ORIGINAL RESEARCH

Genotoxicity of hydroquinone in A549 cells

Cheng Peng & Dionne Arthur & Faye Liu & Jongwha Lee &

Qing Xia & Martin F Lavin & Jack C. Ng

Received: 19 December 2012 /Accepted: 20 May 2013# Springer Science+Business Media Dordrecht 2013

Abstract Hydroquinone (HQ) is found in natural andanthropogenic sources including food, cosmetics, cig-arette smoke, and industrial products. In addition toingestion and dermal absorption, human exposure toHQ may also occur by inhaling cigarette smoke orpolluted air. The adverse effects of HQ on respiratorysystems have been studied, but genotoxicity HQ onhuman lung cells is unclear. The aim of this study wasto investigate the cytotoxicity and genotoxicity of HQin human lung alveolar epithelial cells (A549). Wefound that HQ induced a dose response in cell growthinhibition and DNA damage which was associatedwith an increase in oxidative stress. Cytotoxicity re-sults demonstrated that HQ was most toxic after 24 h(LC50=33 μM) and less toxic after 1 h exposure(LC50=59 μM). Genotoxicity of HQ was measuredusing the Comet assay, H2AX phosphorylation, andchromosome aberration formation. Results from thecomet assay revealed that DNA damage was highestduring the earlier hours of exposure (1 and 6 h) and

thereafter was reduced. A similar pattern was ob-served for H2AX phosphorylation suggesting thatdamage DNA may be repaired in later exposure hours.An increase in chromosomal aberration correspondedwith maximal DNA damage which further confirmedthe genotoxic effects of HQ. To investigate whetheroxidative stress was involved in the cytotoxic andgenotoxic effects of HQ, cellular glutathione and 8-Oxo-deoguanisone (8-Oxo-dG) formation were mea-sured. A decrease in the reduced glutathione (GSH) andan increase oxidized glutathione (GSSG) was observedduring the early hours of exposure which correspondedwith elevated 8-Oxo-dG adducts. Together these resultsdemonstrate that HQ exerts its cytotoxic and genotoxiceffects in A549 lung cells, probably through DNAdamage via oxidative stress.

Keywords Hydroquinone (HQ) . Oxidative stress .

Genotoxicity . Comet assay . γH2AX . A549 cells

Introduction

Hydroquinone (HQ) has been widely used in variousindustries including the manufacturing of rubber, paints,varnishes, motor fuels, oils, and as a reagent in photo-graphic developers and in cosmetic products (McGregor2007). In addition to industrial sources, HQ exists in freeform and as β-D-glucopyranoside conjugate (arbutin) insome bacteria, plants, coffee, red wine, and wheat ce-reals (Deisinger et al. 1996) as well as in cosmetics(Olumide et al. 2008). HQ is also one of the majorcomponents found in cigarette smoke (Gopalakrishna

Cell Biol ToxicolDOI 10.1007/s10565-013-9247-0

C. Peng :D. Arthur : F. Liu : J. Lee :Q. Xia : J. C. Ng (*)National Research Centre for Environmental Toxicology-Entox,The University of Queensland, 39 Kessels Road,Coopers Plains, Brisbane 4108, Australiae-mail: j.ng@uq.edu.auURL: www.entox.uq.edu.au

C. Peng :D. Arthur : F. Liu : J. Lee :Q. Xia : J. C. NgCooperative Research Centre for ContaminationAssessment and Remediation of the Environment(CRC-CARE), Adelaide 4095, Australia

M. F. LavinRadiation Biology and Oncology, Queensland Instituteof Medical Research, Brisbane 4006, Australia

et al. 1994). Hence, humans may be exposed to HQ viaingestion of plant-derived dietary sources, dermal ab-sorption, and inhalation in occupational settings andfrom cigarette smoke. Toxicity information of HQ inanimals and humans is limited (McGregor 2007).Animal studies using rats and mice have shown thatHQ causes renal tubular and hepatocellular adenomas(Hard et al. 1997; Kari et al. 1992; Shibata et al. 1991).Epidemiological studies performed in occupational set-tings did not provide enough evidence on the associa-tion between HQ exposure and increased cancer inci-dence and as such HQ was not classable as to its carci-nogenicity to humans (IARC 1999).

