, time-bound salaried job. No women worked in and commuted regularly to urban areas. So they are not exposed to traffic derived ambient air pollution that exists in urban areas. Biomass users were poorer and usually resided in houses with mud walls and a hay/tin/terracotta roof in contrast to the houses with brickwalls with a concrete/terracotta roof of LPG-users. Motor vehicles were rare in these villages; bicycles, cycle rickshaws and bullock carts were the principal mode of transport. The participants were included in the study after giving written informed consent in accordance with the Declaration of Helsinki. The study was carried out from January 2010 to April 2012 in the summer, autumn, and winter months. Monsoon (June to September) was avoided because the relative humidity during monsoon often rises above 95% when laser photometers cannot function properly (Ramachandran et al., 2003).

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2.2.

Inclusion and exclusion criteria

Inclusion criteria were (i) apparently healthy, pre-menopausal, married women, (ii) actively engaged in daily household cooking for the past 5 years or more either with BMF or LPG, (iii) having normal menstrual cycle length (28 ± 2days) and blood pressure level (systolic blood pressure <120 and diastolic blood pressure <80 mmHg), (iv) never-smoker and non-chewer of tobacco and betel nut, (v) having a body mass index >15 and <30 kg/m2 . The mixed fuel users (biomass + LPG/kerosene), pregnant or lactating women, diabetics, those currently using oral contraceptive pills or under any other medication, exposed to ionizing radiation (e.g., X-ray) in past 6 months or had a recent or past history of malignancy were excluded. Information about age, habits, education, family size and income, number of smokers in family, cooking time per day, years of cooking, fuel and oven type, kitchen location was obtained during personal interview with female researchers of the study team. The Ethics Committee of Chittaranjan National Cancer Institute, Kolkata approved the study protocol.

2.3.

Chemicals

Low melting agarose, ethidium bromide (EtBr), dimethyl sulfoxide (DMSO), ethylenediamine tetra acetic acid (disodium salt), HEPES, dimethyl formamide and 2 ,7 dichlorofluorescein diacetate and bovine serum albumin (BSA) were purchased from Sigma–Aldrich Chemicals, Saint Louis, MO, USA. DAB, 50X was obtained from Santa Cruz Biotechnology, USA. High melting agarose, 1% Triton X-100, TRIS (hydroxymethyl) aminomethane, nicotinamide adenine dinucleotide reduced disodium salt and dithiotheritol were obtained from SISCO Research Laboratories, India. Phosphatase inhibitor mixture was purchased from Calbiochem, Germany and protease minitab from Roche Biochemicals, Indianapolis, USA. The source of diethanolamine (DEA) and triethanolamide (TEA) was Qualigens fine chemicals, Mumbai. NaCl crystal, NaOH pallets (purified), mercaptoethanol, manganous chloride and hydrogen peroxide were obtained from Merck (India) Limited, Worli, Mumbai and SD-Fine Chem, Mumbai, India. All other chemicals were purchased locally and were of analytical reagent grade.

2.4.

Measurement of PM10 and PM2.5 in indoor air

Particulate matters with an aerodynamic diameter of <10 ␮m (PM10 ) and <2.5 ␮m (PM2.5 ) were measured by real-time laser photometer (DustTrakTM Aerosol monitor, model 8520, TSI Inc., Shoreview, MN, USA). The instrument was calibrated to the standard ISO 12103-1 A1 test dust and operates at a flow rate of 1.7 L/min. We used two monitors for simultaneous measurement of PM10 and PM2.5 . Air sampling was carried out in each household for 3 consecutive days, 8 h/day (07:00–15:00 h), covering both cooking and non-cooking hours. For biomassusing women who cook in a sitting position 2–3 ft away from the oven, the monitor was placed in the breathing zone of the cook, 2.5 ft above floor level on a wooden stool, 3 ft away from the chullah. LPG users, on the other hand, cook in a standing position and the monitor was placed accordingly at a height

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of 4.5 ft. Real-time laser photometers overestimate PM levels. Because of this, raw data obtained from laser photometers were reduced by dividing it with internationally accepted correction factor of 2.77 for PM2.5 (Siddiqui et al., 2009), and 2.50 for PM10 (Chung et al., 2001; Lehocky and Williams, 1996).

