RESEARCH ARTICLE

Reduction of DNA mismatch repair protein expressionin airway epithelial cells of premenopausal women chronicallyexposed to biomass smoke

Bidisha Mukherjee & Anindita Dutta & Saswati Chowdhury &

Sanghita Roychoudhury & Manas Ranjan Ray

Received: 4 April 2013 /Accepted: 4 October 2013 /Published online: 22 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Biomass burning is a major source of indoor airpollution in rural India. This study examined whether chronicinhalation of biomass smoke causes change in the DNAmismatch repair (MMR) pathway in the airway cells. For this,airway cells exfoliated in sputum were collected from 72premenopausal nonsmoking rural women (median age34 years) who cooked with biomass (wood, dung, crop resi-dues) and 68 control women who cooked with cleaner fuelliquefied petroleum gas (LPG) for the past 5 years or more.The levels of particulate matters with diameters less than 10and 2.5 μm (PM10 and PM2.5) in indoor air were measured byreal-time aerosol monitor. Benzene exposure was monitoredby measuring trans ,trans -muconic acid (t ,t -MA) in urine byhigh-performance liquid chromatography with ultraviolet de-tector. Generation of reactive oxygen species (ROS) and levelof superoxide dismutase (SOD) in airway cells were measuredby flow cytometry and spectrophotometry, respectively.Immunocytochemical assay revealed lower percentage of air-way epithelial cells expressing MMR proteins mutL homolog1 (MLH1) and mutS homolog 2 (MSH2) in biomass-usingwomen compared to LPG-using controls. Women whocooked with biomass had 6.7 times higher level of urinary t ,t -MA, twofold increase in ROS generation, and 31 % deple-tion of SOD. Indoor air of biomass-using households hadthree times more particulate matters than that of controls.ROS, urinary t ,t-MA, and particulate pollution in biomass-using kitchen had negative correlation, while SOD showed

positive correlation with MSH2 and MLH1 expression. Itappears that chronic exposure to biomass smoke reducesMMR response in airway epithelial cells, and oxidative stressplays an important role in the process.

Keywords Biomass fuel . Particulate matter . Benzene .

Oxidative stress .Mismatch repair . Airway cells

Introduction

The genome of the eukaryotic cells is under constant attackfrom various environmental agents as well as from the prod-ucts of normal cellular metabolism. This may result in avariety of changes in the DNA including single strand breaks,double strand breaks, mismatches, and chemical adducts. Ifthe damaged DNA is left unrepaired, it causes genomic insta-bility (Lengauer et al. 1998; Hoeijmakers 2001; Colnaghiet al. 2011) that may ultimately lead to, among others, cancer(Hanahan and Weinberg 2000). To overcome this problem,cells have evolved different DNA repair mechanisms to main-tain the integrity of the genome (Kolodner et al. 2002; Sancaret al. 2004). There are five main DNA repair mechanisms inhuman body—homologous recombination, nonhomologousend joining (NHEJ), nucleotide excision repair, base excisionrepair, and mismatch repair (MMR). Each mechanism is con-trolled by several proteins. Overall, at least 130 genes areinvolved in DNA repair (Christmann et al. 2003).

Indoor air pollution (IAP) from cooking with traditionalbiomass fuel (wood, dung, and crop residues) is a major healthproblem in rural India. A majority of poor, rural women of thecountry still cook with highly polluting biomass. Biomasssmoke itself is a “probable carcinogen,” and some of itsconstituents like benzene, 1,3-butadiene, and benzo(a )pyreneare carcinogenic to humans according to the International

Responsible Editor: Philippe Garrigues

B. Mukherjee :A. Dutta : S. Chowdhury :M. R. Ray (*)Department of Experimental Hematology, Chittaranjan NationalCancer Institute, 37, S. P. Mukherjee Road, Kolkata 700 026, Indiae-mail: manasrray@rediffmail.com

S. RoychoudhuryCentral Pollution Control Board, New Delhi, India

Environ Sci Pollut Res (2014) 21:2826–2836DOI 10.1007/s11356-013-2218-4

Agency for Research on Cancer (Zhang and Smith 2003;Reddy et al. 2004; Sinha et al. 2006; Naeher et al. 2007;Danielsen et al. 2009). In agreement with this, we havereported an excess of micronucleus formation, an indicatorof chromosomal breakage, and DNA damage in peripheralblood lymphocytes (Mondal et al. 2010) and in airway epi-thelial cells (Mukherjee et al. 2013) of a group of never-smoking Indian women in their child-bearing age who cookeddaily with biomass. Besides, a deficiency in NHEJ type ofDNA repair was observed in these women (Mondal et al.2010). In view of these findings, we felt it is important toexamine other DNA repair pathways in the airway cells ofbiomass-using women. TheMMR pathway seemed especiallyrelevant in this context because the metabolites of benzene, ahuman carcinogen abundantly present in biomass smoke,generate DNA mismatches requiring MMR activation (Ságiet al. 1998).

