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International Journal of Hygiene and Environmental Health

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Polycyclic aromatic hydrocarbons and their quinones modulate the metabolic prole and induce DNA damage in human alveolar and bronchiolar cells
Deepak Gurbani a,g , Santosh Kumar Bharti f , Ashutosh Kumar b , Alok K. Pandey b , Godson R.E.E. Ana e , Ambrish Verma c , Altaf Husain Khan c , Devendra K. Patel d , M.K.R. Mudiam d , Swatantra K. Jain g , Raja Roy f , Alok Dhawan a,b,h,
Systems Toxicology & Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, PO Box 80, Lucknow 226001, Uttar Pradesh, India Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, PO Box 80, Lucknow 226001, Uttar Pradesh, India c Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, PO Box 80, Lucknow 226001, Uttar Pradesh, India d Regulatory Toxicology Group, CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, PO Box 80, Lucknow 226001, Uttar Pradesh, India e Department of Environmental Health Sciences, Faculty of Public Health, University of Ibadan, Ibadan, Nigeria f Centre of Biomedical Magnetic Resonance, SGPGIMS Campus, Lucknow 226014, Uttar Pradesh, India g Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India h Institute of Life Sciences, Ahmedabad University, University Road, Ahmedabad 380009, Gujarat, India
b a

a r t i c l e

i n f o

a b s t r a c t
The release of particulate pollutants into the air through burning of coal, crude oil, diesel, coal tar, etc. raises concerns of potential health hazards to the exposed human population. Polycyclic aromatic hydrocarbons (PAHs) are major toxic constituents of particulate matter (PM), which upon ingestion get metabolized to even more toxic metabolites such as quinones. The PAHs levels were assessed in both respirable particulate matter (RSPM, <10 M size) and suspended particulate matter (SPM, >10 M size) of urban ambient air (UAA) and that of major contributors viz. diesel exhaust particles (DEPs) and coal tar combustions emissions (CTCE). Seven US Environmental Protection Agency (USEPA) prioritized PAHs in RSPM and 10 in SPM were detected in UAA. Ten and 15 prioritized PAHs, respectively, were also detected in diesel exhaust particles (DEP) and coal tar combustion emission (CTCE) evidencing their release in the air. These PM associated PAHs for UAA, DEP and CTCE showed signicant increase (p < 0.05) in mutagenicity and mammalian genotoxicity in the order CTCE > DEP > UAA. Human lung alveolar (A549) and bronchiolar (BEAS-2B) cells when treated with PAH-metabolites viz. 1,4-benzoquinone (1,4-BQ), hydroquinone (HQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ) and 9,10-phenanthroquinone (9,10-PQ) showed metabolic modulation in these cell lines with signicant depletion of principal cellular metabolites viz. NADP, uracil, asparagines, glutamine, and histidine and accumulation of di-methyl amine and beta-hydroxybutyrate, identied using 1 H NMR spectroscopy. These results suggest that PAH-quinones induce genotoxic effects by modulating the metabolic machinery inside the cells by a combined effect of oxidative stress and energy depletion. Our data for metabolic proling of human lung cells could also help in understanding the mechanism of toxicity of other xenobiotics. 2013 Elsevier GmbH. All rights reserved.

Article history: Received 14 April 2012 Received in revised form 2 April 2013 Accepted 8 April 2013 Keywords: Polycyclic aromatic hydrocarbons (PAHs) Quinones Genotoxicity Human lung cells Metabolic proling

Introduction Particulate matter (PM) is continuously released into the air through burning of coal, crude oil, diesel, bitumen, coal tar, etc. This has resulted in the deterioration of air quality, leading to exposure of the general population to these toxic air pollutants. The principal components of PM particles include polycyclic aromatic

Corresponding author at: Institute of Life Sciences, School of Science and Technology, Ahmedabad University, Opp. University Bus Stand, University Road, Ahmedabad 380009, Gujarat, India. Tel.: +91 79 26302414 18; fax: +91 79 26302419. E-mail address: alok.dhawan@ahduni.edu.in (A. Dhawan). 1438-4639/$ see front matter 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijheh.2013.04.001

hydrocarbons, metals, etc. (Andreou and Rapsomanikis, 2009; Ko and Hui, 2009; Tredaniel et al., 2009). It has also been shown that PAHs (naphthalene, phenanthrene, pyrene, uoranthene, etc.) persist in the rural and urban environment and have been linked to chronic respiratory diseases such as asthma, severe bronchitis, lung cancer, etc. (Aubier, 2009; Taguchi et al., 2007; Valavanidis et al., 2008). These PAHs undergo metabolic transformations through cytochrome P450 enzymes (CYP450), epoxide hydrolases, aldoketo reductases, etc. resulting in the formation of more toxic metabolites viz. 1,4-benzoquinone, naphthoquinone, phenanthroquinone, anthraquinone, etc. (Courter et al., 2007; Miranda et al., 2006; Oh and Chung, 2006; Spencer et al., 2009; Shultz et al.,

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2011; Zhang et al., 2012). These compounds have the potential to interact with macromolecules such as cellular enzymes responsible for transcription, DNA replication, protein synthesis and cellular metabolism eventually leading to mutations and cancer (Courter et al., 2008; Misaki et al., 2008; Sumi and Kumagai, 2007). It should however be noted that more directed toxicity studies are required to dissect the diverse toxicological effects of these PAH-quinones in lung cells. Diesel exhaust from vehicles and small power generators is one of the major contributor of PAH-quinones such as 1,4-benzoquinone (1,4-BQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), 9,10-phenanthroquinone (9,10PQ) and 1,4-anthraquinone (1,4-AQ) as identied by gas chromatographymass spectroscopy (GCMS) (Jakober et al., 2007; Sumi and Kumagai, 2007). Due to high concentrations of DEPs especially at trafc junctions, policemen, drivers, and commuters are routinely exposed to PAHs (Cavallo et al., 2006; Huang et al., 2007; Ryno et al., 2006; See et al., 2006). Taguchi et al. (2007) have reviewed the pulmonary toxicity of two PAH-quinones (1,2-NQ and 9,10-PQ) present in diesel exhaust particles (DEPs) in relation to the onset of cardiovascular and pulmonary diseases. Exposure to PAHs also occurs through coal tar, which is widely used for road construction, water proong of roofs and ooring and has been shown to cause carcinogenic and genotoxic effects (Karaman and Pirim, 2009). Epidemiological studies have also demonstrated that occupational exposure to soot, coal tar, and other PAH-containing mixtures is carcinogenic to humans (Boffetta et al., 1997; Bosetti et al., 2007; Raulf-Heimsoth et al., 2008). Several European Union (EU) countries have restricted the use of coal tar pitch due to their toxic effects. Developing countries, such as India and China are undergoing economic reforms and rapid industrialization. This has led to an increase in the levels of the particulate matter in the environment. Recent air monitoring has revealed that in most cities in India, the annual average concentrations of RSPM and SPM exceeded the National Ambient Air Quality Standards (NAAQS) limits (100 g/m3 for RSPM; 200 g/m3 for SPM; India) (CPCB, India 2009). Thus, presence of these carcinogenic PAHs in respirable particulate fractions (<10 M size), poses a serious threat to human health as these particles have the potential to reach the innermost portions of the lung. A comprehensive lung cell toxicity assessment would provide a framework of evidences and more detailed understanding supporting epidemiological studies describing adverse health effects. Based on these observations, the present study was aimed to ascertain the presence and release of carcinogenic PAHs in the ambient air particulate matter in a city of Northern India. Assessment of DNA damaging potential of collected particulate fractions using the Ames test in bacteria and micronucleus assay in human lung alveolar cells. Further, to unveil the complexity of PAHquinone induced toxicological effects, metabolic proling in human adenocarcinoma (A549) and human bronchiolar derived (BEAS-2B) lung cells was done using 1 H NMR.

