Polycyclic Aromatic Hydrocarbons and Their Quinones Modulate the Metabolicprofile and Induce DNA Damage in Human Alveolar and Bronchiolar Cells

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Please cite this article in press as: Gurbani, D., et al., Polycyclic aromatic hydrocarbons and their quinones modulate the metabolic profile and

induce DNA damage in human alveolar and bronchiolar cells. Int. J. Hyg. Environ. Health (2013), http://dx.doi.org/10.1016/j.ijheh.2013.04.001

ARTICLE IN PRESSGModel

IJHEH-12685; No.of Pages13

International Journal of Hygiene and Environmental Health xxx (2013) xxxxxx

Contents lists available at SciVerse ScienceDirect

InternationalJournal ofHygiene andEnvironmental Health

journa l homepage: www.elsevier .com/ locate / i jheh

Polycyclic aromatic hydrocarbons and their quinones modulate the metabolic

profile and induce DNA damage in human alveolar and bronchiolar cells

Deepak Gurbania,g, Santosh Kumar Bharti f, Ashutosh Kumar b, Alok K. Pandey b,Godson R.E.E. Ana e, Ambrish Vermac, AltafHusain Khan c, Devendra K. Patel d,M.K.R. Mudiam d, Swatantra K.Jain g, Raja Roy f, Alok Dhawan a,b,h,

a Systems Toxicology & HealthRisk Assessment Group, CSIR-Indian Institute of Toxicology Research,Mahatma GandhiMarg, PO Box80,Lucknow 226001, Uttar Pradesh, Indiab Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research,Mahatma GandhiMarg, PO Box80, Lucknow 226001, Uttar Pradesh, Indiac Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research,MahatmaGandhiMarg, PO Box80, Lucknow 226001, Uttar Pradesh, Indiad Regulatory Toxicology Group, CSIR-Indian Institute of Toxicology Research,Mahatma GandhiMarg, PO Box80, Lucknow 226001, Uttar Pradesh, Indiae Department of Environmental HealthSciences,Faculty of Public Health, University of Ibadan, Ibadan, NigeriafCentre of Biomedical Magnetic Resonance, SGPGIMS Campus, Lucknow 226014, Uttar Pradesh, Indiag Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, NewDelhi 110062, Indiah Institute of Life Sciences, AhmedabadUniversity,University Road,Ahmedabad380009, Gujarat, India

a r t i c l e i n f o

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 cellsMetabolic profiling

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 ofparticulate 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, 10M size)

ofurban ambient air (UAA) and that ofmajor contributors viz. diesel exhaust particles (DEPs) and coal tar

combustions emissions (CTCE). Seven US Environmental Protection Agency (USEPA) prioritized PAHs in

RSPM and 10in 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 significant increase (p< 0.05) in mutagenicity

and mammaliangenotoxicity 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 significant depletion ofprincipal cellular metabo-

lites viz. NADP, uracil, asparagines, glutamine, and histidine and accumulation ofdi-methyl amine and

beta-hydroxybutyrate, identified using 1H NMRspectroscopy. These results suggest that PAH-quinones

induce genotoxic effects by modulating the metabolic machinery inside the cells by a combined effect

ofoxidative stress and energy depletion. Our data for metabolic profiling ofhuman lung cells could also

help in understanding the mechanism oftoxicity ofother xenobiotics.

2013 Elsevier GmbH. All rights reserved.

Introduction

Particulate matter (PM) is continuously released into the air

through burning of coal, crude oil, diesel, bitumen, coal tar, etc.

This hasresulted in thedeterioration of airquality,leading to expo-

sure 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.: +9179 26302414 18;fax: +9179 26302419.

E-mail address: [email protected](A. Dhawan).

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, fluoranthene, etc.) per-

sist in the rural and urban environment and have been linked to

chronic respiratory diseases such as asthma, severebronchitis, 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, aldo-

keto reductases, etc. resulting in the formation of more toxic

metabolites viz. 1,4-benzoquinone, naphthoquinone, phenanthro-

quinone, anthraquinone, etc. (Courter et al., 2007; Miranda et al.,

2006; Oh and Chung, 2006; Spencer et al., 2009; Shultz et al.,

1438-4639/$ see front matter 2013 Elsevier GmbH. All rights reserved.

