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Atmospheric Environment 92 (2014) 384e393

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Atmospheric Environment

journal homepage: www.elsevier .com/locate/atmosenv

Characteristics of trace metals in fine (PM2.5) and inhalable (PM10)particles and its health risk assessment along with in-silico approachin indoor environment of India

P. Gursumeeran Satsangi a,*, Suman Yadav a, Atar Singh Pipal a, Navanath Kumbhar b

aDepartment of Chemistry, University of Pune, Pune 411 007, Indiab Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India

h i g h l i g h t s

� Measurements of indoor air quality in terms of PM concentration in Pune.� Characteristics of trace metals and its bioavailability in PM2.5 and PM10.� Assessment of health risk for carcinogenic metals in urban and rural environments.� Correlation matrix is employed for source identification of the trace metals.� In-silico study of Ni metal with nucleosomal protein.

a r t i c l e i n f o

Article history:Received 26 February 2014Received in revised form21 April 2014Accepted 22 April 2014Available online 25 April 2014

Keywords:IAQPM concentrationTrace metalsMetal toxicityHealth risk assessmentIn-silico study

* Corresponding author.E-mail address: pgsatsangi@chem.unipune.ac.in (P

http://dx.doi.org/10.1016/j.atmosenv.2014.04.0471352-2310/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Indoor concentrations of fine (PM2.5: aerodynamic diameter � 2.5) and inhalable (PM10: aerodynamicdiameter � 10 mm) particles and its associated toxic metals are of concern now-a-days due to its effectson human health and environment. PM10 and PM2.5 samples were collected from indoor microenvi-ronments on glass fiber and PTFE filter paper using low volume air sampler in Pune. The average con-centration of PM2.5 and PM10 were 89.7 � 43.2 mg m�3 and 138.2 � 68.2 mg m�3 at urban site while it was197.5 � 84.3 and 287 � 92 mg m�3 at rural site. Trace metals such as Cd, Co, Cr, Cu, Fe, Mn, Pb, Sb and Zn inparticulate matter were estimated by ICPeAES. Concentrations of crustal metals were found to be higherthan the carcinogenic metals in both the microenvironments. On the contrary the soluble and bio-availability fraction of carcinogenic metals were found higher thus it may cause the higher risk to hu-man health. Therefore, cancer risk assessment of carcinogenic metals; Cr, Ni and Cd was calculated.Among the carcinogenic metals, Ni showed highest cancer risk in indoor PM. The higher cancer riskassessment of Ni has been supported by In-silico study which suggested that Ni actively formed co-ordination complex with histone proteins (i.e. H3eNi/H4eNi) by maintaining strong hydrogenbonding interactions with Asp and Glu residues of nucleosomal proteins. Present In-silico study of Ni-histone complexes will help to emphasize the possible role of Asp and Glu residues in DNA methyl-ation, deacetylation and ubiquitinations of nucleosomal proteins. Hence, this study could pave the way tounderstand the structural consequence of Ni in nucleosomal proteins and its impact on epigeneticchanges which ultimately cause lung and nasal cancer.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Indoor air quality (IAQ) has received considerable attention inrecent years due to rapid economic growth and urbanization indeveloping countries. It is ubiquitous which emitted smoke from

.G. Satsangi).

solid fuel combustion in households (Zhang and Smith, 2003).The U.S. Environment Protection Agency (USEPA) has ranked in-door air pollution among the top five environmental risks. Morethan 40% of the world population relies on unprocessed solidbiomass and other solid fuels for their daily household cookingneeds (Taneja et al., 2008). In India, larger part of population livesin rural areas, and meeting their energy requirements in a sus-tainable manner which continues to be a major challenge for the

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393 385

country (Lawrence et al., 2005). Traditional biomass resourcesstill meet an overwhelming proportion of the rural energy de-mand. The main reason of this continued dependence on biomassis the failure of the commercial fuels such as kerosene and LPG topenetrate the rural areas on account of high costs and lowaccessibility (Khare and Gupta, 2000; Balakrishnan et al., 2003).Hence, the indoor air quality issue is more important for Asiancountries due to its diversity, high population density, diverseliving habit, and growing economy resulting urbanization andindustrialization.