2.5.

Collection of sputum samples

The participants were given sterile plastic cups to collect the early morning spontaneously expectorated sputum for three consecutive days. Spontaneously expectorated sputum is considered as surrogate for airway cells as it contains samples from upper and lower respiratory tracts and even alveoli. The thick viscous parts of the sputa were smeared on clean glass slides. Seven slides were prepared from sputa of each participant. One slide was immediately fixed in 95% ethanol for cytology and the remaining 6 slides were fixed with cold methanol at the site of collection for immunocytochemical localization of 8-oxoguanine, OGG1 and APE1 respectively. The remaining sputum samples were transferred to sterile plastic containers containing 20 ml of phosphate-buffered saline (PBS) with 0.1% dithiothreitol (Sigma Chem, USA), and transported to the laboratory in ice box. The sputum samples were centrifuged at 2500 rpm for 10 min and the supernatant was discarded. The cell pellet was washed with PBS for 2–3 times, suspended in PBS and stored at −20 ◦ C for future analysis.

2.6.

Pap staining for sputum cytology

Cytology of sputum cells was evaluated after staining the slides with Papanicolau (Pap) method following the procedure of Hughes and Dodds (Hughes and Dodds, 1968). Pap-stained slides were coded and examined under light microscope (Leitz, Germany) at 400× and 1000× magnification. At least 10 high power fields (hpf, 40× objectives with 10× eyepiece) per slide were examined and differential count of sputum cells (excluding squamous epithelial cells whose origin is not the inner airways) was made using established morphological criteria (Grubb, 1998).

2.7. Immunocytochemistry (ICC) for 8-oxoG, OGG1 and APE1 expressions Expressions of 8-oxoguanine (8-oxoG), OGG1 and APE1 proteins were detected by ICC using the diagnostic kits manufactured by Abcam, UK and following the established staining protocol (Ghosh et al., 2009). Briefly, the slides containing sputum cells were fixed in cold methanol for 30 min, air dried, washed in PBS thrice and blocked in 3% bovine serum albumin (BSA) for 1 h at room temperature. Thereafter, rabbit polyclonal primary antibodies against OGG1 (ab22766) and APE1 (ab82) diluted 1:80 and 1:100 in 1% BSA respectively, and mouse monoclonal primary antibody against 8-oxoG (ab64548) diluted 1:400 in 1% BSA were added separately to each slide. The slides were placed in a humid box at 4 ◦ C and kept overnight in darkness. After washing with PBS, goat polyclonal anti-rabbit IgG-H & L (HRP) secondary antibody (ab6721; diluted 1:500 in 1% BSA) was added to the slides against rabbit polyclonal primary antibodies and rabbit polyclonal antimouse IgG-H & L (HRP) secondary antibody (ab6728; diluted

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1:500 in 1% BSA) was added to the slides against mouse monoclonal primary antibodies and kept for 90 min. After washing with PBS, the HRP substrate mixture [50× diaminobenzidine (Santa Cruz Biotechnology, USA), 50% H2 O2 , 1 M Tris–HCl and distilled water] was added to the slides and kept for 45 min in darkness. Then the slides were washed with distilled water and counterstained with hematoxylin, dehydrated in graded ethanol and mounted in distyrene plasticizer xylene (DPX) and examined under light microscope. On an average, 400 basal, parabasal and intermediate epithelial cells, 100 alveolar macrophages and 500 sputum neutrophils were examined for the expressions of 8-oxoG, OGG1 and APE1 proteins in each individual.

diacetate (DCFH-DA, Sigma Chemical, USA) solution in dimethyl formamide was added to the 500 ␮l of sputum cell suspension in 1 ml of Hank’s balanced salt solution containing 0.15 M NaCl and 5 mM HEPES, pH 7.35, and incubated at 37 ◦ C for 30 min in darkness. After washing the cells with icecold PBS, 10,000 events were acquired immediately in a flow cytometer (FACS Calibur with sorter, Becton Dickinson [BD], San Jose, CA, USA) using Cell Quest software (BD, USA). Total neutrophils present in sputum samples were gated on dot plot. Respiratory burst and generation of ROS by the cells resulted in green fluorescence that was recorded in fluorescence channel1 and was expressed as mean fluorescence intensity (MFI) in arbitrary unit.