The MMR system repairs the base mismatches after DNAreplication, preventing recombination of nonidentical DNAsequences. TheMMR is accomplished in three steps. First, themismatch is recognized by the MutSα [MSH2 (mutS homo-log 2)/mutS homolog 6 heterodimer] or MutSβ complexes[MSH2/mutS homolog 3 heterodimer]. Second, the mismatchis excised from the DNA. This process is initiated by thebinding of the protein MutLα [a mutL homolog 1 (MLH1)/postmeiotic segregation increased 2 heterodimer] toMutSα orMutLβ [a MLH1/mutL homolog 3 heterodimer] to MutSβand subsequent recruitment of an exonuclease (EXO1) thatsequentially removes the nucleotides between an adjacentsingle strand break up to and beyond the mismatch on thedaughter DNA strand. The third and the final step involvesresynthesis of DNA by DNA polymerase-δ, proliferating cellnuclear antigen, and replication protein A, followed by thesealing of the nick in the daughter strand by a DNA ligase(Kinsella 2009). Defects in MMR pathway, as observed inpetroleumworkers occupationally exposed to benzene (Paz-y-Miño et al. 2008), increase the rate of spontaneous mutations(O'Brien and Brown 2006). Mutations and loss of function ofMSH2 and MLH1 genes have been reported in inheritedhuman colon cancer (Fishel and Kolodner 1995), while defi-cient expressions of MMR proteins have been found in pa-tients with lung cancer (Hansen et al. 2003; Scartozzi et al.2006; Lo et al. 2011).

Considering these reports, the objective of this study was toinvestigate whether chronic inhalation of biomass smoke ad-versely affects the expression of the DNA mismatch repairproteinsMLH1 andMSH2 in cells that are present at the directline of smoke exposure. We evaluated the DNA mismatchrepair system by measuring the expression of the key proteinsMSH2 and MLH1 in exfoliated airway epithelial cells byimmunocytochemistry in a group of never-smoking premen-opausal women who were chronically exposed to high level ofIAP due to daily household cooking with biomass fuel for the

past 5 years or more. Age-matched women from the samelocality who cooked with cleaner fuel liquefied petroleum gas(LPG) were enrolled as controls. Since benzene, a volatileorganic compound and an important constituent of biomasssmoke (Sinha et al. 2006), may affect the DNA mismatchrepair pathway, we estimated the benzene metabolite trans ,trans -muconic acid (t ,t-MA) in urine of the participants as ameasure of benzene exposure (Boogaard and van Sittert 1996;Fustinoni et al. 2005; Carrieri et al. 2010).

Materials and methods

Participants

The participants were recruited from nine villages in Hooghlyand Burdwan districts of West Bengal, a state in eastern Indiathrough village Panchayats (local administration) afterdiscussing the purpose and the protocol of the study with thelocal housewives and their husbands. The villages were se-lected with the consideration that their location should be atleast 5 km away from the national or state highways tominimize vehicular pollution, there should not be any air-polluting industry such as thermal power plant, brick kiln,sponge iron factory, and rice mill within 5 km radius, and thevillagers should use both biomass and liquefied petroleum gasas cooking fuel. The inclusion criteria were premenopausal(age >20 but <44 years) married women who were nonsmokerand non-chewer of tobacco and betel nut with a body massindex of >15 and <30 kg/m2, normal menstrual cycle length of28±2 days, systolic blood pressure of <120 mmHg and dia-stolic pressure of <80mmHg, and used to cook for at least 2 h/day and 6 days in a week for the past 5 years or more eitherwith biomass or LPG. Women who were mixed fuel (biomass+ LPG/kerosene) users, pregnant or breastfeeding, using oralcontraceptive pills or under any other medication, exposed toionizing radiation (e.g., X-ray) in the past 6 months, or had arecent or past history of malignancy were excluded.

A total number of 162 women were screened, and 140 ofthose screened met the study criteria and completed the study.Among the participants, 72 women (age 22–41 years, median34 years) cooked daily with wood, cow dung cake, andagricultural refuse, such as bamboo, jute stick, paddy husk,hay, and dried leaves for the past 5 years or more.Accordingly, they were grouped as biomass users. The re-maining 68 women aged 23–40 years, median age 33 years,were residents of the same locality, but they cooked withcleaner fuel LPG. They were considered as reference or con-trol. The participants were included in the study after givingwritten informed consent in accordance with the Declarationof Helsinki. The study protocol was approved by theInstitutional Ethics Committee of Chittaranjan NationalCancer Institute, Kolkata.

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Chemicals

Ethylenediaminetetraacetic acid (disodium salt), t ,t -MA,(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), dimethyl formamide, and 2′,7′-dichlorofluoresceindiacetate and bovine serum albumin (BSA) were purchasedfrom Sigma-Aldrich Chemicals, Saint Louis, MO, USA.Diaminobenzidine (DAB), 50×, was obtained from Santa CruzBiotechnology, USA. Tris(hydroxymethyl)aminomethane, nico-tinamide adenine dinucleotide-reduced disodium salt,and dithiothreitol were obtained from SISCO ResearchLaboratories, India. Phosphatase inhibitor mixture waspurchased from Calbiochem, Germany and proteaseminitab from Roche Biochemicals, Indianapolis, USA.The source of diethanolamine (DEA) and triethanolamine(TEA)was Qualigens Fine Chemicals, Mumbai. NaCl crys-tal, mercaptoethanol, methanol, manganous chloride, andhydrogen peroxide were obtained from Merck (India)Limited, Worli, Mumbai. All other chemicals were pur-chased locally and were of analytical reagent grade.