Most of the people spend 80e90% of their time indoors, whereexposure to majority air pollution is quite different from those ofoutdoors (Ashmore and Dimitroulopoulou, 2009). Indoor con-centrations of some pollutants are higher than their levels outdoorand most of the time people are suffering from these pollutants,therefore indoor air pollution may pose a greater health threatthan outdoor pollution (Taneja et al., 2008). Fine and inhalableparticles are much more concern now-a-days because they de-posit deep in the respiratory tract and increases respiratory dis-eases in human. On particulate matter (PM), there is a broad rangeof chemical species, ranging from elemental to organic and inor-ganic compounds (Callén et al., 2009). Among the inorganiccompounds, most important are the trace metals which areemitted by various natural and anthropogenic sources, such as,crustal materials, road dust, construction activities, motor vehicleemissions, coal and oil combustion, incineration and other in-dustrial emissions (Shah et al., 2006). In addition, several otherprocesses including smoking, cooking, space heaters, and re-suspension may also contribute to the indoor particulates (Joneset al., 2000). These particulates and its associated trace metalshave been linked with both short and long-term adverse healtheffects which mostly include chronic respiratory diseases such aslung cancer, heart diseases and spoil other organs (Magas et al.,2007; Wild et al., 2009). It is essential to be familiar with thecurrent scenario of indoor air quality and its effects on environ-ment as well as human health, the different studies have beencarried out in worldwide. Several epidemiological studies haveshown a correlation between elevated levels of airborne particu-lates and increased rate of morbidity and mortality (Pope, 2000).Moreover, toxicological studies have suggested that it is the sol-uble trace element content of PM instead of total element contentthat has more direct links to harmful effects. Therefore, in additionto simple determination of particle size and shape, a qualitativeand quantitative description of the particles for detailed toxico-logical evaluation of PM and its associated elements is essential.Some studies have been characterized indoor air quality in northregion of India (Massey et al., 2013; Habil et al., 2013) but not muchwork has done in south west part of India. There are great needsfor better characterization of indoor air quality in terms of par-ticulate matter and its trace metals characterization to perceivethe current scenario and its effects on human health. Thus, thepresent study has been carried out at urban and rural microen-vironments of Pune city to see the present status of indoor airquality covering the following objectives (i) Mass concentrationslevel of fine (PM2.5) and inhalable (PM10) at urban as well as ruralenvironments, (ii) characteristics of trace metals in fine andinhalable particles (iii) bioavailability of trace metals (iv) healthrisk assessment of carcinogenic metals in urban and rural envi-ronments and (v) In-silico study of metal with nucleosomal pro-tein. This study would provide baseline data to the health relatedpollution status of the urban and rural indoor atmosphere of Puneand elsewhere. The data obtained would be useful towards airpollution abatement programs and could motivate some futuristicstudies on comparative metal distribution, source contributionand enhancement relative to the clean background.

2. Materials and methods

2.1. Site description

Pune (18�320 N, 73�510 E) is located 560 m (1840 ft) above sealevel on the western margin of the Deccan plateau. Pune has atropical wet and dry climate with average temperatures rangingbetween 20 and 33 �C. The monsoon lasts from June to Octoberwith good rainfall and temperatures ranging from 10 to 28 �C. Insummer temperature was ranging from 27.4 to 40.3 �C and inwinter from 10.2 to 23.0 �C. Detailed site description has been givenelsewhere (Satsangi and Yadav, 2014).

Depending upon their different geographical locations, vehic-ular activities, population density, two sampling areas wereselected in Pune; Mundhwa and Warje. Mundhwa, is locatedw12 km away and towards east of the main Pune city. Warje islocatedw10 km away and towards the south west of the main Punecity (Fig. 1). These areas include both urban and rural sites. Indoorsamples were collected at 6 sites located in different parts of thesetwo areas i.e. 3 rural and 3 urban sites. Houses were selected on thebasis of their locations i.e. urban and rural. The urban houses wereclosely build houses, modern type, as they have been built recently;usually their environment is of high traffic during the office hours,outdoor environment has shopping complexes and small marketsand less greenery in the surrounding areas. Rural houses are small,basically made of mud, grasses and are situated in areas with littletraffic and lots of greenery around. The occupants were givenquestionnaires to know the daily activity pattern in indoor such ascooking, cleaning, heating, the number of occupants, other activ-ities carried in the indoor environment of the homes along with thesurvey of the houses to know the sources responsible for the par-ticulate emission and related health effects. Table 1 depicts generalactivity schedule in indoor microenvironments of both urban andrural sites of two different sampling locations.

2.2. Sample collection

The PM2.5 and PM10 samples were collected with the help of lowvolume air sampler (Mini Vol TAS) during the period of June 2012eMay 2013 from urban and rural environments of Pune. PM2.5 andPM10 samples were collected on 47 mm diameter PTFE and quartzfiber filters, respectively, for 24 h thrice in a week. Filter paperswere weighed thrice before the sampling and after sampling foraccuracy by using four digits microbalance (Shimadzu AUX 220;sensitivity � 0.1 mg). Filters were handled only with tweezerscoated with teflon tape to reduce the possibility of contamination.After weighing, samples were stored in refrigerator at 4 �C till thefurther analysis.

Prior to extraction, all the plastic and glassware used forextraction and storage were cleaned by soaking in 2% HNO3 over-night followed by rinsing with de-ionized and double distilledwater several times. The exposed filters were extractedwithmilli-Qwater followed by acid (HNO3) extraction for determination ofmetals by using ultrasonicator and hot plate, respectively.

2.3. Quality control

The unexposed filter papers were used for monitoring blank testbackground contamination, which were processed with fieldsamples. The blank filters were taken thrice during the studyperiod. Background contamination of heavy metals was deter-mined by subtracting the field blank values from concentrations.Field blank values were very low, typically below or around themethod detection limits. In this study, the background contami-nation was used to correct measurements.