HQ is one of the major metabolites of benzene, ahuman carcinogen associated with various types ofleukemia and lymphomas (McGregor 2007; Snyderet al. 2002; Yin et al. 1996). It is believed that benzeneexerts its carcinogenesis through its metabolites(McHale et al. 2012). In this context, HQ has beenstudied in vitro in hematopoietic cells and lympho-cytes. HQ was found to induce genotoxicity such asDNA damages mediated by oxidative stress in thesecell models (Andreoli et al. 1999; Ishii et al. 2008;Peng et al. 2012; Wan and Winn 2007). Similar find-ings were also found in other cell models such as V79cells (Silva et al. 2003) and Chinese hamster ovary(CHO) cells (Winn 2003). HQ readily undergoes auto-oxidation to H2O2 and semiquinone radicals, whichare highly reactive molecules (Bolton et al. 2000; Luoet al. 2008), leading to oxidative stress (Bolton et al.2000; Rubio et al. 2011) and subsequent oxidativedamage (Gut et al. 1996; Luo et al. 2008; Winn2003). In addition, HQ induces caspase-3, caspase-9activation, and PARP cleavage (Kim et al. 2009; Leeet al. 2007), which are essential steps in apoptosis.Apoptosis induced by HQ in these cells appears to bemediated by ROS generation since the cytotoxicity ofHQ and caspase activation is reduced in the presenceof the antioxidant N-acetyl-L-cysteine. Moreover, thenuclear factor (erythroid-derived 2)-like 2 (Nrf2) tran-scription factor, a stress-responsive transcription factorwell-known to be part of a cellular adaptive defensemechanism in response to various oxidative stressors(Kang et al. 2005) has been recently reported to beinduced by HQ (Rubio et al. 2011). Together thesestudies confirm the role of ROS production and oxi-dative stress in HQ cytotoxicity.

As a constituent in cigarette and industrial contam-inant, HQ has drawn attention for its potential adverse

effects in other systems. HQ has been associated withan increased incidence of age-related macular degen-eration in human smokers (Bertram et al. 2009;Pons and Marin-Castaño 2011). A study investigat-ing the effects of HQ on the respiration systemindicated that HQ can affect lung tissue homeosta-sis and impair immune defense through reducingmonocyte chemoattractant protein-1 and monocyterecruitment and stimulating the tumor necrosis fac-tor secretion by tracheal epithelial cells in mice(Shimada et al. 2012). Additionally, Rubio et al.(2011) demonstrated an increase in cytoxicity and celldeath following HQ exposure in human lung epithelialcells (Beas-2B) that lack expression of the nuclearfactor (erythroid-derived 2)-like 2 (Nrf2). These stud-ies suggest that HQ toxicity affects cells of the respi-ratory system. However, the possible genotoxic effectsof HQ on lung cells are unknown. A very recent studyinvestigated the gene expression changes by cigarettesconstituents in A549 cells and found that HQ induceddifferential expressions in 55 genes (Cheah et al.2013). Most of these genes are involved in DNA dam-age responses, apoptosis, cell cycle arrest, and chro-mosome structure, which may suggest the potentialgenotoxic effects of HQ in lung cells. Given that theHQ-induced mutation and oxidative stress have beenfound in other in vitro cell models, there is a need toassess these effects in lung cells to understand a widertoxicological spectrum of this environmental contami-nant. Therefore, the aim of this study was to investigatethe potential genotoxic effects of HQ in human lungcells. To this end, we evaluated the cytotoxicity andgenotoxicity of HQ in A549 cells, a human lung ade-nocarcinoma epithelial cell line. The A549 cell line isalveolar type II cells of the pulmonary epithelium andhas been widely used as an in vitro model for assessingthe adverse effects produced by inhaled chemicals intoxicological and pharmaceutical studies (Castell et al.2005). Since HQ exerts its toxicity via ROS resultingin oxidative stress, levels of glutathione (reduced andoxidized forms) were determined as an indicator ofcellular redox disruption. HQ has been reported toinduce oxidative damage to DNA in cell-free system(Leanderson and Tagesson 1990), recombinantEscherichia coli (Horita et al. 2005), and HepG2 cells(Luo et al. 2008). To confirm such effects in lung cells,we also measured 8-oxo-2-deoxyguanosine (8-OH-dG) lesions, a reliable marker for oxidative DNA dam-age using fluorescent immunostaining.