Measurement of particulate pollution in indoor air

Real-time measurement of airborne particulate matter concen-tration was measured by a portable, battery-operated, laserphotometer (DustTrakTM aerosol monitor, model 8520, TSIInc., Shoreview, MN, USA). The instrument contains 10-mmnylonDor-Oliver cyclone, operates at a flow rate of 1.7 L/min,and measures particle load in the concentration range of 1 μg–100 mg/m3. We measured particulate matters with an aerody-namic diameter of <10 μm (PM10) and <2.5 μm (PM2.5). Themonitor was calibrated to the standard ISO 12103-1 A1 testdust. We used two monitors for simultaneous measurement ofPM10 and PM2.5. PM2.5 values were reduced by a correctionfactor of 2.77 (Siddiqui et al. 2009), while a correction factorof 2.50 was used for PM10 (Chung et al. 2001; Lehocky andWilliams 1996). Air sampling was carried out in each house-hold for three consecutive days, 8 h/day (0700–1500 hours),covering both cooking and non-cooking hours. Air samplingwas carried out from January 2010 to April 2011 in thesummer, autumn, and winter months when the questionnaireswere administered and the biological samples were taken. Therainy season (middle of June to September) was avoidedbecause the relative humidity during monsoon often risesabove 95 % when laser photometers cannot function properly(Ramachandran et al. 2003).

Particulate matter (PM) measurements in indoor air weredone simultaneously in biomass- and LPG-using householdsof a given village to minimize seasonal variation in air quality.Indoor air quality of a total number of 90 households, 5biomass using and 5 LPG using, from each village wasmonitored. The mean of 3 days was used as the indoor airquality of a single household. For biomass-using women who

cook in a sitting position 2–3 ft away from the open chullah(oven), the monitor was placed in the breathing zone of thecook, 2.5 ft above floor level on a wooden stool and 3 ft awayfrom the chullah. LPG users, on the other hand, cook in astanding position and the monitor was placed accordingly at aheight of 4.5 ft.

Assessment of benzene exposure

Current exposure of benzene was estimated from thelevel of benzene metabolite t ,t -MA in urine by high-performance liquid chromatography with ultraviolet de-tector (HPLC-UV) following the method of Ducos et al.(1990). In brief, urine (0.025–0.050 L) was collectedwithin an hour after completion of cooking for lunch(between 1300 and 1400 hours) into 0.1-L plasticscrew-cap vials. The samples were protected from light,brought to the laboratory, and stored at −20 °C untilanalysis. The samples were thawed and 1-mL aliquotswere passed through a 0.3-g Prep Sep-Sax cartridge(Fisher Scientific, USA) that had been preconditionedwith 0.003 L of methanol and 0.003 L of distilledwater. Then, the t ,t -MA was eluted with 0.003 L of a10 % aqueous acetic acid and measured in HPLC(Waters, USA), 10 μL fraction of the elute was usedper injection in column filled with LiChrosorb C18,5 μM (Waters, USA), and the detector was set at259 nm. The eluent was a mixture of 1 % aqueousacetic acid and methanol (9:1, v /v ). With a flow rateof 0.0012 L/min, the retention time of t ,t -MA was10 min and the duration of an analytical run was20 min. A stock solution of t ,t -MA (0.1 g/L, SigmaChemical, USA) was prepared in 10 % acetic acid.

Collection of sputum samples

The participants were given sterile plastic cups to collect theearly morning spontaneously expectorated sputum for threeconsecutive days. The thick viscous parts of the sputa weresmeared on clean glass slides. Five slides were prepared fromthe sputa of each participant. One slide was immediately fixedin 95 % ethanol for cytology and the remaining four slideswere fixed with cold methanol at the site of collection forimmunocytochemical expression of MSH2 and MLH1, re-spectively. The remaining sputum samples were transferred tosterile plastic containers containing 0.02 L of phosphate-buffered saline (PBS) with 0.1 % dithiothreitol (SigmaChemical, USA) and transported to the laboratory in icebox. The sputum samples were centrifuged at 2,500 rpm for10 min and the supernatant was discarded. The cell pellet waswashed with PBS for two to three times, suspended in PBS,and stored at −20 °C for future analysis.

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Pap staining for sputum cytology

Cytology of sputum cells was evaluated after staining theslides with Papanicolau (Pap) method following the procedureof Hughes and Dodds (1968). Pap-stained slides were codedand examined under light microscope (Leitz, Germany) at×400 and ×1,000 magnification. At least 10 high power fields(hpf, ×40 objectives with ×10 eyepiece) per slide were exam-ined and differential count of sputum cells (excluding squa-mous epithelial cells whose origin is not the inner airways)was made using established morphological criteria (Grubb1988).