Fig. 1. Map showing the sampling sites.

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393386

At least 10% of the samples were evaluated by spiking with aknown amount of metal to calculate the recovery efficiencies.Table 2 represents the recovery test results andminimum detectionlimit (MDL) of all the metals. The range of recovery efficiency was91.2%e110.6%. For reproducibility test, analysis for the same stan-dard solution was repeated 10 times. Reproducibility testsdemonstrated the stability of instruments.

2.4. In-silico analysis

In-silico study was performed to understand the structuralconsequences of Ni metal in context of nucleosomal histone pro-teins (H3 and H4) and demethylase JMJD2A enzyme. The startingcoordinates for H3 and H4 histone proteins were extracted fromcrystal structure of nucleosome core particles, whereas for deme-thylase JMJD2A were adopted from crystal structure of histonedemethylase (Mohideen et al., 2010). The extracted crystal

structures were subjected for 10,000 steps of steepest descentminimizations using Spdbviewer software (Guex and Peitsch,1997). Minimized structures were analyzed for hydrogen bondinginteractions using Chimera software. The pictorial presentation ofH3eNi, H4eNi and JMJD2A-Ni complexes were made usingChimera software (Pettersen et al., 2004).

3. Results and discussion

3.1. Mass concentration of fine (PM2.5) and inhalable (PM10)particles in urban and rural environments

The average concentration of PM2.5 and PM10 were89.7 � 43.2 mg m�3 and 138.2 � 68.2 mg m�3 at urban site while itwas 197.5 � 84.3 and 287 � 92 mg m�3 at rural site. The concen-tration of fine and coarse particles was approximately two timeshigher at rural environment in comparison to urban environment.

Table 1General activity scheduled in indoors sampling locations.

Urban Rural

Building material Houses made up of bricks, cement and well painted Houses made up of bricks, stone, mud, bamboo and grassesand not well painted

Fuel used in kitchen L.P.G, microwaves, electric ovens Wood, thrash/grass, crop residues, cow-dung cakes, keroseneOther activities Incenses sticks, mosquito coils/room fresheners Incenses sticks, mosquito coilsVentilation in living room Acceptable (large windows and exhaust fans) Not acceptable (small windows)Outdoor activities Heavy as well as light vehicular emissions, road dust,

agricultural and dust, agricultural and wood burningAgricultural waste burning, biomass burning, suspension of dustdue to dry open fields construction activities

Cooking frequency 2e3 times/day (No use of electrical chimney) 3e4 times/dayPopulation (family member) Average 5 members/family Average 8e10 members/familyDusting/vacuuming/Sweeping 2 times/day 2 times/day

200

300

400 Urban

3 )

PM10 PM2.5

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393 387

Higher concentration in rural environment may be due to the do-mestic activities such as coal, biomass burning and other inferiorfuels (dry thrashes and crop residues) which are used for cookingpurpose due to socioeconomic status. The indoor air quality mea-surement standards are still not available therefore present resultsare compared with ambient standards which are given by differentnational and international bodies. The concentrations of PMobserved in this study are considerably higher than the annualaverages standards stipulated by the National Ambient Air Quality(PM2.5 ¼ 40 mg m�3 and PM10 ¼ 100 mg m�3) and WHO standards(PM2.5¼10 mgm�3 and PM10¼ 20 mgm�3). Massey et al. (2013) wasobserved PM levels (PM10: 242.53 mg m�3 and PM2.5:164.60 mg m�3) in north Indian rural houses which was lower thanour present study. Smith et al. (1994) also observed higher con-centration of inhalable (PM10: 2000 mg m�3) in indoor environmentof Pune from cooking activities using wood fuel. Similarly, Pandeyet al. (1990) in Nepal found the PM2.5 concentration(8200 mg m�3) from cooking using wood/crop residues fuels.Albalak et al. (1999) did also observe higher PM10 concentration(1830 mg m�3) in Bolivia Kitchens.

Fig. 2 shows the monthly trends of PM2.5 and PM10 mass con-centration at urban as well as rural microenvironments. Higherconcentration of PM2.5 (166.7 mgm�3) and PM10 (236.1 mgm�3) wasobserved in the month of Feb., while lower was in the month ofAug. (PM2.5: 46.3 mg m�3 and PM10: 55.5 mg m�3) at urban envi-ronment (Fig. 2). Whereas at rural site it was higher in the month ofOct. (PM2.5: 261 mg m�3) and July (PM10: 398.1 mg m�3) and lowerwere in May (PM2.5: 23.9 mg m�3, PM10: 83.3 mg m�3) (Fig. 2). Theseasonal trend of PMmass concentration indicates that the fine andinhalable particles concentration were higher during the winterseason (Nov. to Feb.) in comparison to monsoon (June to Sept.) aturban environment but in the case of rural environment, it washigher in both monsoon and winter seasons. This higher concen-tration of PM was observed at rural microenvironments in com-parison to urban microenvironment which may be due to little

Table 2Detection limits, recovery rate and reproducibility for different metals.