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2 D. Gurbani et al. / International Journal of HygieneandEnvironmental Healthxxx (2013) xxxxxx

2011; Zhang et al., 2012). These compounds have the potential to

interact with macromolecules such as cellular enzymes responsi-

ble 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 genera-

tors 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 (9,10-

PQ) and 1,4-anthraquinone (1,4-AQ) as identified by gas

chromatographymass spectroscopy (GCMS) (Jakober et al., 2007;

Sumi and Kumagai, 2007). Due to high concentrations of DEPs

especially at traffic 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 pulmonarytoxicityof 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 proofing of roofs and flooring 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

NationalAmbientAir Quality Standards(NAAQS) limits (100g/m3

forRSPM; 200g/m3 forSPM;India) (CPCB,India 2009). Thus,pres-

ence of these carcinogenic PAHs in respirable particulate fractions(


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D. Gurbani et al. / International Journal of Hygieneand Environmental Healthxxx (2013) xxxxxx 3

Table 1

Individual PAHs and their concentrations in 1ml mixture, dilutions1 and 2, 20l injectionvolume forEPA-610kit used as standard in HPLC analysis.

EPA 610 PAH mixture 1 ml mixture (g/ml) 25 times dilution 1 (g/ml) 25 times dilution 2 (g/ml) Concentration (ng) (in 20l)

1. Napthalene 1014 40.56 1.62 1.30

2. Acenaphthylene 2006 80.24 3.21 2.57

3. Acenapthene 1012 40.48 1.62 1.30

4. Flourene 201.2 8.048 0.32 0.26

5. Phenanthrene 100.9 4.036 0.16 0.13

6. Anthracene 101 4.04 0.16 0.13

7. Fluoranthene 202 8.08 0.32 0.268. Pyrene 104.4 4.176 0.17 0.13

9. Benzo(a)anthracene 99.7 3.988 0.16 0.13

10. Chrysene 101.8 4.072 0.16 0.13

11. Benzo(b)fluorenthene 201 8.04 0.32 0.26

12. Benzo(k)fluorenthene 103.2 4.128 0.17 0.13

13. Benzo(a)pyrene 97.6 3.904 0.16 0.12

14. Dibenzo(ah)anthracene 214.6 8.584 0.34 0.27

15. Benz(g,h,i)perylene 207.5 8.3 0.33 0.27

16. Indeno(1,2,3,-CD)pyrene 105.6 4.224 0.17 0.14

with a Turbo Mass detector (Perkin-Elmer Singapore Pvt. Ltd.,

Singapore). Analytical separation was achieved on a capillary col-

umn DB5-MS (30 m0.25mm i.d., 0.25m film thicknesses). The

carrier gas was helium (99.99%) at a constant flow of 1.5ml/min.The sample (1L) was injected in a pulsed splitless mode. Pulse

pressure was 12.64psi for 0.30min. 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 1200C,

again rampedat 8 C/min to a final temperature of 316 C. Total run

time was 21.50min. The mass detector operated in electron impact

at70 eVin full scan, acquiringionsofm/z178to252atthescreening

level.Checking thepresence of diagnostic ions at theexpectedrela-

tiveretentiontimes monitoringthe possible presence in the sample

ofeach compoundwas considered in this study.Heliumtarget com-

pounds were identified by GCMS in the selection ion-monitoring

mode using molecular ion and quantification as shown in Table 2.

Mutagenicity test multi plate format ames test

The mutagenicity testing was performed according to a modi-

fied Ames test using a multiplate format (MPF) assay (Xenometrix

AG, Allschwil, Switzerland). This assay is based on the microfluc-

tuation 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 qualifier ions used in the analysis of PAHs by gas

chromatographymass spectrometry (GCMS).

Compound Molecular ion Quantifier ion

Naphthalene 128 127

Acenaphthylene 152 151

Acenaphthene 154 153

Flourene 166 165

Phenanthrene 178 176

Anthracene 178 176

Flouranthene 202 101

Pyrene 202 101

Benz(a)anthracene 228 114

Chrysene 228 114

Benzo(b)flouranthene 252 126

Benzo(k)flouranthene 252 126

Benzo(a)pyrene 252 126

Indeno(1,2,3,-CD)pyrene 276 138

Dibenz(a,h)anthracene 278 139

Benzo(g,h,i)perylene 276 138

Anthracene d10 (Internal Standard, IS) 164 162

and TA100 were purchased from Xenomatrix AG ( Allschwil,

Switzerland) and the experiments were conducted according to

the manufacturers protocol. Liver S9 fraction was initially pre-

pared from male SpragueDawley rats treated with Phenobarbitalsodium (liver enzyme inducer). The final components of S9 mix

used were, 8 mM magnesium chloride, 33 mM potassium chloride,

5 mM glucose-6-phosphate (G6P), 4 mM NADP, 100mM 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 3g/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 final volume of 0.250ml exposure medium and incubated

at 37 C for 90min at 250rpm in an incubator shaker (Innova 42,

New Brunswick Scientific, 175 Freshwater Blvd., Enfield, CT, USA).