2.8. Alkaline single cell gel electrophoresis (comet assay)

2.10. Spectrophotometric measurement of SOD in sputum cell lysate

The alkaline comet assay was used to detect DNA damage in airway cells following the procedure of Singh et al. (1988). In brief, frosted slides coated with 1% high melting agarose (Sigma Chem, USA) were kept overnight for drying and to form the base layer. An intermediate layer, containing a mixture of 20 ␮l of sputum cell suspension and 80 ␮l of 0.6% low melting agarose (Sigma Chem, USA) was applied over the first layer and cover glasses were immediately placed over the second layer and the slides were kept at 4 ◦ C for 30 min to solidify the agarose. After removing the cover glass, a third layer of 0.8% low melting agarose was applied and the agarose was allowed to solidify at 4 ◦ C for 30 min. Thereafter the slides were immersed in freshly prepared chilled lysis buffer containing 2.5 M NaCl, 0.3 M NaOH, 0.1 M Na2 EDTA, 10 mM TRIS and distilled water, pH 10.5 (5 ml of 10% dimethyl sulfoxide and 50 ␮l of Triton X-100 were added to 50 ml of lysis solution just before use). After lysis, the slides were placed in a horizontal gel electrophoresis tank, and electrophoresis was conducted in ice-cold alkaline electrophoresis buffer (0.3 M NaOH, 1 mM Na2 EDTA and distilled water, pH > 13) for 20 min at 25 V and 300 mA. Then the slides were washed with neutralization buffer containing 0.4 M TRIS and distilled water, pH 7.5, thrice. Washed slides were stained for 5 min with 50 ␮l of 20 ␮g/ml ethidium bromide, dipped into chilled distilled water to remove excess stain, and covered with a cover glass. The slides were analyzed in a fluorescence microscope (Leica DM 4000B, Germany) with image analysis software (Komet 5.5; Kinetic Imaging, Liverpool, UK) for measuring the tail DNA (%), tail length in ␮m, and Olive tail moment in arbitrary unit.

Airway cells present in sputum were lysed in 500 ␮l of lysis buffer containing 0.05 M Tris (pH 7.4), 0.15 M NaCl, 1% Nonidet P-40, with added protease and phosphatase inhibitors: 1 protease minitab (Roche Biochemicals, Indianapolis, USA)/10 ml and 1× phosphatase inhibitor mixture (Calbiochem, Germany) on ice for 20 min. The lysates were then sonicated for 20 s, kept at 4 ◦ C for 30 min, and spun at 15,000 × g for 10 min, and the supernatant was collected and stored at −70 ◦ C until use. The activity of antioxidant enzyme superoxide dismutase (SOD) was measured spectrophotometrically (Paoletti et al., 1986) at 340 nm in a spectrophotometer (Shimadzu, Japan).

2.9.

Flow cytometric measurement of ROS

The major cell types that are present in the sputum samples are basal and intermediate epithelial cells, ciliated and nonciliated columnar epithelial cells, goblet cell, Type I and Type II pneumocytes, alveolar macrophages, neutrophils, eosinophils and lymphocytes. Among them the rise in the number of neutrophils causes airway inflammation where reactive oxygen species (ROS) is produced through its vigorous respiratory burst activity (Vaitkus et al., 2013). Generation of ROS in airway neutrophils exfoliated in sputum was measured by flow cytometry following established procedure (Rothe and Valet, 1990). In brief, 20 ␮l of 0.5 mM 2 ,7 -dichlorofluorescein

2.11.

Statistical analysis of data

The results were statistically analyzed using SPSS statistical software (Statistical Package for Social Sciences for windows, release 17.0; SPSS Inc., Chicago, IL, USA). Statistical differences were determined by using Chi-square test, Student’s t-test, and Mann–Whitney U test, as applicable. The impact of all variables on the expression of base excision repair proteins was first examined by logistic regression analysis to identify the potential confounders. Then, the cumulative impact of these factors on the base excision repair parameters was evaluated by stepwise multivariate logistic regression analysis. Any measured parameter was treated as a variable, either continuous (when computing univariately for correlation) or dichotomous (when examining association). Univariate analysis was carried out using Spearman’s rank correlation test to find out the relation between two measurable parameters as continuous variables, and the result was expressed as rho value. Statistical significance was assigned at p < 0.05.