Immunocytochemistry for MSH2 and MLH1 detection

Expression of MSH2 and MLH1 proteins was detected byimmunocytochemistry (ICC) using the diagnostic kitsmanufactured by Abcam, UK following the established stain-ing protocol (Ghosh et al. 2009). Briefly, the slides containingsputum cells were fixed in cold methanol for 30 min, air dried,washed in PBS thrice, and blocked in 3%BSA for 1 h at roomtemperature. Thereafter, rabbit polyclonal primary antibodiesagainst MSH2 (ab47076) and MLH1 (ab47703) diluted 1:500and 1:200 in 1 % BSA, respectively, were added separately toeach slide. The slides were placed in a humid box at 4 °C andkept overnight in darkness. After washing with PBS, goatpolyclonal anti-rabbit IgG-H&L [horseradish peroxidase(HRP)] secondary antibody (ab6721; diluted 1:500 in 1 %BSA) was added to the slides and kept for 90 min at roomtemperature. After washing with PBS, the HRP substratemixture [50× diaminobenzidine (Santa Cruz Biotechnology,USA), 50 % H2O2, 1 M Tris–HCl, and distilled water] wasadded to the slides and kept for 45 min in darkness. Then, theslides were washed with distilled water and counterstainedwith hematoxylin, dehydrated in graded ethanol, and mountedin distyrene plasticizer xylene and examined under light mi-croscope. Slides were evaluated for the presence of DAB-stained golden brown nuclei for detection of MSH2- andMLH1-positive cells, and the results were expressed as per-centage of MSH2- and MLH1-positive cells for each individ-ual. On an average, 300 basal, parabasal, and intermediateepithelial cells, 100 alveolar macrophages, and 500 sputumneutrophils were examined for the expressions of MSH2 andMLH1 proteins in each individual.

Flow cytometric measurement of reactive oxygen species

Generation of reactive oxygen species (ROS) in sputum cellswas measured by flow cytometry following the procedure ofRothe and Valet (1990). In brief, 20 μL of 0.5 mM 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma Chemical,USA) solution in dimethyl formamide was added to the500 μL of sputum cell suspension in 0.001 L of Hank's

balanced salt solution containing 0.15 M NaCl and 5 mMHEPES, pH 7.35, and incubated at 37 °C for 30 min indarkness. After washing the cells with ice-cold PBS, 10,000events were acquired immediately in a flow cytometer (FACSCalibur with a sorter, Becton Dickinson [BD], San Jose, CA,USA) using Cell Quest software (BD, USA). Total cellspresent in sputum samples were gated on dot plot.Respiratory burst and generation of ROS by the cells resultedin green fluorescence that was recorded in fluorescencechannel-1 and was expressed as mean fluorescence intensity(MFI) in arbitrary unit.

Spectrophotometric measurement of antioxidant enzymesuperoxide dismutase

Sputum cells were lysed in 500 μL of lysis buffer containing0.05 M Tris (pH 7.4), 0.15 MNaCl, and 1 % Nonidet P-40 andadded with protease and phosphatase inhibitors: 1 proteaseminitab (Roche Biochemicals, Indianapolis, USA)/0.010 Land 1× phosphatase inhibitor mixture (Calbiochem,Germany)] on ice for 20 min. The lysates were then sonicatedfor 20 s, kept at 4 °C for 30 min, and spun at 15,000×g for10 min, and the supernatant was collected. Cell lysates werestored at −70 °C until use. The activity of antioxidant enzymesuperoxide dismutase (SOD) in sputum cell lysate was mea-sured spectrophotometrically following the procedure ofPaoletti et al. (1986). In a spectrophotometric cuvette, 800 μLof TEA–DEA buffer containing 100 mM each of TEA andDEA; 40 μL of 7.5 mM nicotinamide adenine dinucleotide-reduced disodium salt, pH 7.4; 25 μL of a mixture (1:1, v /v) of0.2 M ethylene diaminetetraacetic acid disodium salt; and0.1 M manganous chloride and 100 μL of sample (sputum celllysate) were added and mixed well. The absorbance [opticaldensity (OD)] was measured immediately (0 min) and at 1, 2, 3,4, and 5 min after addition of mercaptoethanol at 340 nm in aspectrophotometer (Shimadzu, Japan). SOD activity (units permilliliter) was calculated from the ODs at different timeintervals.

Statistical analysis

The fundamental hypothesis of this study was that the cumu-lative biomass smoke exposures would be associated with thealterations in DNA mismatch repair protein expression inairway epithelial cells of premenopausal women of ruralIndia. The results were statistically analyzed using SPSSstatistical software (Statistical Package for Social Sciencesfor Windows, release 17.0; SPSS Inc., Chicago, IL, USA).Statistical differences were determined by using chi-squaretest, Student's t test, and Mann–WhitneyU test, as applicable.The impact of all variables onmismatch repair parameters wasfirst examined by logistic regression analysis to identify thepotential confounders. Then, the cumulative impact of these

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factors on the mismatch repair parameters was evaluated bystepwise multivariate logistic regression analysis. Any mea-sured parameter was treated as a variable, either continuous(when computing univariately for correlation) or dichotomous(when examining association). Univariate analysis was carriedout using Spearman's rank correlation test to find out therelation between two measurable parameters as continuousvariables, and the result was expressed as rho value(Spearman's rank correlation coefficient). Statistical signifi-cance was assigned at p <0.05.