Envirotech Instruments Pvt. Ltd., New Delhi] and a high-volume sampler [(HVS), APM-410; Envirotech Instruments Pvt. Ltd., New Delhi) were used to collect particulate matter (RSPM and SPM) from different sources. The samples were collected at an average ow rate of 1.01.1 m3 /min from the respirable zone, i.e. 1.5 m above the ground level. The sampling duration for urban ambient air (UAA) quality was 8 h while that for coal tar combustion emission (CTCE) and diesel exhaust particulate (DEP) was 2 h. The UAA samples for RSPM and SPM were collected in an open area, 10 m away from the road. For diesel exhaust particles, three air samples at 2 h intervals were collected by operating two buses in idle conditions. Three samples each of RSPM and SPM were also collected at 2 h intervals from coal tar after heating it for 6 h. The mass of the collected fractions was estimated gravimetrically. Extraction of PAHs PAH extraction was performed according to USEPA Method 8310 (www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8310. pdf). The lter papers containing particles rich in PAHs were subjected to Soxhlet extraction for 16 h with dichloromethane (HPLC grade, SigmaAldrich Co., St. Louis, MO, USA). The extracts were concentrated up to 10 ml using rotary vacuum evaporator (Rotavapor R-210/R-215; V-850, Buchi, Switzerland). The PAH extracts were further cleaned using heat activated silica gel (100200 mesh) packed in glass column (25 mm 10 mm), pre-equilibrated with n-pentane, at a ow rate of 2 ml/min. The bound PAHs were eluted with 25 ml dichloromethane/n-pentane mixture (2:3, v/v) and concentrated using rotary vacuum evaporator followed by solvent exchange with acetonitrile (HPLC grade, SigmaAldrich Co., St. Louis, MO, USA) to a nal volume of 5 ml. PAH analysis Identication and quantication of PAHs in the samples were carried by High Performance Liquid Chromatography (HPLC; Waters Miliford, MA, USA) using PAH column [LCPAH column C-18 (5 cm 4.6 mm, 3 m) Supelco, USA] and UV detector-2487 at 254 nm. EPA-610 PAH kit (SigmaAldrich Co., St. Louis, MO, USA) containing 1 ml mixture of 16 USEPA prioritized PAHs (Naphthalene; Acenaphthylene; Acenaphthene; Flourene; Phenanthrene; Anthracene; Flouranthene; Pyrene; Benzo(a)anthracene; Chrysene; Benzo(b)uoranthene; Benzo(k)uoranthene; Benzo(a)pyrene; Dibenz(a,h)anthracene; Benzo(g,h,i)perylene; Indeno(1,2,3,-CD)pyrene) was used as a standard reference. This 1 ml mixture was serially diluted two times in 25 ml acetonitrile and labeled as dilutions 1 and 2 as described in Table 1. The individual concentrations are also listed. 20 l of dilution 2 was injected for HPLC analysis of different PAHs. Elution was performed by gradient method using acetonitrile and water (50% acetonitrile, 0 min to 100% acetonitrile, 30 min) at the ow rate of 1 ml/min with total run time of 30 min. Data acquisition and processing were carried out on Empower 2 chromatography manager software (Waters, Miliford, MA, USA). The total PAHs (TPAHs ) were calculated as:
Concentration in ppm(g/ml) = area of sample amount of standard injected(ng) made up volume(ml) area of standard sample volume injected(g) total amount of sample(ml)

Materials and methods Air sampling The sampling of RSPM and SPM was carried out as per standard methodologies [Indian Standard, 2006; IS: 5182 (Part IV). 1973]. The complete details regarding sampler conguration, sample collection, calculations for true air volume, sample volume conversion, corrections for temperature and pressure, calculations for SPM and RSPM may be found elsewhere (https://law.resource.org/pub/in/bis/is.5182.04.1999.pdf). Briey, A dichotomous respirable dust sampler [(RDS), APM460NL;

GCMS validation of different PAHs in EPA 610 STD and samples The TPAHs identied by HPLC were conrmed using Gas ChromatographyMass Spectrometry (GCMS). The GCMS analyses were performed on autosystem XL gas chromatograph coupled

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D. Gurbani et al. / International Journal of Hygiene and Environmental Health xxx (2013) xxxxxx 3 1 ml mixture (g/ml) 1014 2006 1012 201.2 100.9 101 202 104.4 99.7 101.8 201 103.2 97.6 214.6 207.5 105.6 25 times dilution 1 (g/ml) 40.56 80.24 40.48 8.048 4.036 4.04 8.08 4.176 3.988 4.072 8.04 4.128 3.904 8.584 8.3 4.224 25 times dilution 2 (g/ml) 1.62 3.21 1.62 0.32 0.16 0.16 0.32 0.17 0.16 0.16 0.32 0.17 0.16 0.34 0.33 0.17 Concentration (ng) (in 20 l) 1.30 2.57 1.30 0.26 0.13 0.13 0.26 0.13 0.13 0.13 0.26 0.13 0.12 0.27 0.27 0.14

Table 1 Individual PAHs and their concentrations in 1 ml mixture, dilutions 1 and 2, 20 l injection volume for EPA-610 kit used as standard in HPLC analysis. EPA 610 PAH mixture 1. Napthalene 2. Acenaphthylene 3. Acenapthene 4. Flourene 5. Phenanthrene 6. Anthracene 7. Fluoranthene 8. Pyrene 9. Benzo(a)anthracene 10. Chrysene 11. Benzo(b)uorenthene 12. Benzo(k)uorenthene 13. Benzo(a)pyrene 14. Dibenzo(ah)anthracene 15. Benz(g,h,i)perylene 16. Indeno(1,2,3,-CD)pyrene

with a Turbo Mass detector (Perkin-Elmer Singapore Pvt. Ltd., Singapore). Analytical separation was achieved on a capillary column DB5-MS (30 m 0.25 mm i.d., 0.25 m lm thicknesses). The carrier gas was helium (99.99%) at a constant ow of 1.5 ml/min. The sample (1 L) was injected in a pulsed splitless mode. Pulse pressure was 12.64 psi for 0.30 min. The injector temperature was 300 C, transfer line temperature, 280 C, and the source and quadruple were kept at 300 C and 180 C, respectively. The oven was held at 50 C for 1 min then ramped at 25 C/min 1200 C, again ramped at 8 C/min to a nal temperature of 316 C. Total run time was 21.50 min. The mass detector operated in electron impact at 70 eV in full scan, acquiring ions of m/z 178 to 252 at the screening level. Checking the presence of diagnostic ions at the expected relative retention times monitoring the possible presence in the sample of each compound was considered in this study. Helium target compounds were identied by GCMS in the selection ion-monitoring mode using molecular ion and quantication as shown in Table 2. Mutagenicity test multi plate format ames test The mutagenicity testing was performed according to a modied Ames test using a multiplate format (MPF) assay (Xenometrix AG, Allschwil, Switzerland). This assay is based on the microuctuation method cited in the OECD Guideline 471 (OECD, 1997) for testing chemicals. It uses 384-well microtiter plates having a colorimetric read-out. Salmonella typhimurium strains TA98
Table 2 Molecular ion and qualier ions used in the analysis of PAHs by gas chromatographymass spectrometry (GCMS). Compound Naphthalene Acenaphthylene Acenaphthene Flourene Phenanthrene Anthracene Flouranthene Pyrene Benz(a)anthracene Chrysene Benzo(b)ouranthene Benzo(k)ouranthene Benzo(a)pyrene Indeno(1,2,3,-CD)pyrene Dibenz(a,h)anthracene Benzo(g,h,i)perylene Anthracene d10 (Internal Standard, IS) Molecular ion 128 152 154 166 178 178 202 202 228 228 252 252 252 276 278 276 164 Quantier ion 127 151 153 165 176 176 101 101 114 114 126 126 126 138 139 138 162