3.

Result

3.1. Demographic and socio-economic characteristics of the participants Demographic and socio-economic characteristics of the participants are summarized in Table 1. The control and BMF-using women were comparable with respect to age, body mass index (BMI), cooking years, cooking hours per day, number of smokers in family, spouse smoking habit, use of

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Table 1 – Demographic and socio-economic characteristics of biomass and LPG-using rural women. Variable Age in year, median (range) Body mass index (kg/m2 ), median (range) Cooking years, median (range) Cooking hours per day (mean ± SD) Years of schooling, median (range) Homes with separate kitchen (%) Smoking husband (%) Number of cigarettes/bidia smoked per day by the husbands (mean ± SD) Use of mosquito repellant at home (%) Food habit, mixed (%) Members in family, median (range) Family income per month in US $ (mean ± SD)

LPG-using control (n = 70)

Biomass user (n = 80)

p value

33.0 (23–40) 23.3 (19.5–26.5) 15 (5–17) 3.5 ± 0.8 9 (4–14) 85.7 42.8 15.8 ± 5.7

34.0 (22–42) 22.9 (19.2–25.8) 16 (5–20) 3.7 ± 1.5 3 (0–9) 62.5 43.7 16.7 ± 3.6

NS NS NS NS <0.05** <0.01* NS NS

64.3 100 5 (3–6) 105 ± 14

62.5 100 5 (4–6) 51 ± 10

NS NS <0.0001***

n, number of subjects; NS, statistically not significant. Local-made cigarette with inferior quality of tobacco wrapped on Tendu leaves. ∗ Significantly different from LPG-using control in Chi-square test. ∗∗ Significantly different from LPG-using control in Mann–Whitney U test. ∗∗∗ Significantly different from LPG-using control in Student’s t-test. a

were 150.9 and 315.7 ␮g/m3 , respectively, in BMF-using households against 47.0 and 72.1 ␮g/m3 in LPG using homes.

3.3.

Fig. 1 – Histogram showing the mean 8-h concentrations of PM10 and PM2.5 in cooking areas of biomass-and LPG-using households. Bars represent standard deviation.

mosquito repellant at home, food habit, marital status, number of family members. However, the two groups differed significantly with respect to education (p < 0.05) and family income (p < 0.0001) which were lower in BMF users, and lack of separate kitchen (p < 0.01) which was more prevalent among BMF users (Table 1).

3.2.

Particulate pollution in indoor air

The 8-h mean concentration of corrected PM10 values in BMF-using kitchen was 3.6-times more than that of LPG-using kitchen (arithmetic mean 454.3 ± 256.5 (SD) vs. 126.9 ± 32.1 ␮g/m3 , p < 0.0001). Similarly, the corrected concentration of PM2.5 in BMF-using kitchen was 4.2-times higher than that of LPG using kitchen (247.2 ± 99.6 vs. 59.2 ± 14.2 ␮g/m3 , p < 0.0001; Fig. 1). The 25th and 75th percentile levels of PM10 in BMF-using homes were 215.0 and 655.0 ␮g/m3 , respectively, against 96.1 and 146.0 ␮g/m3 in LPGusing households. The 25th and 75th percentile levels of PM2.5

Sputum cytology

Compared with control, the sputa of BMF-using women were more cellular (89.9 ± 24.1 vs. 62.9 ± 11.2 cells per high power field [hpf] of microscope in control, p < 0.0001) and contained an excess of airway epithelial cells (8.1 ± 2.3 vs. 6.2 ± 2.2 cells/hpf, p < 0.0001), neutrophils (61.3 ± 18.6 vs. 46.6 ± 9.2 cells/hpf, p < 0.0001), eosinophils (1.6 ± 0.8 vs. 0.4 ± 0.2 cells/hpf, p < 0.0001), lymphocytes (4.8 ± 1.3 vs. 3.3 ± 1.1 cells/hpf, p < 0.0001), and alveolar macrophages (13.2 ± 6.9 vs. 5.9 ± 3.1 cells/hpf, p < 0.0001). The findings suggest greater prevalence of airway inflammation among biomass users.