Results

Demographic and socioeconomic characteristics

Demographic and socioeconomic characteristics of 140 wom-en participants of this study are summarized in Table 1. TheLPG-using control women and biomass users were compara-ble with respect to age, body mass index, cooking years,cooking hours per day, tobacco smoking or chewing habit,use of mosquito repellant at home, food habit, marital status,number of family members, and environmental tobaccosmoke exposure for presence of smokers in family.However, biomass fuel (BMF) users were less educated (p <0.05 in Mann–Whitney U test), and their family income wassignificantly lower than that of their neighbors who used tocook with LPG (p <0.001 in Student's t test). Moreover,62.5 % of BMF-using households lacked a separate kitchenagainst 85.3 % of LPG-using households, and the differencebetween these two groups in this regard was significant (p <0.01) in chi-square test.

Particulate pollutants in biomass-using households

The 8-h mean concentration of PM10 in biomass-usingkitchen was 3.2 times more than that of LPG-usingkitchen (arithmetic mean 414.3±213.5 (SD) vs. 130.5±38.5 μg/m3, p <0.0001) covering both cooking and non-cooking hours. Similarly, the concentration of PM2.5 inbiomass-using kitchen was 3.4 times higher (210.7±104.6 vs. 61.3±16.3 μg/m3, p <0.0001) than LPG-using kitchen. The ranges and median values of PM10

and PM2.5 concentrations in biomass and LPG-usinghouseholds are shown in Fig. 1. The 25th and 75thpercentile levels of PM10 in biomass-using homes were204.0 and 625.0 μg/m3, respectively, against 92.75 and169.0 μg/m3 in LPG-using households. The 25th and75th percentile levels of PM2.5 were 108.0 and313.2 μg/m3, respectively, in biomass-using householdsagainst 45.0 and 77.0 μg/m3 in LPG-using homes.

Concentration of t ,t -MA (milligrams per liter) in urine

The concentration of t ,t -MA in urine, a biomarker ofbenzene exposure, was 7.96±5.74 mg/L in biomass-using women in contrast to 1.19±0.8 mg/L in controlsubjects (Fig. 2, p <0.0001 in Student's t test). Therange of urinary t,t -MA was 0.3–2.9 mg/L with a me-dian of 0.9 mg/L in control women, whereas biomass-usingwomen had a range of 1.5–20.5 mg/L with a median of6.5 mg/L (p <0.0001 in Mann–Whitney U test). The 25thand 75th percentile levels of t ,t-MA were 5.1 and 7.7 mg/L,respectively, in biomass-using households against 0.6 and1.7 mg/L in LPG-using homes.

Table 1 Demographic and so-cioeconomic characteristics ofbiomass and LPG-using women

n number of subjects, NS statisti-cally not significanta Locally made cigaretteb Chi-square test, significantly dif-ferent from controlcMann–Whitney U test, signifi-cantly different from controld Student's t test, significantly dif-ferent from control

Variable LPG-using control(n =68)

Biomass user(n =72)

p value

Age in years, median (range) 33.0 (23–40) 34.0 (22–41) NS

Body mass index (kg/m2), median (range) 23.2 (19.5–25.6) 22.6 (18.3–25.5) NS

Cooking years, median (range) 15 (5–18) 16 (5–20) NS

Cooking hours per day, median (range) 3.0 (2–5) 3.0 (3–6) NS

Cooking hours per day (mean ± SD) 3.5±0.7 3.7±1.0 NS

Years of schooling, median (range) 9 (4–15) 3 (0–9) <0.05c

Homes with separate kitchen (%) 85.3 62.5 <0.01b

Smoker in family (%) 42.6 44.4 NS

Number of bidia smoked per day by the husbands(mean ± SD)

15.7±8.7 16.6±7.6 NS

Use of mosquito repellant at home (%) 64.7 62.5 NS

Food habit, mixed (%) 100 100

Members in family, median (range) 5 (3–6) 5 (4–6) NS

Family income per month in US$ (mean ± SD) 104±14 51±10 <0.001d

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Sputum cytology

Biomass users had elevated number of total cells in sputum[89.6±24.5 vs. 62.8±11.2 cells/hpf of microscope,p <0.0001] than controls. Sputa of biomass fuel userscontained more airway epithelial cells (7.9±2.3 vs. 5.9±2.2cells/hpf, p <0.0001), neutrophils (60.1±18.6 vs. 46.6±11.8cells/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 (14.9±6.9 vs. 6.7±3.1cells/hpf, p <0.0001) than that of control.

Expression of MSH2 and MLH1 proteins by ICC

In ICC, the expressions of MSH2 and MLH1 proteins werefoundmainly in the nuclei of airway epithelial cells, especiallythe basal and parabasal cells (Figs. 3 and 4). The proteins werenot detected in other cell types such as the airway neutrophilsand the alveolar macrophages.