and TA100 were purchased from Xenomatrix AG (Allschwil, Switzerland) and the experiments were conducted according to the manufacturers protocol. Liver S9 fraction was initially prepared from male SpragueDawley rats treated with Phenobarbital sodium (liver enzyme inducer). The nal components of S9 mix used were, 8 mM magnesium chloride, 33 mM potassium chloride, 5 mM glucose-6-phosphate (G6P), 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and 10% S9 fraction (v/v). The experiment was designed for a total of three test concentrations along with negative and positive controls. The tester strains were freshly prepared by culturing them overnight at room temperature and treated with 0.5, 1 and 3 g/ml concentration of each sample with and without the presence of liver S9 fraction based on cytotoxicity data (data not shown). The treatment of the compounds was given in the nal volume of 0.250 ml exposure medium and incubated at 37 C for 90 min at 250 rpm in an incubator shaker (Innova 42, New Brunswick Scientic, 175 Freshwater Blvd., Eneld, CT, USA). After incubation, 2.8 ml of indicator media (histidine decient) was added to it and dispensed into 48 wells of a 384-well microplate. After 48 h of incubation, the number of positive (yellow) wells per replicate and dose were compared with the number of spontaneous revertants obtained in the negative control. The average number of wells containing revertants per culture and concentration was calculated from the triplicate sections, and the increases above the negative control were determined at each concentration of the respective test chemicals. A twofold increase in the revertant colonies was considered signicant as reported earlier (Kumar et al., 2011). The color of indicator media changes from purple (Bromocresol purple) to yellow due to drop in pH (pK1 = 5.2) by the catabolic activity of revertant cells, which grow in the absence of histidine. Cell lines and cell culture Human lung adenocarcinoma epithelial (A549, ATCC number: CCL-185) and human bronchial epithelial (BEAS-2B, ATCC number: CRL-9609) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, USA). A549 and BEAS-2B cells were cultured in DMEM/HF12 (1:1, v/v; GIBCO, Invitrogen, Auckland, NZ) and RPMI1640 medium (GIBCO, Invitrogen, Auckland, NZ), respectively, supplemented with 10% heat inactivated fetal bovine serum (FBS; GIBCO, Invitrogen, Auckland, NZ). The cells were cultured as monolayers in 25 cm2 and 75 cm2 tissue culture asks depending on the experiment and kept at 37 C in a humidied atmosphere in 5% CO2 incubator. Quinones viz. 1,4-BQ, 1,2-NQ, 1,4-NQ, 9,10-PQ (DMSO) and HQ (Milli Q; de-ionized water) was freshly prepared.

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Final concentration of DMSO in each treatment group was less than 1%. Micronucleus assay using ow cytometry The in vitro micronucleus assay using ow cytometry was carried out by the method of Nusse and Marx (1997) and modied by Pandey et al. (2009). Briey, A549 cells were treated in 1 ml of the serum-free medium for 3 h with 0.5, 1 and 3 g/ml of RSPM and SPM fractions of CTCE, UAA and DEP. Ethyl methane sulphonate (EMS; 6 mM) was used as a positive control. After exposure, the treatment was aspirated and cells were washed with serum-free medium and cultured for 48 h in complete DMEM/F12 medium. Cells were harvested with 500 l of Trypsin (0.125%) and centrifuged at 250 g for 10 min. Cells were resuspended in 1 ml of solution I (containing 10 mM NaCl, 3.4 mM sodium citrate, 10 mg/L RNAse, 0.3 mg/L Igepal, 25 mg/L EtBr), vortexed and incubated at room temperature for 1 h. To this, 1 ml of solution II (1.5% citric acid, 0.25 M sucrose, 40 mg/L EtBr) was added. After 15 min, the suspension was ltered through a 53 m nylon mesh into polystyrene round bottom tube (BD BioSciences, NJ, USA) and kept on ice till analysis. The suspensions of nuclei and micronuclei were analyzed using a ow cytometer (FCM; BD FACSCanto II, BD Biosciences, San Jose, CA, USA) with an argon ion laser (20 mW; excitation wavelength: 488 nm). Fluorescence was detected with 590 nm long pass band lter and data acquisition was conducted with BD FACS Diva 6.1.2 software (BD Biosciences, San Jose, CA, USA). A log scale was used to register DNA and side scatter (SSC) signals. G1-phase nuclei were stored around channel 20,000 and micronuclei were counted in the region between 5% and 40% of the DNA content of G1-phase nuclei. A minimum of 30,000 events per replicate were analyzed. Percent MN per nucleus (% MN/N) was analyzed by dividing the number of micronuclei (events in P4 gate) with the number of nuclei (P3). The apoptotic cells were excluded by gating of populations. Three independent experiments were conducted with two replicates. The statistical analysis was carried out by one way analysis of variance (ANOVA). The post hoc comparisons of the mean of independent groups were performed using Dunnets test (p < 0.001) for evaluating statistical signicance.
1H

NMR spectroscopy

The cells were cultured in 75 cm2 asks till they were 8090% conuent. The cells were then treated with a concentration [1 M (1 h)] of the test compounds depending on their cytotoxicity experiments (MTT and PI uptake assay) published previously in a related study (Gurbani et al., 2012). Based on the observation from both MTT and PI uptake assay, 1 M was taken as the concentration for further comparative study of all these quinones. The control and treated cells were harvested by trypsinization and washed three times with PBS to remove the extra cellular components and growth medium. Cell pellets were obtained by centrifuging at 1500 rpm for 5 min, weighed and stored at 80 C until NMR analysis. All cell pellets samples were thawed at room temperature, mixed with 0.5 ml of distilled water, sonicated for 3 min under ice cooled condition and lyophilized. The whole cell lysate was then reconstituted in deuterium oxide (D2 O, SigmaAldrich Co., St. Louis, MO, USA) and thoroughly mixed by vortexing the sample. The total volume of cell lysate was make-up to 0.6 ml. All samples were centrifuged at 3000 rpm to remove the cell debris and 0.5 ml of sample was taken in 5 mm WILMAD NMR tube for NMR spectral acquisition. NMR spectra were recorded on a Bruker Biospin Avance-III 800 MHz NMR (Bruker GmBH, Germany) spectrometer operating