3.4.

DNA damage in the airway cells

The comet assay of airway cells showed remarkable increase in DNA damage among BMF users. Compared with control, they showed 5-fold increase in tail percent DNA, 1.3-fold increase comet tail length, and 6-fold increase in Olive tail moment (p < 0.0001; Fig. 2 and Table 2). Spearman’s rank correlation test showed positive correlation between concentrations of PM10 and PM2.5 in indoor air and the tail % DNA (rho = 0.688, p = 0.01; rho = 0.723, p = 0.01 respectively), tail length (rho = 0.611, p = 0.01; rho = 0.608, p = 0.01 respectively) and tail moment (rho = 0.487, p = 0.01; rho = 0.513, p = 0.01 respectively) measured in comet assay.

3.5. ICC

Expression of 8-oxoG, OGG1 and APE1 proteins by

8-oxoG, OGG1 and APE1 proteins were found localized mainly in the nuclei of airway epithelial cells, especially the basal and the parabasal cells. OGG1 and APE1 proteins were not detected in other cell types such as the airway neutrophils and the alveolar macrophages. The percentage of airway epithelial

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Fig. 2 – Comet assay showing varying degrees of DNA damage in (A) inflammatory cells and (B) epithelial cells in sputum of biomass-using women compared with LPG-using control women (C and D respectively).

cells expressing 8-oxoG was significantly higher in BMF users when compared with that of control (p < 0.0001). In contrast, the percentages of airway epithelial cells expressing OGG1 and APE1 in BMF users were 50% lower than the control women (p < 0.0001; Table 3 and Fig. 3).

3.6.

ROS and SOD level in airway cells

The MFI of DCFH-DA of sputum cells collected from BMF users was 53.2% higher than that of control (728.7 ± 84.9 vs. 341.2 ± 48.1, p < 0.0001), suggesting increased ROS

generation by the airway cells of BMF users. In contrast, the level of SOD in sputum cells was 1.4-times lower than that of control (555.30 ± 115.18 U/ml in BMF users compared with 788.35 ± 83.77 U/ml in control, p < 0.0001; Fig. 4).

3.7. Indoor air pollution, ROS generation, DNA damage and BER Generation of ROS in sputum cells showed positive correlation with PM10 (rho = 0.461, p < 0.01) and PM2.5 levels in indoor air (rho = 0.512, p < 0.01) in Spearman’s rank correlation test. The

Table 2 – Comparison of the parameters of DNA damage of sputum cells between biomass- and LPG-using women. Comet parameters Tail percent DNA Mean ± SD Median (range) Comet tail length (␮m) Mean ± SD Median (range) Olive tail moment (in arbitrary unit) Mean ± SD Median (range)

LPG-users (n = 70)

BMF-users (n = 80)

10.1 ± 3.2 7.2 (3.5–14.2)

36.2 ± 8.2 36.3 (21.7–49.8)

<0.0001 <0.0001

29.3 ± 4.6 32.4 (22.8–40.1)

45.2 ± 5.5 43.4 (28.1–53.7)

<0.0001 <0.0001

1.2 ± 0.5 0.9 (0.4–1.7)

6.2 ± 1.9 5.5 (4.3–11.5)

<0.0001 <0.0001

n, number of subjects. Significant in Student’s t-test (comparison of mean) and Mann–Whitney U test (comparison of median).

∗

p value*

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Table 3 – Immunocytochemical localization of proteins associated with DNA base excision repair in airway epithelial cells. Percentage of airway cells expressing 8-Oxoguanine Mean ± SD Median (range) OGG1 Mean ± SD Median (range) APE1 Mean ± SD Median (range)

LPG users (n = 70)

BMF users (n = 80)

p value

3.21 ± 1.3 3.3 (1.4 –5.8)

7.52 ± 1.3 8.2 (4.2–11.3)

<0.0001* <0.0001**

3.14 ± 1.2 3.1 (1.2–5.6)

1.56 ± 0.6 1.5 (1.3–3.2)