After incubation, 2.8 ml of indicator media(histidine deficient) wasadded to it and dispensed into 48 wells of a 384-well microplate.

After 48h of incubation, the number of positive (yellow) wells

per replicate and dose were compared with the number of spon-

taneous revertants obtained in the negative control. The average

number of wells containing revertants per culture and concentra-

tion 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 rever-

tant colonies was considered significant 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), respec-

tively, supplemented with 10% heat inactivated fetal bovine serum

(FBS; GIBCO, Invitrogen, Auckland, NZ). The cells were cultured as

monolayers in 25cm2 and 75cm2 tissue culture flasks depending

on the experiment and kept at 37C in a humidified atmosphere

in5%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 concentrationof DMSO in each treatmentgroupwas less than

1%.

Micronucleus assay using flowcytometry

The in vitro micronucleus assay using flow cytometry was car-

ried out by the method ofNusse and Marx (1997) and modified by

Pandey et al. (2009). Briefly, A549 cells were treated in 1 ml of the

serum-free medium for 3h 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 48h in complete DMEM/F12 medium.

Cells were harvested with 500l of Trypsin (0.125%) and cen-

trifuged at 250g for 10min. Cells were resuspended in 1ml of

solution I (containing 10mM NaCl, 3.4mM sodium citrate, 10mg/L

RNAse, 0.3 mg/L Igepal, 25mg/L EtBr), vortexed and incubated at

room temperature for 1h. To this, 1ml of solution II (1.5% citric

acid,0.25M sucrose,40 mg/L EtBr)was added.After 15min,the sus-

pension was filtered through a 53m 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 flow cytometer (FCM; BD FACSCanto II, BD Biosciences, San Jose,

CA, USA) with an argon ion laser (20mW; excitation wavelength:

488 nm). Fluorescence was detected with 590 nm long pass band

filter and data acquisition was conducted with BD FACS Diva 6.1.2

software (BDBiosciences, SanJose,CA, USA). A logscalewas used to

register DNA and side scatter (SSC) signals. G1-phase nuclei were

storedaround channel 20,000 andmicronuclei 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 inde-pendent groups were performed using Dunnets test (p< 0.001) for

evaluating statistical significance.

1H NMR spectroscopy

The cells were cultured in 75cm2 flasks till they were 8090%

confluent. The cells were then treated with a concentration [1M

(1h)] of thetest compoundsdepending on their cytotoxicityexper-

iments (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, 1M 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 componentsand growth medium. Cell pellets were obtained by centrifuging

at 1500rpm for 5min, weighed and stored at 80 C until NMR

analysis.

All cell pellets samples were thawed at room temperature,

mixed with 0.5ml of distilled water, sonicated for 3 min under

ice cooled condition and lyophilized. The whole cell lysate was

then reconstituted in deuterium oxide (D2O, 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 3000rpm to remove the cell debris and 0.5 ml

of sample was taken in 5 mm WILMAD NMRtube for NMRspectral

acquisition.

NMR spectra were recorded on a Bruker Biospin Avance-III

800MHz NMR (Bruker GmBH, Germany) spectrometer operating

at proton frequency of 800.21 using 5 mm 1H/13C/15N triple reso-

nance cryoprobe equipped withz-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 D2O was used for quantita-

tive 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 1H NMR spec-

tra with water suppression was acquired using 1D single pulsesequence and CPMG pulse sequence with the following exper-

imental parameters: spectral width of 12,820.5Hz, 65,536 time

domain data points of, effective 90 flip angle, 9.0s, relax-

ation 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 40ms) was performed to remove short

T2 components arising dueto thepresenceof lipidsas well toobtain

a good baseline. All the spectra were processed using line broaden-

ing for exponential window function of 0.3Hz prior to Fouriers

transformation. The 1H NMR spectra of all samples were man-

ually phased, and baseline corrected using TOPSPIN 2.1 (Bruker

Analytik, Rheinstetten, Germany). The 1H NMR spectra for all

samples was referenced to the methyl resonance of alanine at

1.48ppm.