at proton frequency of 800.21 using 5 mm 1 H/13 C/15 N triple resonance cryoprobe equipped with z-gradient accessories. A WILMAD co-axial insert containing known concentration of sodium salt of TSP [sodium salt of 3-trimethylsilyl-(2,2,3,3-d4 )-propionic acid; SigmaAldrich, St. Louis, MO, USA] in D2 O was used for quantitative estimation of metabolites as well as chemical shift referencing. Sample temperature was regulated using a Bruker BCU-05 unit at 298 K during the acquisition of spectra. The 1 H NMR spectra with water suppression was acquired using 1D single pulse sequence and CPMG pulse sequence with the following experimental parameters: spectral width of 12,820.5 Hz, 65,536 time domain data points of, effective 90 ip angle, 9.0 s, relaxation delay 10.0 s acquisition time of 2.55 s, 128 numbers of scan (for zgpr NS = 32) with 4 dummy scans, a constant receiver gain of 203 with a total recording time of 20 min. The 1D CarrPurcellMeiboomGill (CPMG) pulse sequence with water suppression (echo time of 40 ms) was performed to remove short T2 components arising due to the presence of lipids as well to obtain a good baseline. All the spectra were processed using line broadening for exponential window function of 0.3 Hz prior to Fouriers transformation. The 1 H NMR spectra of all samples were manually phased, and baseline corrected using TOPSPIN 2.1 (Bruker Analytik, Rheinstetten, Germany). The 1 H NMR spectra for all samples was referenced to the methyl resonance of alanine at 1.48 ppm. To conrm the assignments, 2D homo nuclear correlation spectroscopy (1 H1 H COSY) and 1 H13 C hetero nuclear single quantum correlation spectroscopy (HSQC) was performed using Brukers standard pulse program library. The parameters used for COSY were: 2048 data points were collected in the t2 domain over the spectral width of 12,820 Hz, 512 t1 increments were collected with 64 transients, relaxation delay of 1.5 s, acquisition time of 95 ms, and pre-saturation of water resonance was carried out during the relaxation delay. The resulting data were zero-lled to 512 in the t1 and as well as t2 dimensions and were weighted with 90 sine window functions in both the dimensions prior to Fouriers transformation. The parameters used for 1 H13 C HSQC were: 2048 data points were collected in t2 dimension over the spectral width of 12,820 Hz, 256 t1 increments were collected with 32 transients, relaxation delay of 2.0 s, acquisition time of 80 ms and a 90 pulse of 9.0 s. The phase sensitive data were obtained by the Anti echo-Time Proportional Phase Increments (Anti echo-TPPI) method. The resulting data were zero-lled to 512 data points in the t1 and t2 dimensions and were weighted with 90 squared sine window functions in both the dimensions prior to Fouriers transformation. The metabolites were quantitated using the following formula:

Concmet =

Imet Nstd Mmet Concstd Istd Nmet Mstd

where I, N, M, and Conc are integral area, number of nuclei in respective NMR peak, molar mass and concentration of analyte (met) and standard (std), respectively. Volume/dilution factor for reference and metabolites was not taken into account because both were dissolved in same sample solution (i.e. internal referencing in NMR). All the metabolites from same reference are quantiable in NMR spectroscopy. To remove the effects of molar mass and number of nuclei, integral area was normalized with molar mass and number of nuclei in NMR peaks (Bharti and Roy, 2012). The statistical signicance (p < 0.05) for the quantied metabolites was determined by one way ANOVA using post hoc Bonferroni multiple comparison test.

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Results Particulate burden in urban ambient air (UAA), coal tar combustion emissions (CTCE) and diesel exhaust particles (DEP) The average RSPM levels for UAA, DEP, and CTCE were found to be 520.75 41.4 g/m3 , 1048.7 184.9 g/m3 , 693.25 103.9 g/m3 and SPM levels were 651.78 104.06 g/m3 , 5219 131.73 g/m3 , 1282 272.60 g/m3 , respectively. Polycyclic aromatic hydrocarbon concentrations in UAA, CTCE, and DEP All the fractionated particulate fractions (RSPM and SPM) collected from emission sources and urban air (DEP, CTCE, and UAA) revealed the existence and release of USEPA prioritized PAHs. However, the number and concentration of these PAHs varied in different samples. The PAHs were compared with a standard HPLC chromatogram for the separation of USEPA prioritized 16 PAHs with their respective retention times (Fig. 1A). CTCE revealed the maximum number of carcinogenic PAHs followed by DEP > UAA (Fig. 1BG) which was conrmed by GCMS analysis (Fig. S1; Table 2). Total PAHs (TPAHs ) in SPM of UAA, CTCE and DEP were 80.83 9.20 ng/m3 , 52.2 10.8 ng/m3 , and 207.69 31.91 ng/m3 . However, in RSPM they were 15.53 2.36 ng/m3 , 120.67 11.72 ng/m3 and 269.52 33.06 ng/m3 , respectively (Table 3). Supplementary Fig. S1 related to this article found, in the online version, at http://dx.doi.org/10.1016/j.ijheh.2013.04.001. Ames test for assessment of mutagenicity for UAA, CTCE and DEP The cytotoxicity of PAHs were tested in S. typhimurium strains up to 5 g/ml. This concentration was found to be cytotoxic to S. typhimurium (data not shown). Hence the mutagenicity studies were conducted only up to 3 g/ml (non-cytotoxic concentration). All the collected fractions (SPM and RSPM) from different sources demonstrated a positive mutagenic response in Salmonella (TA98 and TA100) strains in the presence and absence of liver S9 fractions. The mutagenic response was higher in the presence of S9 fraction (metabolic activation) as compared to without S9. The maximum mutagenic response was observed at 3 g/ml. SPM fraction of UAA showed a prominent base pair substitution mutation with S9 fraction when compared to without S9 fraction and control. However, the RSPM fraction showed more than twofold increase in all strains in the presence and absence of S9 fraction (Fig. 2A and B). Similarly, CTCE (Fig. 2C and D) and DEP fractions (Fig. 2E and F) showed more than twofold reversion rates for both the strains, which was much higher in the presence of S9 as compared to without S9 activation. Genotoxicity assessment for UAA, CTCE and DEP using the micronucleus (MN) induction assay in human lung alveolar cells (A549) Treatment of A549 cells with non-cytotoxic concentrations (data not shown) of fractionated TPAHs in DMSO for RSPM and SPM of UAA revealed 36% and 23% increase at 0.5 g/ml and 63% and 115% increase at 1 g/ml in the occurrence of micronuclei over control cells. A statistically signicant (p < 0.05) dose dependent increase in MN frequency in A549 cells was observed at 0.5 g/ml for CTCE and DEP and at 1 g/ml for UAA when compared to the negative control (Table 4). Coal tar emission particulate fractions showed maximum induction of micronucleus followed by DEP > UAA.

Metabolic proling using 1 H NMR spectroscopy in alveolar and bronchiolar cells Several NMR spectra were recorded for A549 and BEAS-2B cells treated with 1 M (1 h) of HQ, 1,4-BQ, 1,2-NQ, 1,4-NQ and 9,10-PQ. Typical expansions of the 1 H NMR spectra stacked plots showing assignments for control along with the treatment of 1 M (1 h) for 1,4-BQ in both cell lines, A549 and BEAS-2B (Figs. 3A and B and 4A and B). Several resonances were detected corresponding to chemical shifts of different metabolites across all samples analyzed. Metabolites that were signicantly affected in both lung cell lines upon exposure to quinone compounds include nicotinamide adenine dinucleotide phosphate (NADP), formate (For), fumarate (Fum), isoleucine (Ile), myoinositol (mIno), tyrosine (Tyr), leucine (Leu), valine (Val), taurine (Tau), lactate (Lac), alanine (Ala), creatine (Cr) asparagine (Asn), glutamate (Glu), glutamine (Gln), succinate (Suc), uridine (Urdn), creatine (Cr), choline (Cho), phosphocholine (Pcho), taurine (Tau), uracil (Ura), glycine (Gly), phenylalanine (Phe), nucleotides(NTP/NDP) (Figs. 3A and B and 4A and B). A doublet signal at 1.2 ppm of betahydroxybutyrate was observed only in treated cells. Similarly, Figs. S4 and S5 show the collective stack plots of altered metabolic proles in response to exposure of different quinones in A549 and BEAS-2B cell lines. Tables 5 and 6 show the mean values with standard deviations of all metabolites identied over the spectra of the both cell types A549 and BEAS-2B. The concentrations are expressed as micromoles per milligram of total cell pellet obtained. Supplementary Figs. S4 and S5 related to this article found, in the online version, at http://dx.doi.org/10.1016/j.ijheh.2013.04.001. The data analyzed for all the compounds revealed a statistically signicant (p < 0.05, p < 0.01, p < 0.001) variation in the content of all identied metabolites in both the cell lines at 1 M (Tables 5 and 6). The most pronounced effect was on NADP, Uracil, asparagines, glutamine and histidine, where very low signal intensity was observed or was beyond the NMR detection limit, when compared to control in both cell lines. It was also observed that there was no signicant change in glucose content of BEAS-2B cells; however it increased signicantly in A549 cells. Dimethyamine (DMA) levels also increased signicantly in all treatment groups for all compounds tested in both cell lines indicative of a possible role of this metabolite in quinone-mediated toxicity.