Detection limit (ppb) Recovery (%) Reproducibility (ppm)

Cu 0.8 102.3 1.3Zn 0.2 96.2 0.45Mn 0.1 98.2 0.85Fe 0.5 108.2 0.84Ca 0.3 103.8 0.43Cr 0.7 106.2 0.34K 30.7 97.4 1.81Na 12 91.2 1.41Ni 0.6 103.9 0.59Cd 0.2 101.8 0.14Al 0.1 110.6 1.19Mg 0.1 99.2 1.18

ventilation and cooking activities on traditional stoves, open-firethree stone stoves (i.e. brick stoves) by the use of wood, coal, cowdung and biomass. In rural environment, cooking is done outsidethe houses during the dry season whereas in monsoon season thecooking activities are carried out inside the houses which result inhigher concentration of PM. It is also observed that in Pune, ruraland suburban households have a number of cooking arrangements.Moreover, these fuels are very less energy efficient and create morepollutants. According to survey of distribution of cooking fuels usedin urban and rural environment of India indicates that more than80% solid fuels are use for cooking purpose in rural environmentwhile in urban environment 50% LPG and 40% solid fuels are usedfor cooking (Kumar, 2009). Apart from this, infiltration of ambientpollution is also responsible for higher concentrations in indoorenvironment via building leakage, and air exchange rate (ventila-tion) in the building from re-suspension of road as well as soil dustand emission from solid waste burning.

3.2. Characteristics of metals in urban and rural environment

Fig. 3 shows the average concentrations of metals (Na, Mg, K, Ca,Fe, Mn, Cu, Zn, Cr, Cd, and Ni) in fine and inhalable particles at urbanand rural microenvironments in the entire study period. In thepresent study, analyzed metals are distributed into three categoriesi.e. crustal, (Na, Mg, K, Ca and Fe) trace (Mn, Cu and Zn) andcarcinogenic (Cr, Ni and Cd). Crustal elements were found to bedominant in comparison to carcinogenic metals influencing theindoor urban and rural micro-environment in fine and inhalable

June July Aug Sep Oct Nov Dec Jan Feb March April May0

100

200

300

400

20132012

Rural

0

100

Con

cent

ratio

ns (μ

g m

-

Fig. 2. Monthly average mass concentration of PM10 and PM2.5 at urban and ruralenvironment.

Fig. 3. Total average metal concentration in PM10 and PM2.5 at urban and rural sites.

Table 3Percent water soluble fraction of different metals in urban and rural indoormicroenvironment.

Metals Urban Rural

PM10 PM2.5 PM10 PM2.5

Ca 20 12 14 31Cd 42 21 48 42Cr 14 33 29 42Cu 5 19 9 6Fe 1 1 1 2K 72 40 52 44Mg 3 2 2 9Na 40 43 47 53Ni 13 41 93 36Zn 5 12 6 21Mn 4 10 7 12

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393388

particles. The crustal elemental concentration is affected bydifferent local and transported sources in different season.Amongst the metals, Ca is the most abundant in fine and inhalableparticles at both microenvironments. At urban site, Ca concentra-tion observed 52.44 mg m�3 inhalable and 43.94 mg m�3 in finewhereas 53.97 mgm�3 in inhalable and 47.21 mgm�3 in finemode atrural site followed by Mg 45.83 mg m�3 in inhalable and31.40 mg m�3 in fine particles at rural environment whereas at ur-ban environment it was 43.67 mg m�3 in inhalable and30.89 mg m�3 in fine mode particles. The concentration of Ca andMg were found to be moreover similar in both the environments.This may be due to the Ca and Mg is rich in crust and they aremostly found in the study region. This fact is supported by earlierstudy of Satsangi and Yadav (2014) that Ca and Mg are present inthe form of calcite (CaCO3) and dolomite (CaMg (CO3)2). Among thetrace metals Mn is highest (0.27 mg m�3) in inhalable and0.08 mg m�3 in fine fraction in urbanwhile at rural it is 0.21 mg m�3

in inhalable and 0.12 mg m�3 in fine fraction followed by Cu and Znat both sites (i.e. urban and rural). Contribution of Ni was highestamong the carcinogenic metals in inhalable fraction at rural sitefollowed by Cr and Cd, while at urban site concentration of Cr wasfound to be higher followed by Ni and Cd in both inhalable and finefraction.

Table 3 shows the percent water soluble fraction of differentmetals at urban and rural microenvironments. Na and K in bothinhalable and fine fraction at urban and rural site showed highsolubility but it is not of more concern as these metals are not toxic.Ni (93%) showed highest solubility in the inhalable fraction fol-lowed by Cd (48%) and Cr (29%) while in fine fraction Cr and Cd(42%) showed slightly high solubility than Ni (36%) at rural site. Aturban site Cd (41%) showed high solubility in inhalable fractionwhile in fine fraction Ni showed high solubility and consequently

are especially harmful to the environment and exposed people.These observed results show that soluble crustal metals are lessproblematic with regard to soluble (bioavailable) carcinogenicmetals. Moreover, higher bioavailability index value of carcinogenicmetals (Fig. 4, detail is given in Section 3.4) indicates that it may betotally available to physiological activities once inhaled to lungsystem. The carcinogenic metals such as Cr, Cd and Ni are higher inrural environment for both total and soluble form (Table 3) whichindicates that their toxicity and health risk is more to ruralinhabitants.