To confirm the assignments, 2D homo nuclear correlation spec-

troscopy (1H1H COSY) and 1H13C 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 12,820Hz, 512 t1 increments were collected

with 64 transients, relaxation delay of 1.5 s, acquisition time of

95ms, and pre-saturation of water resonance was carried out dur-

ing the relaxation delay. The resulting data were zero-filled 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 1H13C HSQC

were: 2048 data points were collected in t2 dimension over thespectral width of 12,820 Hz, 256 t1 increments were collected with

32 transients, relaxation delay of 2.0s, acquisition time of 80ms

anda90 pulse of 9.0s. The phase sensitivedata were obtained by

the Antiecho-TimeProportionalPhase Increments(Anti echo-TPPI)

method. The resulting data were zero-filled 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 trans-

formation. The metabolites were quantitated using the following

formula:

Concmet =ImetIstd

NstdNmet

MmetMstd

Concstd

where I,N,M, andConcare integralarea,number ofnuclei inrespec-

tive NMRpeak, molar mass and concentration of analyte (met) and

standard (std), respectively. Volume/dilution factor for reference

and metabolites was nottaken into account because both were dis-

solved in same sample solution (i.e. internal referencing in NMR).

All the metabolites from same reference are quantifiable 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 NMRpeaks (Bharti and Roy, 2012).

The statistical significance (p< 0.05) for the quantified metabo-

lites wasdetermined by one wayANOVAusing 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 520.7541.4g/m3, 1048.7184.9g/m3,

693.25103.9g/m3 andSPM levelswere 651.78104.06g/m3,

5219131.73

g/m3, 1282272.60

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 pri-

oritized PAHs. However, the number and concentration of

these PAHs varied in different samples. The PAHs were com-

pared 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 car-

cinogenic PAHs followed by DEP > UAA (Fig. 1BG) which was

confirmed by GCMS analysis (Fig. S1; Table 2). Total PAHs(TPAHs) in SPM of UAA, CTCE and DEP were 80.839.20ng/m

3,

52.210.8ng/m3, and 207.6931.91 ng/m3. However, in

RSPM they were 15.532.36ng/m3, 120.6711.72 ng/m3 and

269.5233.06 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 5g/ml. This concentration was found to be cytotoxic to

S. typhimurium (data not shown). Hence the mutagenicity studies

were conducted only up to 3g/ml (non-cytotoxic concentration).

All the collected fractions (SPM and RSPM) from different sources

demonstrated a positive mutagenic response in Salmonella (TA98

andTA100) strains in thepresenceand 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 3g/ml. SPM fraction of UAA

showed a prominent base pair substitution mutation with S9 frac-

tion when compared to without S9 fraction and control. However,

theRSPM fraction showedmore than twofold increase in all strains

in the presence and absence of S9fraction (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 0.5g/ml and

63% and115% increase at 1g/ml in the occurrence of micronu-

clei over control cells. A statistically significant (pUAA.

Metabolic profiling using1H NMR spectroscopy in alveolar and

bronchiolar cells

Several NMR spectra were recorded for A549 and BEAS-2B

cells treated with 1M (1 h) of HQ, 1,4-BQ, 1,2-NQ, 1,4-NQ

and 9,10-PQ. Typical expansions of the 1H NMR spectra stacked

plots showing assignments for control along with the treatment

of 1M (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 detectedcorresponding to chemical shifts of different metabolites across

all samples analyzed. Metabolites that were significantly affected

in both lung cell lines upon exposure to quinone compounds

include nicotinamide adenine dinucleotide phosphate (NADP), for-

mate (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 beta-

hydroxybutyrate was observed only in treated cells. Similarly, Figs.

S4 and S5 show the collective stack plots of altered metabolic pro-

files 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 identified over the spectra

of the both cell types A549 and BEAS-2B. The concentrations are

expressed as micromoles permilligram of total cell pelletobtained.

Supplementary Figs.S4 andS5 related tothis 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

significant (p< 0.05,p< 0.01,p< 0.001)variation in thecontent of all

identifiedmetabolites in both thecell lines at1 M (Tables5 and 6).