The percentages of airway epithelial cells expressingMSH2 and MLH1 were significantly lower in women whowere chronically exposed to biomass smoke (Figs. 3 and 4 andTable 2). For instance, the percentages ofMSH2- andMLH1-expressing cells were 42 and 54 % lower than the control,respectively (p <0.0001, Table 2).

Since the total number of epithelial cells exfoliated in sputavaried considerably between the biomass and control women,

Fig. 1 Box–whisker plotsshowing concentrations ofparticulate matter (PM) duringcooking hours inside kitchensusing exclusively LPG orbiomass fuel for cooking purpose.a PM10 level. b PM2.5 level. Thelines across each box plotrepresent the median value. Thelines that extend from the top andthe bottom of each box representthe lowest and highestobservations still inside the lowerand upper limit of confidence.The difference between these twogroups was statisticallysignificant (p <0.0001) in Mann–Whitney U test

Fig. 2 Box–whisker plot showing the concentrations of benzene metab-olite trans ,trans-muconic acid (t ,t-MA) in urine of women who cookedexclusively with either biomass fuel or LPG for the past five years ormore. The lines across each box plot represent the median values. Thelines that extend from the top and the bottom of each box represents thelowest and highest observations still inside the lower and upper limit ofconfidence. The circle outside the box represents outlier. The differencebetween these two groups was statistically significant (p <0.0001) inMann–Whitney U test

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we converted the relative (percent) values to absolute ones(cells per high power field). Still, the numbers of MSH2-(0.22±0.06 vs 0.28±0.06 cells/hpf in control, p <0.05) andMLH1-expressing cells (0.15±0.04 vs 0.25±0.09 cells/hpf incontrol, p <0.05) were significantly lower than the controls inwomen who cooked with biomass fuel.

ROS and SOD levels in sputum cells of biomass users

Flow cytometric analysis showed 50.4 % higher MFI ofDCFH-DA among biomass fuel users (718.7±74.9 vs.356.4±45.1, p <0.001), suggesting doubling of ROS genera-tion in sputum cells of these women. In contrast, there was31.6 % decreased level of superoxide dismutase in sputumcells of biomass-using women (544.30±116.18 vs. 796.35±84.77 U/mL, p <0.001; Fig. 5). The results suggest oxidativestress in the airways of biomass users.

Indoor air pollution and ROS generation

Generation of ROS by sputum cells showed positive correla-tion with PM10 (rho=0.463, p <0.01), PM2.5 in indoor air(rho = 0.498, p < 0.01), and t ,t -MA level in urine(rho=0.429, p <0.01) in Spearman's rank correlation test.The concentration of SOD in sputum, on the other hand,correlated negatively with PM10 (rho=−0.564, p <0.01) and

PM2.5 in indoor air (rho=−0.489, p <0.01) and t ,t -MA levelin urine (rho=−0.312, p <0.05 ).

Relationship between PM10 and PM2.5 levels in indoor airand expression of MSH2 and MLH1 proteins in airway cells

The percentages of MSH2 and MLH1 protein-expressingairway epithelial cells in sputa of women who cooked withwood, dung, and crop residues correlated significantly nega-tively with the PM10 and PM2.5 levels in the kitchen ofbiomass-using households (Table 3).

Association between ROS, SOD, t ,t-MA, and mismatchrepair protein expression

The percentages of MSH2 and MLH1 protein-expressing air-way epithelial cells in biomass-using women were negativelycorrelated with ROS generation by sputum cells and urinarylevel of t ,t-MA. In contrast, SOD showed positive correlationwith both MSH2 and MLH1 expressions (Table 4).

In univariate analysis, the reduction in the percentages ofMSH2- and MLH1-positive cells was positively associatedwith increased oxidative stress, elevated urinary t ,t -MA, low-er education and family income, increasing age and exposureyears to biomass smoke, tobacco smoking habit of the hus-band, and cooking in a space adjacent to living room due tolack of separate kitchen. Even after controlling the influenceof the confounders in multivariate logistic regression analysis,

Fig. 3 Photomicrographs showing immunocytochemical expression ofMSH2 protein in the nuclei of airway epithelial cells exfoliated in spon-taneously expectorated sputum of biomass- (a , b) and LPG-using women(c , d). The frequency of MSH2-expressing basal and parabasal cells wasgreater among LPG users compared to that of biomass users. Carbona-ceous particle-laden alveolar macrophages were negative for MSH2 (b).The cell nuclei were counterstained with hematoxylin. Original magnifi-cation ×1,000

Fig. 4 Photomicrographs showing immunocytochemical expression ofMLH1 protein in the nuclei of airway epithelial cells of biomass- andLPG-using women. Expression of MLH1 was observed in basal andparabasal cells (a , b , d), while particle-laden alveolar macrophages (c)were negative. Note greater frequencies of MLH1-expressing cells inLPG-using control women (d) compared with that of biomass users (a–c). The cell nuclei were counterstained with hematoxylin. Original mag-nification ×1,000

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significant association remained between lower expression ofMSH2 and MLH1 proteins and ROS generation, urinary t ,t -MA, and depletion of SOD (Table 5).