<0.0001* <0.0001**

2.83 ± 0.8 3.0 (1.2–4.7)

1.37 ± 0.7 1.1 (0.6–2.2)

<0.0001* <0.0001**

An average of 400 cells were examined for each participant. Statistically significant compared with LPG users in Student’s t-test. ∗∗ Statistically significant compared with LPG users in Mann–Whitney U test. ∗

concentration of SOD in the airway cells, in contrast, correlated negatively with indoor PM10 (rho = −0.564, p < 0.01) and PM2.5 levels (rho = −0.512, p < 0.01). ROS generation by the sputum cells of BMF-using women correlated positively with the percentage of 8oxoG-expressing cells in Spearman’s rank correlation test (rho = 0.814, p < 0.01). The percentages of OGG1 and APE1 protein-expressing airway cells correlated negatively with the PM10 and PM2.5 levels in indoor air and ROS generation by the airway cells (p < 0.01). In contrast, SOD showed significant positive correlation with the percentage of airway cells expressing these two BER proteins (Table 4). In univariate analysis, the decline in the percentage of OGG1 and APE1-expressing cells, increased percentage of 8oxoG-expressing cells and increased comet assay parameter like tail DNA percentage of sputum cells among BMF users displayed positive association with lower education and family income, increasing age and exposure years to biomass smoke, spouse smoking habit, kitchen location and ROS generation. Even after controlling the influence of these confounders in multivariate logistic regression analysis, a positive association was found between cooking with biomass and reduction in the percentages of OGG1- (odds ratio [OR] = 2.48, 95% confidence interval [95%CI] 1.44–4.21) and APE1-expressing airway cells (OR = 1.72, 95% CI 1.22–3.06). In multivariate logistic regression analysis, a positive association was also found between cooking with biomass and increased DNA damage parameters [percentage of 8-oxoG-expressing cells – (OR = 2.08, 95% CI 1.54–3.01) and tail DNA percentage of airway cells – (OR = 1.36, 95% CI 1.15–4.13)] after controlling the influence of abovementioned potential confounders.

4.

Discussion

Cooking with biomass is a common practice in rural India, especially among the poor people who cannot afford cleaner fuel. Women who cook regularly with BMF get exposed to high level of IAP in the process. Earlier studies from this (Mondal et al., 2011, 2010; Mukherjee et al., 2013) and other laboratories (Musthapa et al., 2004; Pandey et al., 2005) have shown excessive DNA damage with insufficient activation of the nonhomologous end-joining pathway of DNA repair (Mondal et al., 2010) in association with chronic biomass smoke exposures

(Mondal et al., 2010). Herein we show marked increase in the number of 8-oxoG-expressing airway epithelial cells with concomitant decline in the number of cells expressing BER proteins OGG1 and APE1 in women who cooked with biomass, suggesting inactivation of the BER in the face of increased oxidative DNA damage. It appears that a substantial part of the altered DNA bases have been left unrepaired which may cause genomic instability (Colnaghi et al., 2011; Hoeijmakers, 2001; Lengauer et al., 1998) with increased risk of mutagenesis and carcinogenesis (Hanahan and Weinberg, 2000; Loft et al., 2012; Roszkowski et al., 2011; Sheng et al., 2012). Oxidative DNA damage is caused by attacks on the DNA by ROS (Franco et al., 2008; Mena et al., 2009; Sørensen et al., 2003). It generates various modified DNA bases among which 8oxoG is the most abundant (Fortini et al., 2003). Oxidative stress is also a known cause of BER inactivation (Bräuner et al., 2007). Biomass smoke pollutants, especially the particulate matters, generate oxidative stress. ROS can be generated directly from ultrafine particles through the presence of free radicals, transition metals and oxidants adsorbed on the particle surface (Fubini et al., 2004; Fullerton et al., 2009). Indeed, we found a strong positive correlation between PM10 and PM2.5 levels in indoor air with ROS generation and SOD depletion. Alternatively, inhaled pollutants can accelerate ROS production via activation of the inflammatory cells such as the neutrophils and the alveolar macrophages (Franco et al., 2008; Knaapen et al., 2004; Li et al., 2003; Voelkel et al., 2003). This could be distinct possibility, because systemic and airway inflammation are common among biomass users (Banerjee et al., 2012). In the present study also, we documented an excess of inflammatory cells in the sputum of BMF users, suggesting airway inflammation. Earlier we reported oxidative stress-mediated excessive DNA damage in both inflammatory and epithelial cells in the airways (Mukherjee et al., 2013), implying the vulnerability of every cells present in the airways to genotoxic insults from smoke constituents. Menstrual cycle does not influence comet assay responses (Bajpayee et al., 2005). Hence excessive DNA damage among biomass users cannot be explained by the possibility that the samples were collected at different phases of cycle. Although some participants were closer to menopause, no difference in 8-oxoG levels has been reported between periˇ and premenopausal women (Zitnanová et al., 2011). Women in perimenopause are under increased oxidative stress, but they