The most pronounced effect was on NADP, Uracil, asparagines, glu-

tamine andhistidine, where very lowsignal intensitywas observed

or was beyond the NMRdetection limit, when compared to con-

trol in both cell lines. It was also observed that there was no

significant change in glucose content of BEAS-2B cells; however it

increased significantly in A549 cells. Dimethyamine (DMA) levelsalso increased significantly in all treatment groups for all com-

pounds 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 car-

cinogenic 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 com-

prehensively 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) humancell 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 biochem-

ical perturbations upon PAH-quinone exposure, which should

be considered important in the context of their reported geno-

toxicity/carcinogenicity. Such an investigation provides complete

understanding of quinone induced toxic effects, thereby contribut-

ingnew scientificdataand risk assessment toolsthat further help in

redefining air quality standards in context to the current exposure

scenario.

In the air monitoring study, RSPM fraction of UAA showed the

presence of fivefold higher particulate burden in comparison to

NAAQS permissible limit (100g/m3

) in India. DEP and CTCElevels
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Fig. 1. (A) Representative HPLC chromatogram showing separation of U.S. EPA prioritized PAH standard mixture. [S= solvent, 1. Naphthalene (1.30ng), 2. Acenaph-

thylene (2.57ng), 3. Acenaphthene (1.30ng), 4. Flourene (0.26ng), 5. Phenanthrene (0.13ng), 6. Anthracene (0.13ng), 7. Flouranthene (0.26ng), 8. Pyrene (0.13ng),

9. Benzo(a)anthracene (0.13ng), 10. Chrysene (0.13ng), 11. Benzo(b)fluoranthene (0.26ng), 12. Benzo(k)fluoranthene (0.13ng), 13. Benzo(a)pyrene (0.12ng), 14.

Dibenz(a,h)anthracene (0.27 ng), 15. Benzo(g,h,i)perylene (0.27ng), and 16. Indeno (1,2,3,-CD)pyrene (0.14ng).] (B) Representative HPLC chromatogram showing detection

of10 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 frac-

tion 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 HPLCchromatogram showing detection of 14 prioritized PAHsin RSPMfractionof CTCE. [S= solvent, 1. Naphthalene,

3. Acenaphthene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 8. Pyrene, 9. Benzo(a)anthracene, 10. Chrysene, 11. Benzo(b)fluoranthene, 12. Benzo(k)fluoranthene,

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)fluoranthene, 12. Benzo(k)fluoranthene, 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. Ace-

naphthylene, 5. Phenanthrene, 6. Anthracene, 7. Flouranthene, 8. Pyrene, 10. Chrysene, 11. Benzo(b)fluoranthene, 12. Benzo(k)fluoranthene, 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 par-

ticulate matter present in the air. Epidemiological studies have

suggested that exposure to low levels of PM10 particles (mass of

particles with aerodynamic diameter of 10m) are related to the

increased incidence of lung cancer (Pope et al., 2002; Tredaniel

et al., 2009; Valavanidis et al., 2008). Basedon epidemiological data,

US Environmental Protection Agency (USEPA, 2005) revised the

Table 3

Comparison of total PAHlevels in RSPM and SPMfractions of UAA, CTCE and DEP.

TPAHs in RSPM (ng/m3) TPAHs in SPM (ng/m

3) NAQQS limita (ng/m3)

Urban ambient air (UAA) 15.53 2.36b 80.83 9.20b 1

Coat tar combustion emissions (CTCE) 120.67 11.72 52.2 10.8 1

Diesel exhaust particles (DEP) 269.52 33.06 207.69 31.91 1

a NAAQSpermissible limit for Benzo(a)pyrene in India. The values aremeanSD of three individual experiments.b

p< 0.001, when compared to respective controls.


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Fig. 2. Mutagenicity of total PAH levelsin particulatefractions. (A) UAASPM, (B)UAA RSPM, (C)CTCE SPM, (D)CTCE RSPM, (E)DEP SPM, and (F)DEP RSPM.

maximum permissible limit for PM10 as 50g/m3 and concluded

that exposures to PM10 are the contributory factors for adversehealth 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 PM10particles 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 differentemission sources. Therefore, USEPA has prioritized 16 major PAHs

as suspectedcarcinogensto humans. Ourstudyconfirmed thepres-

ence of seven prioritized PAHs in PM10 particles (RSPM fraction)

from UAA. The UK Expert Panel on Air Quality Standards (DEFRA,

2001) has set 0.25ng/m3 as an annual average standard for PAHs

using benzo-[a]-pyrene (BAP) asa markerwhereasin 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 identified in the RSPM and SPM frac-

tions of DEP and CTCE in the present study. However, it was only

detected in theSPM fraction of UAA. Therefore, it is evident that dif-

ferentPAHs arecontributedto the airby diesel exhaust andcoal tar
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Table 4