Discussion In the present study, we have ascertained the release of carcinogenic PAHs in respirable particulate fractions of urban ambient air. Diesel exhaust and coal tar combustions were found to be the major contributors of these prioritized PAHs. The study comprehensively deals in correlating the DNA damaging effects of PAH-quinones along with overall impact on cellular metabolism analyzed in alveolar (A549) and bronchiolar (BEAS-2B) human cell lines. The data explicitly demonstrates that these PAHs associated with respirable particulate matter produce genotoxic effects and portend lung cancer risks to the human population. Also, the data from NMR spectroscopy revealed the biochemical perturbations upon PAH-quinone exposure, which should be considered important in the context of their reported genotoxicity/carcinogenicity. Such an investigation provides complete understanding of quinone induced toxic effects, thereby contributing new scientic data and risk assessment tools that further help in redening air quality standards in context to the current exposure scenario. In the air monitoring study, RSPM fraction of UAA showed the presence of vefold higher particulate burden in comparison to NAAQS permissible limit (100 g/m3 ) in India. DEP and CTCE levels

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Fig. 1. (A) Representative HPLC chromatogram showing separation of U.S. EPA prioritized PAH standard mixture. [S = solvent, 1. Naphthalene (1.30 ng), 2. Acenaphthylene (2.57 ng), 3. Acenaphthene (1.30 ng), 4. Flourene (0.26 ng), 5. Phenanthrene (0.13 ng), 6. Anthracene (0.13 ng), 7. Flouranthene (0.26 ng), 8. Pyrene (0.13 ng), 9. Benzo(a)anthracene (0.13 ng), 10. Chrysene (0.13 ng), 11. Benzo(b)uoranthene (0.26 ng), 12. Benzo(k)uoranthene (0.13 ng), 13. Benzo(a)pyrene (0.12 ng), 14. Dibenz(a,h)anthracene (0.27 ng), 15. Benzo(g,h,i)perylene (0.27 ng), and 16. Indeno (1,2,3,-CD)pyrene (0.14 ng).] (B) Representative HPLC chromatogram showing detection of 10 prioritized PAHs in RSPM fraction of diesel exhaust. [S = solvent, 2. Acenaphhthylene, 3. Acenaphthene, 4. Flourene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 8. Pyrene, 9. Benzo(a)anthracene, 10. Chrysene, and 13. Benzo(a)pyrene.] (C) Representative HPLC chromatogram showing detection of 10 prioritized PAHs in SPM fraction of diesel exhaust. [S = solvent, 2. Acenaphthylene, 3. Acenaphthene, 4. Flourene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 8. Pyrene, 9. Benzo(a)anthracene, 10. Chrysene, and 13. Benzo(a)pyrene.] (D) Representative HPLC chromatogram showing detection of 14 prioritized PAHs in RSPM fraction of CTCE. [S = solvent, 1. Naphthalene, 3. Acenaphthene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 8. Pyrene, 9. Benzo(a)anthracene, 10. Chrysene, 11. Benzo(b)uoranthene, 12. Benzo(k)uoranthene, 13. Benzo(a)pyrene, 14. Dibenz(a,h)anthracene, 15. Benzo(g,h,i)perylene, and 16. Indeno(1,2,3,-CD)pyrene.] (E) Representative HPLC chromatogram showing detection of 15 prioritized PAHs in SPM fraction of CTCE. [S = solvent, 1. Naphthalene, 3. Acenaphthene, 4. Flourene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 8. Pyrene, 9. Benzo(a)anthracene, 10. Chrysene, 11. Benzo(b)uoranthene, 12. Benzo(k)uoranthene, 13. Benzo(a)pyrene, 14. Dibenz(a,h)anthracene, 15. Benzo(g,h,i)perylene, and 16. Indeno(1,2,3,-CD)pyrene.] (F) Representative HPLC chromatogram showing detection of 10 prioritized PAHs in SPM fraction of UAA. [S = solvent, 1. Naphthalene, 2. Acenaphthylene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 8. Pyrene, 10. Chrysene, 11. Benzo(b)uoranthene, 12. Benzo(k)uoranthene, and 13. Benzo(a)pyrene.] (G) Representative HPLC chromatogram showing detection of 7 prioritized PAHs in RSPM fraction of UAA. [S = solvent, 1. Naphthalene, 2. Acenaphthylene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 9. Benzo(a)anthracene, and 10. Chrysene.]

are incomparable as they were taken near the source in order to evidence the release of different PAHs as composition of particulate matter present in the air. Epidemiological studies have suggested that exposure to low levels of PM10 particles (mass of

particles with aerodynamic diameter of 10 m) are related to the increased incidence of lung cancer (Pope et al., 2002; Tredaniel et al., 2009; Valavanidis et al., 2008). Based on epidemiological data, US Environmental Protection Agency (USEPA, 2005) revised the

Table 3 Comparison of total PAH levels in RSPM and SPM fractions of UAA, CTCE and DEP. TPAHs in RSPM (ng/m3 ) Urban ambient air (UAA) Coat tar combustion emissions (CTCE) Diesel exhaust particles (DEP)
a b

TPAHs in SPM (ng/m3 ) 80.83 9.20 52.2 10.8 207.69 31.91

b

NAQQS limita (ng/m3 ) 1 1 1

15.53 2.36 120.67 11.72 269.52 33.06

b

NAAQS permissible limit for Benzo(a)pyrene in India. The values are mean SD of three individual experiments. p < 0.001, when compared to respective controls.

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Fig. 2. Mutagenicity of total PAH levels in particulate fractions. (A) UAA SPM, (B) UAA RSPM, (C) CTCE SPM, (D) CTCE RSPM, (E) DEP SPM, and (F) DEP RSPM.

maximum permissible limit for PM10 as 50 g/m3 and concluded that exposures to PM10 are the contributory factors for adverse health effects in human populations. The study therefore reveals that despite regulatory measures by national agencies, human population is recurrently exposed to particulate associated health risks. Different PAHs are present as chemical constituents of PM10 particles and show continuous variation in composition and activity (Lewtas, 2007). PAHs are known genotoxic agents and induce DNA damaging effects, such as DNA adducts, DNA strand breaks, chromosomal aberr ations, sister chromatid exchanges, and micronucleus formation (Franco et al., 2008; Zidzik et al., 2007). It was therefore considered relevant to routinely identify the levels of carcinogenic PAHs in particulate fractions (RSPM and SPM) of air and major contributory sources, i.e. diesel exhaust and coal tar combustion.