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Fig. 3 – Immunocytochemical localization of 8-oxoG, OGG1 and APE1 proteins in the nuclei of airway epithelial cells exfoliated in spontaneously expectorated sputum of biomass- (A, C, E) and LPG-using women (B, D, F). The relative frequency (%) of 8-oxoG-expressing basal and parabasal cells was greater among biomass users (A) compared to that of LPG-using control women (B). In contrast, OGG1- and APE1-expressing basal and parabasal epithelial cells were lower in biomass users (C and E respectively) than in control (D and F respectively). The cell nuclei were counterstained with hematoxylin, original magnification 1000×.

have increased total antioxidant status, increased DNA repair ˇ compared with women in premenopause (Zitnanová et al., 2011). Long term exposure to air pollution is associated with the incidence of diabetes (Andersen et al., 2012; Dijkema et al., 2011) and hypertension (Dutta et al., 2011). While diabetes reduces OGG1 expression (Simone et al., 2008), hypertension is associated with DNA base oxidation (Kim et al., 2009). For these reasons we excluded diabetic and hypertensive women from our study population. Therefore, hypertension or

diabetes cannot account for the observed changes in BMFusing women. Besides, the ambient air pollution levels in the study areas were within the standard (60 ␮ g/m3 ) as the villages were far from the highways and busy road traffic. Bicycle and cycle rickshaw were the principal mode of transport and there were no air-polluting industries within 5 km radius. More importantly, biomass and LPG users were neighbors; hence it eliminates outdoor air pollution as a possible contributor to the difference in 8-oxoG and BER protein expressions

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Fig. 4 – Histograms showing (a) ROS and (b) SOD levels in airway cells of LPG and biomass using women. Bars represent standard deviation of mean.

between biomass and LPG users. Instead, the major contributor appears to be the significantly higher levels of PM10 and PM2.5 in indoor air of BMF-using homes. However, the present study has certain limitations. For evaluation of BER pathway we did only qualitative estimation of BER proteins by ICC. We did not extend the study to gene or mRNA levels. This is one aspect that needs further investigation. Another point is that we have only partially characterized indoor air pollution by measuring PM10 and PM2.5 levels in cooking areas, leaving emission of other pollutants present in biomass smoke. Incomplete combustion of biomass on an open fire or traditional stove generates large amounts of particulate matter as well as carbon monoxide, hydrocarbons, oxygenated organics, oxides of nitrogen (NOx ) and sulfur (SOx ), free radicals, transition metals, polycyclic aromatic compounds such as benzo(a)pyrene, benzo(a)anthracene and volatile organic compounds such as benzene, toluene and xylene (Naeher et al., 2007). Reactive nitrogen species formed from nitric oxide (NO) or its metabolites, can lead to protein tyrosine nitration, which is elevated in lung cancer (Masri et al., 2005). Benzene, following inhalation, is metabolized by the microsomal cytochrome P-450 monooxygenase system in the alveolar epithelial cells in lung and the hepatic cells in liver and causes carcinogenesis in human (Danielsen et al., 2009). In addition, polycyclic aromatic hydrocarbons are highly mutagenic with tumor promoter activity and are responsible for increased risk to malignant neoplasms, especially lung cancer (Abba et al.,