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

% Micronucleus/nucleus

Control 1.920.53

DMSO control 2.240.27

EMS (6 mM) 13.561.90***

Particulate fractions Concentrations

0.5

g/ml 1

g/ml 3

g/ml

UAA rspm 3.380.97 4.710.69* 7.382.49***

UAA spm 2.840.96 6.840.47*** 8.170.20**

DEP rspm 4.880.98* 10.221.00*** 11.881.21***

DEP spm 4.691.03* 8.020.50*** 8.692.01***

CTCE rspm 4.611.13* 7.612.44*** 12.613.08***

CTCE spm 4.770.26* 8.431.56*** 10.430.56***

Values are meanSEM of three experiments. EMS: ethylmethane sulphonate (6mM) 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) classifies DEP

and CTCE as potential carcinogens. The IARC (2012) monograph

(Volume 105, June) recently classified diesel exhaust as carcino-genic 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 inhala-

tion exposure.

The complete respiratory tract is continuously exposed to

particulate matter and is therefore, the target site. Short- andlong-term exposure to PM10 particles may induce lung carcino-

genicity 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 1H NMR (0.54.6ppm) 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-BetaHydroxy Butyrate.)(B) NMR spectralassignments

for treatment of A549cells with1,4-benzoquinone [continued (5.09.7ppm)]. (Ura-Uracil, Fum-Fumaraticacid, NADP-NicotinamideAdenineDinucleotidePhosphate, Urdn-

Uridine, Tyr-Tyrosine, His-Histidine,Phe-Phenylalanine, NDP/NTP-Nucleotide Di Phosphate/Nucleotide Tri Phosphate,-Glu-alpha-Glucose, and UDP-UridineDi Phosphate.)


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Fig. 4. (A)Stack plots showing 1H NMR(0.54.6ppm) 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-BetaHydroxy Butyrate.)(B) NMRspectral assignments

for treatment of BEAS-2B cells with 1,4-benzoquinone (continued 5.69.7ppm; 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 metabolitesdue to exposure of 1M of quinonesin A549 cells.

Metabolites Treatment

Control 1,4-BQ HQ 9,10-PQ 1,2-NQ 1,4-NQ

NADP 156.577.87 1.790.09***

Formate 154.556.45 54.963.21*** 68.352.91*** 57.573.25*** 59.961.75*** 60.981.72***

Uridine 75.473.90 57.985.10 43.761.63*** 46.283.74** 17.9920.04*** 68.282.93

Phenyl-alanine 51.862.37 21.680.86*** 12.870.40*** 16.650.68*** 18.610.30*** 23.160.27***

Histidine 72.795.18*** 30.644.68*** 23.380.97*** 42.431.62*** 8.202.94*** 35.112.74***

Tyrosine 87.742.64 34.140.76*** 32.370.21*** 29.721.17*** 24.841.58*** 32.990.60***

Fumaric acid 26.542.21 2.243.16*** 0.430.05*** 4.140.16*** 5.410.12*** 4.060.15***

Uracil 176.35.68 14.030.99*** 15.241.01*** 46.371.71***

Glucose 466.8023.06*** 450.6538.97*** 688.4631.00*** 742.3145.42*** 261.4710.21***

Myo-inositol 1128.2957.61 99.387.81*** 46.271.19*** 12.800.72***

Glycine 993.5635.07 371.7712.17*** 233.388.28*** 170.2418.31*** 185.894.41*** 204.163.53***

Taurine 1210.6276.91 236.987.44*** 304.4170.26*** 245.8117.81*** 186.750.96*** 73.972.13***

Phospho-choline 323.0021.96 182.7712.23*** 172.054.91*** 109.957.47*** 80.363.81*** 27.202.31***

Choline 194.306.48 40.095.61*** 39.841.49*** 35.054.03*** 19.961.14*** 27.972.72***

Creatine 1878.85231.59 18.740.52*** 87.987.94*** 16.191.27*** 9.310.65*** 17.322.29***