3.3. Statistical analysis for source identifications

Tables 4a and 4b display the correlation matrix at urban andrural environment in fine and inhalable particles and their r values

Fig. 4. Bioavailability index of trace metals at urban and rural environments.

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393 389

were observed between 0.52 and 0.97 in PM2.5 and 0.56 and 0.96 inPM10 at urban. While at rural environment, it was observed be-tween 0.55 and 0.98 in PM2.5 and 0.51 and 0.99 in PM10. The boldvalues in the parenthesis showed significant correlation amongstthe metals which indicates their origin from similar sources(Tables 4a and 4b). In indoor environment Zn is widely used as acoating for iron, steel and brass utensils. Combustion of coal andother fossil fuels leads to the release of Ni into the atmosphere.Crustal elements are generated from mechanical disruption of theearth’s crust and are more enriched in the coarse mode. Zn is alsoenriched in the coarse mode than in the fine mode at both theenvironment (Singh et al., 2002). In general, it is noticed that thecrustal metals are also emitted from fossil fuel and wood com-bustion (Abiye et al., 2013). While the carcinogenic metals such asCr and Ni are contributed from the ware and tare of utensils. Fe issignificantly correlated with all the metals at urban site except thanK (Table 4a) which indicates similar sources. These metals areassociated with an excessive use of electric appliances,

Table 4aCorrelation matrix amongst the elements in PM10 and PM2.5 at urban.

Ca Cd Cr Cu Fe

PM10 Ca 1Cd �0.02 1Cr �0.30 0.13 1Cu 0.27 �0.15 0.04 1Fe [0.64] �0.09 0.25 [0.92] 1K 0.00 0.04 �0.13 0.16 0.04Mg [0.85] �0.09 �0.28 0.43 [0.60]Na [0.83] 0.04 �0.39 0.28 [0.68]Ni 0.46 �0.27 �0.01 0.30 [0.59]Zn 0.43 �0.15 �0.04 [0.96] [0.95]Mn 0.35 �0.14 �0.10 [0.97] [0.91]

PM2.5 Ca 1Cd �0.38 1Cr �0.65 0.02 1Cu 0.20 �0.14 �0.02 1Fe [0.77] �0.30 �0.43 [0.66] 1K 0.00 �0.27 [0.50] 0.14 0.06Mg [0.81] �0.30 �0.58 0.45 [0.92]Na 0.48 �0.23 �0.05 0.35 [0.56]Ni �0.61 �0.10 [0.53] �0.02 �0.43Zn [0.75] �0.28 �0.43 [0.56] [0.95]Mn [0.52] �0.25 �0.28 [0.85] [0.89]

Bold values in parenthesis indicate that the correlation is significant at the 0.01 level.

metallurgical products and biogenic sources and may be respon-sible for their lopsided contribution in indoor particulates (Shahet al., 2006). Mn is also having good correlation with Ca, Fe, K,Mg, and Zn as it is mainly used in the production of steel, non-ferrous alloys and other products such as porcelains, ceramics,dyes, fungicides, disinfectants and food additives. Apart from this,Cu shows good correlation with metals like Fe, Mg, Zn, Mn and K inboth urban and rural environment. In urban environment, it isbelieved to be associated with excessive use of electric appliances,mainly for heating/cooling purpose while in rural it is probably dueto the high wind blown dust, decaying vegetation, burning of cropresidues and fossil fuels (Martuzevicius et al., 2008).

3.4. Bioavailability assessment of metals

Bioavailability is expressed as the percentage of the solubleelement content extracted which is supposed to be easily assimi-lated to living beings system by water with respect to its totalcontent in PM. (Smichowski et al., 2005). The fraction of the tracemetals which are readily bioavailable once inhaled to the respira-tory systemcauses the highest risk to human health. It is assumedthat the soluble fractions are labile and completely bioavailable, abioavailability index (BI) can be calculated according to followingeq. (1)

BI ¼ CS=CT (1)

where CS and CT are the soluble and total concentrations,respectively.

Fig. 4 shows the bioavailability index of metals at urban andrural environments which indicating that Ni has higher bioavail-able index in inhalable at rural environment while at urban Ni andCd have second most bioavailable index after crustal metals. It isinteresting to note that Ni and Cd were observed as mostbioavailable as well as they showed higher cancer risk in indoor PMat rural and urban environment which indicates that they arecausing short-term as well as long-term health effects to the peoplewho are exposed to these metals. Other studies also exemplifiedthe higher concentration of Cd in the soluble fraction representingthe higher bioavailability index among the toxic metals and suggest

K Mg Na Ni Zn Mn

1�0.04 10.39 [0.61] 10.02 [0.56] 0.41 10.13 [0.64] 0.37 0.37 10.16 [0.56] 0.34 0.29 [0.99] 1

1�0.02 10.39 0.42 1

�0.03 �0.46 �0.27 10.08 [0.97] [0.52] �0.37 10.13 [0.83] 0.44 �0.27 [0.87] 1

Table 4bCorrelation matrix amongst the elements in PM10 and PM2.5 at rural.