Urban ambient air is the repository for particulate matter (including PAHs as major constituents) released from different emission sources. Therefore, USEPA has prioritized 16 major PAHs as suspected carcinogens to humans. Our study conrmed the presence of seven prioritized PAHs in PM10 particles (RSPM fraction) from UAA. The UK Expert Panel on Air Quality Standards (DEFRA, 2001) has set 0.25 ng/m3 as an annual average standard for PAHs using benzo-[a]-pyrene (BAP) as a marker whereas in India, 1 ng/m3 of BAP is set as standard by the Central Pollution Control Board (CPCB, India 2009). Benzo(a)pyrene is a known animal carcinogen and probable human carcinogen (http://monographs.iarc.fr/ENG/Monographs/ vol32/volume32.pdf). It was identied in the RSPM and SPM fractions of DEP and CTCE in the present study. However, it was only detected in the SPM fraction of UAA. Therefore, it is evident that different PAHs are contributed to the air by diesel exhaust and coal tar

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D. Gurbani et al. / International Journal of Hygiene and Environmental Health xxx (2013) xxxxxx % Micronucleus/nucleus 1.92 0.53 2.24 0.27 13.56 1.90*** Concentrations 0.5 g/ml 1 g/ml 4.71 0.69* 6.84 0.47*** 10.22 1.00*** 8.02 0.50*** 7.61 2.44*** 8.43 1.56*** 3 g/ml 7.38 2.49*** 8.17 0.20** 11.88 1.21*** 8.69 2.01*** 12.61 3.08*** 10.43 0.56***

Table 4 % Micronucleus/nucleus using ow cytometry in A549 cells after treatment of different PAH fractions.

Control DMSO control EMS (6 mM) Particulate fractions

UAA rspm UAA spm DEP rspm DEP spm CTCE rspm CTCE spm

3.38 0.97 2.84 0.96 4.88 0.98* 4.69 1.03* 4.61 1.13* 4.77 0.26*

Values are mean SEM of three experiments. EMS: ethylmethane sulphonate (6 mM) was used as positive control. * p < 0.05, when compared to respective controls. ** p < 0.01, when compared to respective controls. *** p < 0.001, when compared to respective controls.

combustion emissions despite current regulatory guidelines. The National Toxicology Program (NTP, 2011a, 2011b) classies DEP and CTCE as potential carcinogens. The IARC (2012) monograph (Volume 105, June) recently classied diesel exhaust as carcinogenic to humans (Group 1). Therefore, the emission and toxic potency of these PAHs further elevates human health risk concerns not only to the workers engaged but also to the general population

owing to their release in the environment, persistence and inhalation exposure. The complete respiratory tract is continuously exposed to particulate matter and is therefore, the target site. Short- and long-term exposure to PM10 particles may induce lung carcinogenicity when these are adsorbed inside the respiratory tract and deliver associated PAHs (Suresh et al., 2009). Lungs are situated

Fig. 3. (A) Stack plots showing 1 H NMR (0.54.6 ppm) spectral assignments for treatment of A549 cells with 1,4-benzoquinone. (Ile-Isoleucine, Leu-Leucine, Val-Valine, Glu-Glutamate, Gln-Glutamine, Meth-Methionine, Ala-Alanine, Lac-Lactate, Thr-Threonine, Cit-Citrate, Asn-Asparagine, Tau-Taurine, DMA-Dimethylamine, For-Formate, Ace-Acetate, Suc-Succinate, pCho-Phosphocholine, Cho-Choline, mIno-Myoinositol, Gly-Glycine, Cr-Creatine, and BHB-Beta Hydroxy Butyrate.) (B) NMR spectral assignments for treatment of A549 cells with 1,4-benzoquinone [continued (5.09.7 ppm)]. (Ura-Uracil, Fum-Fumaratic acid, NADP-Nicotinamide Adenine Dinucleotide Phosphate, UrdnUridine, Tyr-Tyrosine, His-Histidine, Phe-Phenylalanine, NDP/NTP-Nucleotide Di Phosphate/Nucleotide Tri Phosphate, -Glu-alpha-Glucose, and UDP-Uridine Di Phosphate.)

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Fig. 4. (A) Stack plots showing 1 H NMR (0.54.6 ppm) spectral assignments for treatment of BEAS-2B cells with 1,4-benzoquinone. (Ile-Isoleucine, Leu-Leucine, Val-Valine, Glu-Glutamate, Gln-Glutamine, Meth-Methionine, Ala-Alanine, Lac-Lactate, Thr-Threonine, Cit-Citrate, Asn-Asparagine, Tau-Taurine, DMA-Dimethylamine, For-Formate, Ace-Acetate, Suc-Succinate, pCho-Phosphocholine, Cho-Choline, mIno-Myoinositol, Gly-Glycine, Cr-Creatine, and BHB-Beta Hydroxy Butyrate.) (B) NMR spectral assignments for treatment of BEAS-2B cells with 1,4-benzoquinone (continued 5.69.7 ppm; Ura-Uracil, Fum-Fumaratic acid, NADP-Nicotinamide Adenine Di Nucleotide, Urdn-Uridine, Tyr-Tyrosine, His-Histidine, Phe-Phenylalanine, and NDP/NTP; Nucleotide Di Phosphate/Nucleotide Tri Phosphate). Table 5 Changes in cellular metabolites due to exposure of 1 M of quinones in A549 cells. Metabolites Treatment Control NADP Formate Uridine Phenyl-alanine Histidine Tyrosine Fumaric acid Uracil Glucose Myo-inositol Glycine Taurine Phospho-choline Choline Creatine Asparagine DMA Glutamine Glutamate Acetate Alanine Lactate Valine Iso-leucine 156.57 7.87 154.55 6.45 75.47 3.90 51.86 2.37 72.79 5.18*** 87.74 2.64 26.54 2.21 176.3 5.68 1128.29 57.61 993.56 35.07 1210.62 76.91 323.00 21.96 194.30 6.48 1878.85 231.59 611.86 17.00 51.08 1.69 1067.11 50.14 2675.45 98.91 5102.38 269.22 754.22 62.40 649.35 27.19 249.03 25.51 136.30 14.87 1,4-BQ 54.96 3.21*** 57.98 5.10 21.68 0.86*** 30.64 4.68*** 34.14 0.76*** 2.24 3.16*** 14.03 0.99*** 466.80 23.06*** 99.38 7.81*** 371.77 12.17*** 236.98 7.44*** 182.77 12.23*** 40.09 5.61*** 18.74 0.52*** 76.25 2.39*** 438.26 54.87 81.09 0.11*** 846.34 14.11*** 1757.00 158.96*** 73.64 10.79*** 256.70 13.44*** 97.60 13.87*** 85.91 16.36*** HQ 68.35 2.91*** 43.76 1.63*** 12.87 0.40*** 23.38 0.97*** 32.37 0.21*** 0.43 0.05*** 450.65 38.97*** 233.38 8.28*** 304.41 70.26*** 172.05 4.91*** 39.84 1.49*** 87.98 7.94*** 100.00 3.81*** 419.90 8.16 95.18 2.58*** 1006.77 36.43*** 1368.90 29.68*** 114.53 29.63*** 256.85 5.99*** 130.58 9.80*** 120.04 3.24 9,10-PQ 1.79 0.09*** 57.57 3.25*** 46.28 3.74** 16.65 0.68*** 42.43 1.62*** 29.72 1.17*** 4.14 0.16*** 15.24 1.01*** 688.46 31.00*** 46.27 1.19*** 170.24 18.31*** 245.81 17.81*** 109.95 7.47*** 35.05 4.03*** 16.19 1.27*** 587.79 37.76 562.76 35.64*** 1947.91 167.42*** 36.84 2.65*** 640.32 24.45 63.57 0.49*** 44.07 2.08*** 1,2-NQ 59.96 1.75*** 17.99 20.04*** 18.61 0.30*** 8.20 2.94*** 24.84 1.58*** 5.41 0.12*** 46.37 1.71*** 742.31 45.42*** 185.89 4.41*** 186.75 0.96*** 80.36 3.81*** 19.96 1.14*** 9.31 0.65*** 619.75 7.47 30.88 3.17*** 433.77 18.50*** 2400.20 5.86*** 46.82 5.30*** 455.85 20.00*** 70.61 7.60*** 48.74 0.53*** 1,4-NQ 60.98 1.72*** 68.28 2.93 23.16 0.27*** 35.11 2.74*** 32.99 0.60*** 4.06 0.15*** 261.47 10.21*** 12.80 0.72*** 204.16 3.53*** 73.97 2.13*** 27.20 2.31*** 27.97 2.72*** 17.32 2.29*** 653.75 24.64 18.86 1.91*** 547.11 19.89*** 1481.36 60.90*** 68.94 5.85*** 188.41 14.51*** 77.08 1.78*** 66.53 2.43***