Asparagine 611.8617.00 76.252.39*** 100.003.81***

DMA 51.081.69 438.2654.87 419.908.16 587.7937.76 619.757.47 653.7524.64

Glutamine 1067.1150.14 81.090.11*** 95.182.58*** 30.883.17*** 18.861.91***

Glutamate 2675.4598.91 846.3414.11*** 1006.7736.43*** 562.7635.64*** 433.7718.50*** 547.1119.89***

Acetate 5102.38269.22 1757.00158.96*** 1368.9029.68*** 1947.91167.42*** 2400.205.86*** 1481.3660.90***

Alanine 754.2262.40 73.6410.79*** 114.5329.63*** 36.842.65*** 46.825.30*** 68.945.85***

Lactate 649.3527.19 256.7013.44*** 256.855.99*** 640.3224.45 455.8520.00*** 188.4114.51***

Valine 249.0325.51 97.6013.87*** 130.589.80*** 63.570.49*** 70.617.60*** 77.081.78***

Iso-leucine 136.3014.87 85.9116.36*** 120.043.24 44.072.08*** 48.740.53*** 66.532.43***

Values are meanSD 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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Table 6

Changes in cellular metabolitesdue to exposure of 1M of quinonesin BEAS-2B cells.

Metabolites Treatment

Control 1,4-BQ HQ 9,10-PQ 1,2-NQ 1,4-NQ

NADP 51.37 3.03 45.741.32 42.001.47 15.311.59*** 35.631.12 21.804.06**

Formate 100.47 3.12 54.851.47*** 67.651.62*** 56.571.96*** 61.445.19*** 72.116.66***

Uridine 104.35 15.20 46.122.94*** 60.912.12** 38.012.10*** 49.488.40*** 28.019.31***

Uracil 159.73 3.82 29.874.41*** 12.405.15*** 19.251.81*** 26.1215.16*** 101.661.85***

Phenyl-alanine 40.94 1.65 9.131.47*** 11.373.35*** 7.880.13*** 10.070.73*** 13.433.85***

Histidine 41.68 1.59

Tyrosine 65.06 1.70 14.422.21*** 9.330.80*** 13.681.75*** 10.101.12*** 24.973.77***

Fumaric acid 8.15 1.32 7.651.47 3.370.59* 1.380.95** 1.470.08**

Glucose 171.26 6.31 101.169.96 108.6516.61 170.172.96 278.2432.65 851.67107.89***

Myo-inositol 712.23 26.34 206.3514.71*** 129.687.35*** 171.452.12*** 104.5973.31*** 155.4912.91***

Glycine 351.43 29.41 51.231.67*** 30.251.03*** 38.051.37*** 54.8315.99*** 31.910.61***

Taurine 269.28 16.62 87.653.38* 78.604.74* 58.220.76** 78.673.43* 53.8326.38**

Phospho-choline 308.97 3.42 77.556.39*** 50.632.56*** 29.345.47*** 48.852.58*** 54.296.79***

Choline 38.40 2.99 13.980.42** 9.320.34*** 17.580.40** 8.930.25*** 3.930.34***

Creatine 281.52 6.50 80.305.41*** 41.231.05*** 45.053.83*** 17.063.04*** 38.663.90***

Asparagine 121.69 12.98 39.192.29** 15.112.21*** 4.436.26*** 25.926.93*** 5.097.19***

DMA 44.61 1.16 407.5519.44*** 478.715.74*** 929.4874.73*** 487.6792.15*** 1165.48109.04***

Glutamine 248.95 15.35 78.994.68*** 47.232.59*** 101.037.66*** 58.890.98*** 50.2531.67***

Glutamate 1023.28 46.79 677.4518.41*** 468.767.68*** 192.484.19*** 478.4111.53*** 546.0422.91***

Acetate 3379.09 56.11 2539.1941.98*** 2582.035.06*** 1799.29258.23*** 3099.7995.52 3065.8481.86

Alanine 351.28 7.24 24.311.06*** 22.140.60*** 33.461.55*** 15.753.52*** 37.5110.73***

Lactate 2928.87 36.07 391.4410.79*** 236.376.11*** 115.4216.44*** 264.2755.33*** 90.7411.72***

Valine 134.61 4.00 30.881.35*** 27.450.49*** 30.990.02*** 29.127.43*** 39.1713.36***Iso-leucine 123.90 1.39 44.812.03*** 40.291.03*** 42.901.90*** 60.012.93*** 15.300.38***