Ca Cd Cr Cu Fe K Mg Na Ni Zn Mn

PM10 Ca 1Cd �0.56 1Cr �0.70 0.45 1Cu 0.03 0.03 0.44 1Fe [0.57] �0.31 �0.18 [0.51] 1K 0.36 �0.10 0.03 0.36 [0.80] 1Mg [0.83] �0.46 �0.62 0.10 0.44 [0.58] 1Na 0.36 �0.20 0.07 0.22 0.36 [0.63] 0.23 1Ni 0.20 �0.16 �0.19 �0.14 �0.13 �0.22 �0.09 0.07 1Zn [0.75] �0.40 �0.55 0.26 [0.78] [0.61] [0.96] 0.21 �0.15 1Mn 0.49 �0.24 �0.30 0.34 [0.94] [0.80] [0.71] 0.30 �0.13 [0.77] 1

PM2.5 Ca 1Cd �0.30 1Cr �0.34 [0.60] 1Cu 0.39 �0.24 �0.30 1Fe [0.57] �0.38 �0.42 [0.87] 1K 0.12 0.23 0.33 0.03 0.01 1Mg [0.73] �0.43 �0.49 [0.74] [0.93] 0.01 1Na 0.21 �0.35 �0.11 0.27 0.18 0.08 0.17 1Ni �0.13 0.38 0.29 �0.06 �0.08 0.07 �0.13 �0.16 1Zn [0.67] �0.24 �0.31 [0.83] [0.80] 0.07 [0.86] 0.23 �0.11 1Mn [0.55] �0.27 �0.30 [0.90] [0.97] 0.09 [0.87] 0.20 0.01 [0.79] 1

Bold values in parenthesis indicate that the correlation is significant at the 0.01 level.

Table 5Excess Cancer Risk (ECR) assessment of carcinogenic metals in urban and rural environment.

Site Elements PM size Concentration mg m�3 Inhalable unit risk (mg m�3)�1 ECR � 10�6

Urban Cr PM2.5 0.0286 1.8 � 10�3 51.48PM10 0.0579 1.8 � 10�3 104.2

Ni PM2.5 0.0085 1.2 � 10�2 102PM10 0.0228 1.2 � 10�2 273.6

Cd PM2.5 0.0013 2.4 � 10�4 0.31PM10 0.0015 2.4 � 10�4 0.36

Rural Cr PM2.5 0.0312 1.8 � 10�3 56.1PM10 0.0301 1.8 � 10�3 54.1

Ni PM2.5 0.0158 1.2 � 10�2 189.6PM10 0.167 1.2 � 10�2 2006.4

Cd PM2.5 0.0028 2.4 � 10�4 0.67PM10 0.0017 2.4 � 10�4 0.4

40

50

60

70

80

e di

strib

utio

n

Urban Rural

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393390

it is harmful to the environment and exposed person (Feng et al.,2009; Yadav and Satsangi, 2013). In this study, it is clear that Niand Cd have high proportion in the water soluble fraction (ultra-sonic digestion) indicating high mobility of these metals whichresulted that they have high bioavailability. Therefore, Ni and Cdcan be considered as important elements when assessing thetoxicity of rural and urban dust particles. The study done bySchleicher et al. (2011) also found that Cd has high mobility inwater-extractable fraction and bioavailability in ambient air ofBeijing. In the present study, it is clearly indicating that thesebioavailable metals are easily intake by people who spend their 80%time in indoor environment.

0

10

20

30

Health effects

Perc

enta

g

Nause

a and

dizzin

essMen

tal

fatigu

eSinus

Skin

proble

ms

Allergi

es

Cold an

d flu

Eye an

d

nose

irrita

tion

Heada

ches

Fig. 5. General symptoms of diseases based on survey analysis during sampling.

3.5. Cancer risk assessment of toxic metal in PM for indoorenvironment

Table 5 shows the cancer risk assessment of carcinogenic metal(Cr, Ni and Cd) in this study which creates more health problems tohuman if they are exposed to indoor air pollutants. In order todemonstrate the risks of the PM10 and PM2.5 and its bound metalssuch as Cd, Cr and Ni in terms of excess cancer risks (ECRs) werecalculated using the following formula (Hieu and Lee, 2010).

Fig. 6. Depicted the energy minimized conformation of NieH3 (metal-histone) nucleosomal protein complex.

Table 6Intermolecular hydrogen bonding interactions between H3eNi/H4eNi and JMJD2A-Ni complexes.