2012; Ohnishi and Kawanishi, 2002; Olsson et al., 2010). Again iron released from airborne PM or other redox metals can stimulate the generation of hydroxyl radicals (HO• ) by Fentontype reactions, causing extensive oxidative damage to cellular macromolecules (Donaldson et al., 1997; Valavanidis et al., 2000). Thus, the observed increased oxidative DNA damage and decrease in BER protein expression among biomass users could be due to the action of other pollutants that we did not measure. However, PM data is recognized as a proxy of polycyclic aromatic hydrocarbons in air (Bonner et al., 2005). PM have a carbonaceous core with adsorbed inorganic and organic materials that can cause adverse health effects (Oberdörster, 2000). In spite of the fact that organic carbon and elemental carbon constitute a considerable part of particle mass, there are large uncertainties over precise contributions to health effects of individual component owing primarily to its complex, heterogeneous nature, which is not well characterized in most geographical settings (Kelly and Fussell, 2012). Although there are thousands of chemicals that have been detected in PM in different locations, some of the more common constituents include nitrates, sulfates, elemental and organic carbon, organic compounds (e.g., polycyclic aromatic hydrocarbons), biological compounds (e.g., endotoxin, cell fragments), and a variety of metals (e.g., iron, copper, nickel, zinc, and vanadium) (Aneja et al., 2006). Thus, PM is considered as the single best indicator of potential harm. Each 10 ␮g/m3 increase in PM2.5 concentrations has been associated

Table 4 – Correlation between the percentage of base excision repair protein-expressing airway epithelial cells and the level of particulate pollution in indoor air. Correlation PM10 with OGG1 PM2.5 with OGG1 PM10 with APE1 PM2.5 with APE1 ROS with OGG1 ROS with APE1 SOD with OGG1 SOD with APE1 ∗ ∗∗

Statistically significant, p < 0.05. Statistically significant, p < 0.01.

Pearson’s coefficient −0.375 −0.399** −0.458** −0.445 ** −0.665** −0.798** 0.321** 0.425** **

Kendall’s tau b −0.351 −0.362** −0.401** −0.426** −0.642** −0.752** 0.299* 0.381** **

Spearman’s rho value −0.349** −0.312** −0.389** −0.435** −0.640** −0.761** 0.271* 0.373**

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with a 15–27% increase in lung cancer mortality (Turner et al., 2011). So we have restricted to measurement of concentrations of PM in this study for assessment of oxidative DNA damage and consequent DNA BER mechanism among biomass-users compared with cleaner fuel LPG-users. Another limitation is that this study did not attempt to identify potential differences in BER pathway between different types of biomass fuel users as varying biomass fuels are emitting different compositions. We have treated all types of biomass-users as exposed group altogether in this study. We carried out indoor measurements and biological sampling for a limited number of days. Therefore, seasonal variation and climatic changes could have influenced the measured parameters. However, monitoring of PM and sputum sampling were done in LPG- and biomassusing homes of a village simultaneously. Thus, seasonal effect does not appear to be a significant confounder. Besides, the control and biomass-exposed women of our study were well matched, the difference in BER protein expression between the comparable groups was highly significant and the sample size of the study seemed adequate for validation of the data to a larger population. In essence, the study has shown that rural women who cook with BMF are highly exposed to IAP that causes oxidative DNA damage and inactivation of BER pathway. Inappropriate repair and accumulation of altered bases to DNA may lead to the development of various diseases including cancer. Indeed, lung cancer is the 5th leading site of cancer among non-smoking women in eastern India where the study has been conducted (Nandakumar et al., 2004). Women in this part of the world get involved in cooking in their late teens when they start helping their mothers in the kitchen. After marriage they take charge of the kitchen of their own. As observed in this and earlier studies (Mondal et al., 2010, 2011), cooking with biomass can lead to cellular injury at the level of nucleic acids in women in their child-bearing age. As millions of poor women of the country are exposed to biomass smoke, efforts should be made by all concerned to reduce smoke exposure by improving kitchen ventilation and by increasing combustion efficiency of the oven. In addition to installation of improved cook stoves, installation of solar devices as a clean and alternative energy source for domestic use is another and perhaps a better option in the villages of India (Achudume, 2009).

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgements The study was supported by funds received from Central Pollution Control Board under Ministry of Environment and Forests, Government of India, and Council of Scientific and Industrial Research, India.

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