Discussion

In a majority of poor households in rural India, unprocessedsolid biomass is still used as fuel for cooking family meals.Biomass burning emits smoke that remains in the cookingareas for long as the kitchens in general are poorly ventilated.A woman who cooks with biomass daily for at least 2 h getsexposed to this smoke that contains a host of health-damagingpollutants (Zhang and Smith 2003; Sinha et al. 2006; Naeher

et al. 2007). This study has shown that chronic exposure tobiomass smoke in these women can affect the expression ofmismatch repair proteins MLH1 and MSH2 in cells at thedirect route of air pollution exposure.

We used spontaneously expectorated sputum as a source ofairway epithelial cells because it contains a large number ofexfoliated airway cells. Besides, the process is simple, cost-effective, and noninvasive and therefore well suited for thepoorly educated women in remote villages who were notenthusiastic about giving biological samples through invasiveprocedures. Compared with age-matched LPG-using womenfrom the same locality, the percentages of airway epithelialcells expressingMSH2 andMLH1 proteins were significantlyreduced in biomass users, suggesting a deficiency in DNAmismatch repair in these women. Although we have notinvestigated the underlying mechanism of the observed defi-ciency of MMR pathway among biomass users, it may arisefrom loss of function of hMLH1 and hMSH2 genes due tomutation as reported in women with ovarian tumors(Stasikowska-Kanicka et al. 2009). Alternatively, epigeneticinactivation of hMLH1 and hMSH2 genes via promoterhypermethylation, as documented in sporadic colorectal can-cer (Kámory et al. 2003; Colombino et al. 2003;Chaksangchaichot et al. 2007; Vilkin et al. 2009; Vlaykovaet al. 2011), non-small cell lung tumors (Wang et al. 2003),and endometrial cancer (Zighelboim et al. 2007), may result in

Table 2 Immunocytochemical expression of proteins associated withDNA mismatch repair in airway epithelial cells of biomass- and LPG-using women

Parameters LPG-usingcontrol (n =68)

Biomass users(n =72)

p value

Percentage of MSH2-expressing cells

Mean ± SD 4.91±1.85 2.85±1.49 <0.0001a

Median (range) 6 (1–7) 3 (1–5) <0.0001b

Percentage of MLH1-expressing cells

Mean ± SD 4.22±1.68 1.96±0.81 <0.0001a

Median (range) 5 (1–6) 2 (1–3) <0.0001b

An average of 300 cells were examined for each participanta Statistically significant compared with LPG users in Student's t testb Statistically significant compared with LPG users in Mann–WhitneyU test

Fig. 5 Reactive oxygen species (ROS) and superoxide dismutase (SOD)levels in airway cells of LPG- and biomass-using women. Bars representstandard deviation of the mean values. The differences in ROS and SODlevels between the two groups were statistically significant (p <0.001) inStudent's t test

Table 3 Correlation between the percentage of mismatch repair protein-expressing airway epithelial cells and the level of particulate pollution inindoor air of biomass-using households

Correlation Pearson'scoefficient

Kendall'stau_b

Spearman's rhovalue

PM10 with MSH2 −0.364** −0.347** −0.352**PM2.5 with MSH2 −0.380** −0.351** −0.313**PM10 with MLH1 −0.424** −0.377** −0.359**PM2.5 with MLH1 −0.441** −0.412** −0.401**

**p <0.01, statistically significant

Table 4 Correlation between mismatch repair protein expression inairway cells, oxidative stress, and biomarker of benzene exposure

Correlation Pearson'scoefficient

Kendall'stau_b

Spearman's rhovalue

ROS with MSH2 −0.718** −0.650** −0.640**t ,t-MAwith MSH2 −0.539** −0.456** −0.512**SOD with MSH2 0.230* 0.219* 0.271*

ROS with MLH1 −0.595** −0.460** −0.473**t ,t-MAwith MLH1 −0.475** −0.587** −0.554**SOD with MLH1 0.249* 0.129* 0.148*

*p <0.05, **p<0.01

Environ Sci Pollut Res (2014) 21:2826–2836 2833

reduced expression of the MMR proteins. In view of thereports, future studies should explore genetic or epigeneticchanges to explain biomass smoke-induced MMR deficiency.