Sr. No. Residues involved Distance in �A

Histone H3 with Ni1 Asp 77 OD1 . Ni 2.2672 Glu 73 OE1 . Ni 2.136

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393 391

ECRðinhalationÞ ¼ ambient concentrationof pollutant�mgm�3

�

�unit risk�mgm�3

��1

(2)

The information on the carcinogenic types and the unit risks ofthe metals was obtained from the USEPA data base for IRIS (Inte-grated Risk Information System) (available at http://www.epa.gov/iris/). It should be noted that the usual goal of cancer risk is set at1 � 10�6 which corresponds to a lifetime exposure to unpollutedambient environment. Calculated excess cancer risks of Cr and Niwere found to be higher than such usual goal suggesting potentialhealth risks for the human (Table 5). On the basis of this it can beconcluded that these metals are very harmful and causing healthproblems for indoor people. Indoor air pollution is associated witha variety of health effects of both an immediate and long-termnature (Colome et al., 1992). During the time of the indoor sam-pling, questionnaire was also filled by the occupants. According tothe responses by the respondents in our survey, the mostfrequently occurring symptoms are shown in Fig. 5. The resultshows that these problems were dominant where the use oftraditional stoves with biomass fuel like wood, cow dung cakes andkerosene were more common in practice. Many toxic heavy metalswere observed predominantly in fine particles which have a moredirect link to human health than those in coarse particles. The effectof heavy metals depends on the concentration, toxicity and dura-tion of exposure.

3 Gln76 OE1 . Ni 3.762Histone H4 with Ni4 Asp 90 OD1 . Ni 1.9125 Asp 90 OD2 . Ni 2.9486 Glu 92 OE1 . Ni 2.049Demethylase with Ni7 His 276 NE2 . Ni 2.2088 His 188 NE2 . Ni 2.5319 Glu190 OE2 . Ni 1.88410 OGA 1 O2 . Ni 2.06711 OGA 1 O20 . Ni 2.14712 Ser 196 OG . Ni 3.878

3.6. In-silico study of Ni with nucleosomal histone proteins H3 andH4

Most potent activity of metals deposited on particulate matter(PM) causing human cancer via inhalation has been observed byearlier studies (International Agency for Research on Cancer, 1990;Doll et al., 1977). In the present study, among the carcinogenicmetals, Ni has shown higher cancer risk in indoor PM at rural andurban environment as discussed in previous sections. Therefore, In-

silico study has been performed only for Ni metal with nucleosomalhistone proteins such as H3 and H4. Many studies have reportedthat higher concentration of Ni metal compounds is carcinogenicand is associated with lung and nasal cancers (International Agencyfor Research on Cancer, 1990; Doll et al., 1977). Different mecha-nisms have been proposed for nickel carcinogens; mutagenesis andepigenetic. Ni particles contained in PM are localized close to thecell nucleus by phagocytosis. Inside the cell, Ni particles are free tointeract with inner cellular components and selectively targetnucleosomal proteins by interactingwith specific coordination sites(Sen and Costa, 1985). Besides this, Ni is also involved in otherepigenetic changes includes aberrant DNA methylation, deacety-lation of histone H3/H4 proteins and increase in dimethylation ofH3K9. These epigenetic changes produce adverse effect on chro-matin folding consequently leads to gene silencing in normal cells(Peana et al., 2013). Similarly, alternations in gene expressions andchanges in chromatin architecture were noticed in cells exposed toNi compounds (West et al., 2012; Qingdong et al., 2006; Peana et al.,2013). The increase in di-methylation of H3 lysine (K) 9 in Chinesehamster G12 cells exposed to Nickel chloride and its impact on gene

Fig. 7. Depicted the energy minimized conformation of Ni-H4 (metal-histone) nucle-osomal protein complex.

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393392

silencing and heterochromatin formation was studied using geneexpression and cell cytotoxicity assays (Qingdong et al., 2006).Crystallographic study was made to understand the mechanism ofdemethylation of H3 lysine (K) 9 in complex with Ni and othercofactors (Mohideen et al., 2010).

Since the discovery of Ni-metal in epigenetic mechanism, itsstructural role has not been studied yet in detail. Hence, in pre-sent scenario we have tried to explore the detailed mechanisms ofNi-metal co-ordination interactions with nucleosomal proteins(H3/H4) and H3K9 demethylase enzyme using In-silico study.Fig. 6 depict the energetically minimized conformation of Ni incomplex with histone (H3) protein. The Ni forms strong inter-molecular hydrogen bonding interactions with carboxyl oxygen ofglutamic (Glu 73) and aspartic acids (Asp 77) residues of H3protein. Additionally, Ni is also involved in weak interaction withGln 76 of H3 protein in order to stabilize H3eNi complex (Fig. 6,Table 6). These co-ordination interactions between Asp 77 OD1 .Ni, Glu 73 OE1 . Ni and Gln 76 OE1 . Ni are maintained duringminimization study (Fig. 6, Table 6). Similar kinds of metal-histone co-ordination interactions were also noticed in earliercrystallographic studies of nucleosomal proteins (Peana et al.,