Values are mean SD of three experiments expressed in micromolar concentration. DMA = di-methyl amine. Indicates very low or no measurable intensity for the metabolite. ** p < 0.01, when compared to respective controls. *** p < 0.001, when compared to respective controls.

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D. Gurbani et al. / International Journal of Hygiene and Environmental Health xxx (2013) xxxxxx 1,4-BQ 3.03 3.12 15.20 3.82 1.65 1.59 1.70 1.32 6.31 26.34 29.41 16.62 3.42 2.99 6.50 12.98 1.16 15.35 46.79 56.11 7.24 36.07 4.00 1.39 45.74 1.32 54.85 1.47*** 46.12 2.94*** 29.87 4.41*** 9.13 1.47*** 14.42 2.21*** 7.65 1.47 101.16 9.96 206.35 14.71*** 51.23 1.67*** 87.65 3.38* 77.55 6.39*** 13.98 0.42** 80.30 5.41*** 39.19 2.29** 407.55 19.44*** 78.99 4.68*** 677.45 18.41*** 2539.19 41.98*** 24.31 1.06*** 391.44 10.79*** 30.88 1.35*** 44.81 2.03*** HQ 42.00 1.47 67.65 1.62*** 60.91 2.12** 12.40 5.15*** 11.37 3.35*** 9.33 0.80*** 3.37 0.59* 108.65 16.61 129.68 7.35*** 30.25 1.03*** 78.60 4.74* 50.63 2.56*** 9.32 0.34*** 41.23 1.05*** 15.11 2.21*** 478.71 5.74*** 47.23 2.59*** 468.76 7.68*** 2582.03 5.06*** 22.14 0.60*** 236.37 6.11*** 27.45 0.49*** 40.29 1.03*** 9,10-PQ 15.31 1.59*** 56.57 1.96*** 38.01 2.10*** 19.25 1.81*** 7.88 0.13*** 13.68 1.75*** 170.17 2.96 171.45 2.12*** 38.05 1.37*** 58.22 0.76** 29.34 5.47*** 17.58 0.40** 45.05 3.83*** 4.43 6.26*** 929.48 74.73*** 101.03 7.66*** 192.48 4.19*** 1799.29 258.23*** 33.46 1.55*** 115.42 16.44*** 30.99 0.02*** 42.90 1.90*** 1,2-NQ 35.63 1.12 61.44 5.19*** 49.48 8.40*** 26.12 15.16*** 10.07 0.73*** 10.10 1.12*** 1.38 0.95** 278.24 32.65 104.59 73.31*** 54.83 15.99*** 78.67 3.43* 48.85 2.58*** 8.93 0.25*** 17.06 3.04*** 25.92 6.93*** 487.67 92.15*** 58.89 0.98*** 478.41 11.53*** 3099.79 95.52 15.75 3.52*** 264.27 55.33*** 29.12 7.43*** 60.01 2.93*** 1,4-NQ 21.80 4.06** 72.11 6.66*** 28.01 9.31*** 101.66 1.85*** 13.43 3.85*** 24.97 3.77*** 1.47 0.08** 851.67 107.89*** 155.49 12.91*** 31.91 0.61*** 53.83 26.38** 54.29 6.79*** 3.93 0.34*** 38.66 3.90*** 5.09 7.19*** 1165.48 109.04*** 50.25 31.67*** 546.04 22.91*** 3065.84 81.86 37.51 10.73*** 90.74 11.72*** 39.17 13.36*** 15.30 0.38***

Table 6 Changes in cellular metabolites due to exposure of 1 M of quinones in BEAS-2B cells. Metabolites Treatment Control NADP Formate Uridine Uracil Phenyl-alanine Histidine Tyrosine Fumaric acid Glucose Myo-inositol Glycine Taurine Phospho-choline Choline Creatine Asparagine DMA Glutamine Glutamate Acetate Alanine Lactate Valine Iso-leucine 51.37 100.47 104.35 159.73 40.94 41.68 65.06 8.15 171.26 712.23 351.43 269.28 308.97 38.40 281.52 121.69 44.61 248.95 1023.28 3379.09 351.28 2928.87 134.61 123.90

Values are mean SD of three experiments expressed in micromolar concentration. DMA = di-methyl amine. indicates very low or no measurable intensity for the metabolite. * p < 0.05, when compared to respective controls. ** p < 0.01, when compared to respective controls. *** p < 0.001, when compared to respective controls.

at the air/blood interface and possess 40 different cell types each carrying out specic function. Epithelial cells are involved in gaseous exchange, ciliated secretory cells for transport of particles to the bronchial tract and then to endothelial cells lining the vasculature (i.e. connection between the blood and respiratory cells). All these types of cells are potential targets for particulate associated PAHs. PM10 particles have increased Brownian motion due to their decreased size and get deposited at the alveolar and bronchiolar region. They are metabolized to PAH-derived quinones (naphthoquinone from naphthalene, phenanthroquinone from phenanthrene, etc.) inside the lungs as a result of three major detoxication pathways (formation of radical cations, diol epoxides and electrophilic and redox-active o-quinones) involving cellular enzymes viz. CYP450s, mono-oxygenases, epoxide hydrolases, aldo-keto reductases and myeloperoxidases (Baulig et al., 2003; Bolton et al., 2000; Miranda et al., 2006; Spencer et al., 2009; Zhao et al., 2009). Our HPLC analysis detected naphthalene, acenaphthylene, phenanthrene, anthracene, ouranthene, benzo(a)anthracene, chrysene in the respirable fraction of urban ambient air. These ndings gain support from previous studies that have revealed the presence and release of quinones such as 1,4-BQ; 1,2-NQ; 1,4-NQ; 9,10-PQ and 1,4-AQ in respirable particulate matter from diesel vehicle exhaust, gasoline vehicle exhaust and wood combustion (Jakober et al., 2007; Oh et al., 2011; Shinyashiki et al., 2009; Solomon and Sioutas, 2008). We would like to mention that since our study was conned only to the unsubstituted PAHs and therefore the possibility of presence of nitro-PAHs and/or their associated effects cannot be ruled out. It is, therefore, hypothesized that, upon pulmonary deposition, these quinones cause epithelial cell injury leading to increased ROS, DNA damage, and chromosomal breakage thereby potentiating lung cancer. The collected fractions were further subjected to mutagenicity and genotoxicity testing. Our experimental ndings on these