Values are meanSD 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 specific function. Epithelial cells are involved

in gaseous exchange, ciliated secretory cells for transport of par-

ticles 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 partic-ulate associated PAHs. PM10 particles have increased Brownian

motion due to their decreased size and get deposited at the alve-

olar and bronchiolar region. They are metabolized to PAH-derived

quinones (naphthoquinonefrom naphthalene, phenanthroquinone

from phenanthrene, etc.) inside the lungs as a result of three

major detoxification pathways (formation of radical cations, diol

epoxides and electrophilic and redox-active o-quinones) involving

cellular enzymes viz. CYP450s, mono-oxygenases, epoxide hydro-

lases, 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 naphtha-

lene, acenaphthylene, phenanthrene, anthracene, flouranthene,

benzo(a)anthracene, chrysene in the respirable fraction of urban

ambient air. These findings 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 particu-

late 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 confined 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 mutageni-

city and genotoxicity testing. Our experimental findings on these

fractions(both RSPM andSPM) demonstratedthat they arecapable

of eliciting mutagenic andgenotoxic responses with the maximum

being present in CTCE followed by DEP >UAA. Ames test findings

suggested that the PM-associated PAHs are capable of causing

frame shift and base pair mutations in DNA. These observations

are significant as the chemical compounds exhibiting a positiveresponse 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 eukary-

otic systems capable of metabolizing these PAHs more efficiently

than bacterial systems.

Micronucleus induction assay for genotoxicity assessment of

different particulate-PAHs fractions of UAA, CTCE and DEP frac-

tions 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

findings. 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 chro-

mosomal damage due to particulate associated PAHs affects the

genomic integrity, human exposure to these fractions could poten-

tially lead to the development of respiratory diseases such as lung

cancer (Karaman and Pirim, 2009). These assays re-affirmed that

the PAHs released into the ambientair in the current scenario por-

tend 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 five different types of quinones viz. 1,4-BQ; HQ 1,2-NQ;

1,4-NQ; and9,10-PQ. However, we do notrule outthe possibility of

formation and implications of other metabolites producing specific

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, NMRstudies were conducted in alveolar and bronchiolar

derived cells. The importance of NMRspectroscopy lies in the fact

that other experimental approaches measure gene/protein expres-

sion levels to provide significant information regarding metabolic

dysfunctions in lung cells. However, such experiments may not

present a complete profile picture of metabolic changes respon-

sible for the observed toxicological effects. The reason behind is

that cellular processes such as posttranslational modifications of

enzymes, role of inhibitors including allosteric regulation, differ-

entgene functions definitelyplay a role in modulating metabolism.

The use of NMR spectroscopy eliminates these factors allowing

detection and quantitation of metabolites providing complete bio-

chemical 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 profile 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 difficult. Also,

high particle doses used in such studies may not be strictly

relevant to human exposure. Therefore, bronchial and alveo-

lar 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 car-

cinogenesis (Don Porto Carero et al., 2001). The A549 and

BEAS-2B cells used in this study are metabolically compe-

tent (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 onthe literature value (Markley et al.,

2007; Wishart et al., 2009) and 2D homonuclear (COSY) and het-

eronuclear (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. Suchketone bodies (beta-hydroxybutyrate, acetone etc.) are known to

be produced in cells under oxidative stress conditions (Pavlides

et al., 2010). Consistent with these findings, ourstudyrevealedthat

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 1M. A similar effect

was observed in the BEAS-2B cells following exposure quinones.

It should also be noted that cell-type specific responses occur for

these reducing equivalents (NADPH production) as a result for

differential utilization of pathways for glucose metabolism (gly-

colysis 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 histi-

dine, although uracil, asparagine and glutamine were significantlydepleted in A549 cells.The depletions in thelevels of these metabo-

lites indicate the prominent effect on glycolysis, and Krebs cycle

whose intermediates provide anabolic precursors for the biosyn-

thesis 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 (Matobaet al., 2006). Ourfindingsare also sup-

ported 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 resultof altered metabolism imposed dueto quinone

toxicity.

Supplementary Figs. S2A, 2B and S3 related

to this article found, in the online version, at

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 inflammatory responses, necrosis and/or

carcinogenesis. This in turn also reflects 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 thatthe depletionof 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 exposurewhichin turn mayleadto development of cancer.

We emphasize that this information describes potential hazards

posedby PAH-quinones in general population. RegulatoryEnviron-

ment Agencies in developingcountries,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.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We gratefully acknowledge the funding from the Council of Sci-

entificand Industrial Research (CSIR), New Delhi under itsnetwork

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.

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