Fig. 8. Energy minimized structure of Ni-JMJD2A enzyme co

2013; Qingdong et al., 2006). The next energy minimized struc-ture of histone H4eNi complex is depicted in Fig. 7, which isstabilized by hydrogen bonding interactions between Asp 90 OD1. Ni and Glu 92 OE1 . Ni (Fig. 7, Table 6). Further, metal-histonecomplex stability was also expected from weak co-ordinationbonding between second carboxyl oxygen (OD2) of Asp 90 andNi metal ion (Table 6). The Asp and Glu residues were activelyinvolved in co-ordination bonding with Ni in both (NieH3 andNieH4) metal-histone complexes (Figs. 6 and 7). Therefore, Nimay inhibit the activity of nucleosomal protein by forming H3eNi/H4eNi complexes, which might be stabilized by above dis-cussed interactions. Hence, present In-silico study of Ni-histonecomplexes would be helpful to emphasize the possible role ofAsp and Glu residues in proper functioning of nucleosomal pro-teins and its impact on normal gene regulations.

Similarly to explore the inhibition mechanism of H3K9 deme-thylase (JMJD2A) enzyme by Ni metal, energy minimization wasperformed over JMJD2A-Ni complex. Various co-ordinationbonding interactions between Ni with JMJD2A and 2-oxoglutarate(OGA) JMJD2A co-factor have been observed (Fig. 8). Imidazolenitrogen of two histidine residues (His 188 and His 276) and car-boxylic oxygen of glutamate (Glu 90) of JMJDAwere involved in co-ordination interaction with Ni metal in order to give structuralstability to JMJD2A-Ni complex (Table 6). Similarly, the 2-oxoglu-tarate (OGA) JMJD2A co-factor is also accessible for two metal co-ordination bonding with Ni. All these minimized complexes(Figs. 6e8) showed close resemblance with available crystallo-graphic information of nucleosomal protein in respect to structuralgeometry as well as co-ordination bonding interactions (Qingdonget al., 2006; Mohideen et al., 2010). Thereby forming co-ordinationinteractions with histone proteins and cofactors (Table 6), Ni metalion can efficiently inhibit demethylase activity of JMJD2A enzyme,which is necessary for normal gene expressions.

Therefore, structural studies of histone H3eNi/H4eNi andJMJD2A-Ni complexes may help understand the structural conse-quence of Ni in DNAmethylation, deacetylation and ubiquitinationsof nucleosomal proteins. Furthermore considering the role ofnucleosomal protein in normal gene regulation and its defect proneto human diseases (lung and nasal cancer), present structural studycould pave the way to understand the structural consequence ofhigh Ni concentrations in gene silencing and heterochromatinformations in human diseases.

mplex showing various hydrogen bonding interactions.

P.G. Satsangi et al. / Atmospheric Environment 92 (2014) 384e393 393

4. Conclusions

In the present study, indoor air quality of microenvironment hasbeen characterized in terms of mass concentration level and metalscomposition of PM along with its health risk assessment to humanhealth. In-silico study has also been performed for Ni metal to un-derstand the binding of metal with nucleosomal proteins. Averageconcentration of PM2.5 and PM10 were 89.7 � 43.2 mg m�3 and138.2 � 68.2 mg m�3 at urban site while 197.5 � 84.3 and287 � 92 mg m�3 at rural site. It reveals that the urban and ruralindoor environment of Pune city is currently under a continuedsevere pollution threat. Crustal metals were found to be dominantin comparison to carcinogenic metals influencing the indoor urbanand rural microenvironment in fine and inhalable particles at bothsites. On the contrary, the soluble and bio-availability fraction ofcarcinogenic metals were found higher at both the microenviron-ment and causes the higher risk to human health once inhaled. Thecancer risk assessment of carcinogenic metals; Cr, Ni and Cd wascalculated along with bioavailability index of metals which in-dicates that Ni and Cd were most bioavailable and showed highestcancer risk in indoor PM at rural and urban environment.Bioavailable carcinogenic metals have higher cancer risk thuscausing first short-term diseases and after continuous exposure ofthese they may cause long-term disease. Further, In-silico studyperformed over nucleosomal protein in complex with Ni metalsuggests that Ni is actively involved in co-ordination bonding withGlu and Asp residues of histone proteins and may inhibit theiractivity by forming H3eNi/H4eNi complexes. This ultimately re-sults the structural consequence of Ni in DNA methylation, deace-tylation and ubiquitinations of nucleosomal proteins and causes tohuman diseases such as lung and nasal cancer. Hence, present In-silico study of Ni-histone complexes would be helpful to emphasizethe possible role of Asp and Glu residues in proper functioning ofnucleosomal proteins and its impact on normal gene regulations.This study has further supported to higher cancer risk assessmentof Ni.

Acknowledgments

Authors wish to thank UGC New Delhi (No: 41-324/2012 (SR))and BCUD (Finance/2013-14/776(2)), Pune for financial assistance.Authors express their gratitude to SAIF, Mumbai for analysis on ICP-AES and Head, Department of Chemistry, University of Pune for hisencouragement.

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