fractions (both RSPM and SPM) demonstrated that they are capable of eliciting mutagenic and genotoxic responses with the maximum being present in CTCE followed by DEP > UAA. Ames test ndings suggested that the PM-associated PAHs are capable of causing frame shift and base pair mutations in DNA. These observations are signicant as the chemical compounds exhibiting a positive response in the Ames test are likely to be carcinogenic in nature (Mortelmans and Zeiger, 2000). Exposure to these PAH mixtures, could therefore, elicit carcinogenic responses in complex eukaryotic systems capable of metabolizing these PAHs more efciently than bacterial systems. Micronucleus induction assay for genotoxicity assessment of different particulate-PAHs fractions of UAA, CTCE and DEP fractions in human lung adenocarcinoma (A549) cells revealed that SPM and RSPM fractions of CTCE are highly genotoxic followed by DEP > UAA. These results were consistent with our Ames test ndings. One would expect a similar result with BEAS-2B cells (also a lung cell line) as similar metabolism exists for PAHs inside these cells (Nichols et al., 1995; van Agen et al., 1997). Since chromosomal damage due to particulate associated PAHs affects the genomic integrity, human exposure to these fractions could potentially lead to the development of respiratory diseases such as lung cancer (Karaman and Pirim, 2009). These assays re-afrmed that the PAHs released into the ambient air in the current scenario portend genotoxic effects and routinely exposed human population is at risk. Since, responses to PAH-quinone induced toxic effects may be complex at cellular levels, we selected and limited our further studies to ve different types of quinones viz. 1,4-BQ; HQ 1,2-NQ; 1,4-NQ; and 9,10-PQ. However, we do not rule out the possibility of formation and implications of other metabolites producing specic genotoxic effects. In order to unveil the complexity between DNA damaging effects and their association with indigenous alterations in metabolic pathways upon PAH-quinone induced genotoxic

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insult, NMR studies were conducted in alveolar and bronchiolar derived cells. The importance of NMR spectroscopy lies in the fact that other experimental approaches measure gene/protein expression levels to provide signicant information regarding metabolic dysfunctions in lung cells. However, such experiments may not present a complete prole picture of metabolic changes responsible for the observed toxicological effects. The reason behind is that cellular processes such as posttranslational modications of enzymes, role of inhibitors including allosteric regulation, different gene functions denitely play a role in modulating metabolism. The use of NMR spectroscopy eliminates these factors allowing detection and quantitation of metabolites providing complete biochemical perturbations even in crude extracts. To the information available till date, in particular to those related in affecting metabolism inside cells, very little is known regarding the role of modulation of the metabolic prole in bronchiolar and alveolar cells upon quinone exposure. It could be due to the fact that utilizing animal models to conduct studies on the trachea-bronchial region is very difcult. Also, high particle doses used in such studies may not be strictly relevant to human exposure. Therefore, bronchial and alveolar cell lines could be used as they are easily maintained in culture (as their doubling time is relatively short) and could be used for mechanistic investigations for quinone-induced carcinogenesis (Don Porto Carero et al., 2001). The A549 and BEAS-2B cells used in this study are metabolically competent (Nichols et al., 1995; Park et al., 2008; van Agen et al., 1997). The data from our NMR studies suggests that exposure of BEAS-2B and A549 to these selective quinones exerts both direct as well as indirect control over pathways involved in energy metabolism and glucose utilization inside the cells. The spectra obtained were reproducible and assignments of the metabolites were further reinstated based on the literature value (Markley et al., 2007; Wishart et al., 2009) and 2D homonuclear (COSY) and heteronuclear (HSQC) experiments (Figs. S2A, 2B and S3). Signals for beta-hydroxybutyrate (at 1.20 ppm) appeared in treated A549 and BEAS-2B cells indicative of oxidative stress inside the cells. Such ketone bodies (beta-hydroxybutyrate, acetone etc.) are known to be produced in cells under oxidative stress conditions (Pavlides et al., 2010). Consistent with these ndings, our study revealed that NADP, the electron donor used in oxidative phosphorylation and used in quinone metabolism by aldo-keto reductases, was depleted completely in A549 cells at concentration of 1 M. A similar effect was observed in the BEAS-2B cells following exposure quinones. It should also be noted that cell-type specic responses occur for these reducing equivalents (NADPH production) as a result for differential utilization of pathways for glucose metabolism (glycolysis versus pentose phosphate pathway). However, it is worth pointing out that reduced NADPH could also result in increased apoptosis. The most affected metabolite in BEAS-2B cells was histidine, although uracil, asparagine and glutamine were signicantly depleted in A549 cells. The depletions in the levels of these metabolites indicate the prominent effect on glycolysis, and Krebs cycle whose intermediates provide anabolic precursors for the biosynthesis of fatty acids, nucleic acids, and proteins. This may also be attributed to the fact that PAH-quinones induce DNA damage, which would trigger p53 activation inside cells. It has been shown that p53 directly regulates genes involved in glycolysis (Kondoh et al., 2007) and indirectly affects respiration via modulation of cytochrome oxidase (Matoba et al., 2006). Our ndings are also supported by previous studies which show that upon oxidative DNA damage, Poly (ADP-ribose) polymerase (PARP) activation occurs, leading to depletion of cellular energy currencies and resulting in cell death (Bai et al., 2001; Gagne et al., 2006). Increase in DMA levels in both cell lines indicates that this metabolite might be

produced as a result of altered metabolism imposed due to quinone toxicity. Supplementary Figs. S2A, 2B and S3 related this article found, in the online version, at to http://dx.doi.org/10.1016/j.ijheh.2013.04.001. In our previous and related study, we have shown that high levels of DNA-DSBs accumulate upon quinone exposure in human lung cells due to inhibition of topoisomerase II thereby affecting DNA replication, repair (Gurbani et al., 2012). In response to the cellular capacity of DNA repair and maintaining genome integrity, changes occur in growth, energy reservoirs, protein metabolism, lipid metabolism and glucose homeostasis. Such a mechanism would therefore initiate inammatory responses, necrosis and/or carcinogenesis. This in turn also reects that upon DNA damage in cultured lung cells, perturbations occur at the level of gene expression, signal transduction and protein interaction networks thereby affecting pathways regulating cellular growth and energy metabolism. We therefore hypothesize that the depletion of certain metabolites inside the cells along with accumulation of novel ones may lead to development of neoplasmic effects in lungs provided that DNA repair and/or apoptosis fail inside the cells. In conclusion, our study establishes a connection between the DNA damage in human lung cells and changes in metabolism upon quinone exposure which in turn may lead to development of cancer. We emphasize that this information describes potential hazards posed by PAH-quinones in general population. Regulatory Environment Agencies in developing countries, should frame new emission norms and laws to reduce emissions so that preventive measures may put forth to minimize the risk of detrimental effects observed in human population. Conict of interest The authors declare that there are no conicts of interest. Acknowledgements We gratefully acknowledge the funding from the Council of Scientic and Industrial Research (CSIR), New Delhi under its network project (NWP34) and Supra institutional Project (SIP-08) and IITR communication No. 2932. Deepak Gurbani thanks the CSIR, New Delhi for the award of Senior Research Fellowship. References
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