All participants of this study were never smokers and non-chewers of tobacco or betel nut, and exposure to environmen-tal tobacco smoke was similar among biomass and LPG users.Besides, they had no history of malignancy, were not currentlyunder medication, and were not exposed to ionizing radiationlike X-ray in recent times. Since cooking with biomass in-creases the risk of hypertension (Dutta et al. 2011) that altershMSH2 expression (Yeager et al. 2002), we excluded womenwith hypertension from this study. Therefore, it seems thatenvironmental or lifestyle factors did not play a significantrole in mediating MMR deficiency among biomass users.Similarly, it is difficult to attribute the change to ambient airpollution because the ambient air pollution levels in the studyareas were negligible as the villages were far from the high-ways and busy road traffic. Bicycle and cycle rickshaw werethe principal mode of transport and there were no air-pollutingindustries within 5 km radius. More importantly, since bio-mass and LPG users were neighbors, the influence of outdoorair pollution on DNA repair mechanism seems uniformamong the participants. The major differences between thesetwo groups were significantly higher exposure to particulateair pollution and benzene among biomass users. Indoor airpollution might have played a role in mediating MMR defi-ciency as the levels of PM10 and PM2.5 in indoor air and theconcentration of urinary t ,t -MA, a biomarker of benzeneexposure, were significantly elevated in biomass users.Biomass smoke constituents can influence the MMR pathwayvia generation of oxidative stress, a known cause of MMRinactivation (Chang et al. 2002). In support of this hypothesis,we found a strong and positive association between PM andurinary t ,t -MA levels with the generation of oxidative stress(excess ROS generation and SOD depletion) that correlatedwith deficient MMR protein expression in the airways.

The sputa of biomass-using women had increased thenumber of inflammatory cells, and their airway cells generatedmore ROS than that of LPG users. Like the present

observation, activation of inflammatory cells like neutrophilsand alveolar macrophages and generation of excess ROS havebeen observed following inhalation of PM (Li et al. 2003;Voelkel et al. 2003; Knaapen et al. 2004; Franco et al. 2008).Besides, the ultrafine particles (diameter <100 nm) which areabundant in biomass smoke (Kleeman et al. 2009) can directlygenerate ROS because of the presence of free radicals andoxidants adsorbed on their surface (Fubini et al. 2004). Inessence, our results suggest airway inflammation and conse-quent oxidative stress among biomass users. This can result inMMR deficiency because chronic inflammation and resultantoxidative stress have been shown to mediate MMR inactiva-tion (Chang et al. 2002). Therefore, inflammation can be a linkbetween biomass smoke exposure, oxidative stress, andMMRdeficiency. The consequence of decreased expression ofMSH2 and MLH1 proteins among biomass-using womencan be harmful as it may lead to microsatellite instability,initiation and promotion of carcinogenesis (Karihtala et al.2006), and consequent increased risk of developing cancerin the lung (Wang et al. 2003) and other organs (Akoum et al.2009; Stasikowska-Kanicka et al. 2009).

We acknowledge certain limitations of this study. For theevaluation of MMR pathway, we did only qualitative estima-tion ofMMRproteins by ICC, but could not extend the study togene or mRNA levels. This is one aspect that needs furtherinvestigation. Another point is that biomass smoke containshundreds of pollutants, but we only measured PM andbiomonitored benzene exposure. Thus, the observed declinein MMR protein expression in the airways of biomass userscould be due to the action of other pollutants that we did notmeasure. However, PM data is recognized as a proxy of poly-cyclic aromatic hydrocarbons in air (Bonner et al. 2005).Besides, the LPG-using control and biomass-exposed womenof our study were well matched, the difference in MMR proteinexpression between the comparable groups was highly signif-icant, and the sample size of the study seemed adequate forvalidation of the data to a larger population.

After adjusting for potential confounders, this study hasshown that chronic inhalation of smoke during daily house-hold cooking with biomass fuel was associated with deficien-cy in DNAmismatch repair protein expression in the airways.The change can be attributed to a great extent to high level ofindoor air pollution, airway inflammation, and resultant oxi-dative stress. Deficiency in mismatch repair causes microsat-ellite instability and increased risk of cancer. In agreementwith this, lung cancer is the fifth leading site of cancer amongnonsmoking women in eastern India (Nandakumar et al.2004). Since millions of poor women of the country still usebiomass for cooking in poorly ventilated kitchen, reduction ofsmoke exposure by improving kitchen ventilation and byincreasing combustion efficiency of the oven are urgentlyneeded. These simple measures can do wonders as installationof improved cook stoves has resulted in 85 % reduction of

Table 5 Association between oxidative stress, benzene exposure, andlower expression of mismatch repair proteins in airway epithelial cells ofbiomass-using women

Lower expressionof MSH2

Lower expressionof MLH1

ROS generation 2.22 (1.46–4.02) 2.02 (1.35–3.48)

Depletion of SOD 1.40 (1.05–2.53) 1.85 (1.19–2.97)

t ,t-MA in urine 1.64 (1.04–2.36) 1.44 (1.02–2.10)

Results are expressed as odds ratio with 95 % confidence interval inparentheses after controlling education, family income, age, exposureyears, husband's smoking habit, and adjacent kitchen as potential con-founders in multivariate logistic regression analysis

2834 Environ Sci Pollut Res (2014) 21:2826–2836

indoor concentration of PM2.5 in Guatemala (Albalak et al.2001) andmore than 70% reductions of carbonmonoxide andPM2.5 in Mexico (Cynthia et al. 2008). Switching to cleanerfuel such as solar energy is another and perhaps a betteroption. India being a tropical country with 9 months of brightsunshine in a year is a fit case for introduction of solar devicesas a clean and alternative energy source for domestic use.

Acknowledgments The study was supported by funds received fromCentral Pollution Control Board under the Ministry of Environment andForests, Government of India and Council of Scientific and IndustrialResearch, India.

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