Cell Biol Toxicol

Materials and methods

Chemicals and cell culture

Hydroquinone (H9003), PIPES (P6757), and lowmelting point (LMP) agarose were obtained fromSigma–Aldrich Pty Ltd (Castle Hill NSW Australia).Dulbecco’s Modified Eagle’s Medium (DMEM),minimum essential media (MEM), fetal bovine se-rum (FBS), L-glutamine, and penicillin/streptomycinwere from GIBCO BRL. All other reagents were ofanalytical grade. Although HQ is water soluble, weobserved a color change over time when usingwater as a solvent. This color change may reflectinstability of HQ and interfere with the results.Therefore we used DMSO as a solvent based onexperiments previously reported by others (Sharmaet al. 2012). No color change in this stock solutionwas observed using DMSO, which was preparedfresh and in the dark prior to use. Human lungadenocarcinoma epithelial cell line, A549 cells (ATCC:CCL-185) cultured in DMEM, containing 10 % FCS,2 mM L-glutamine, 100 U/mL penicillin/streptomycinand maintained in a humidified incubator at 37 °Cunder 5 % CO2.

Cell survival assay by MTS

Cytotoxicity of HQ was measured using the PromegaCellTiter 96 AQueous Non-Radioactive CellProliferation (MTS) assay (Promega, Alexandria,Australia). The assay was performed according to themanufacturer’s instructions (Promega) with slightmodifications. Briefly, 8×103 cells/well were seededinto each well of a 96-well plate and cultured for 20 hbefore treatment. The cells were treated with differentconcentrations of HQ over different incubation pe-riods (1, 6, 12, and 24 h). Control groups were treatedwith the reagent vehicle DMSO only. At the end ofeach incubation time, the cells were washed threetimes with the medium without serum followed bythe addition of 120 μL medium containing 20 μL ofMTS and further incubated for 2 h. The absorbance ofthe formazan product was then recorded at 490 nm usinga microplate reader (FLUOstar OPTIMA by BMGLabtech, Offenburg, Germany). Each experiment wasperformed in triplicate and repeated independently threetimes. Results are expressed as the percentage growthinhibition with respect to the untreated cells. IC50 was

calculated using the GraphPad Prism version 5(GraphPad, La Jolla, USA) for each time point. EachIC50 measurement was taken as the mean ± SD fromthree independent experiments.

The single cell gel electrophoresis assay (comet assay)

HQ-induced DNA damage was detected using theComet assay kit (Trevigen, Cat# 4250-050-K,Gaithersburg, MD, USA), according to the manufac-turer’s instructions with slight modifications. Briefly,2×105 cells/well were seeded in six-well plates andincubated for 18 h followed by treatment with differ-ent concentrations of HQ over different incubationperiods (1, 6, 12, and 24 h). At the end of each timepoint, cells were washed with Ca2+- and Mg2+-freePBS (Trevigen). The cell suspension was mixed withmolten LMP agarose (Trevigen) at a 1:10 (v/v) ratio.The mixture (60 μL) was immediately transferred ontothe slide provided which was placed flat at 4 °C for20 min. After cell lysis at 4 °C for 40 min, slides weretreated with alkali solution (0.3 M NaOH, 1 mMEDTA) for 40 min at room temperature followed byelectrophoreses at 1 volt/cm for 25 min. After stainingwith SYBR green dye (Trevigen), slides were visual-ized and photographed by a digital camera (AxioCamMRm; Carl Zeiss MicroImaging, Inc.) attached to afluorescent microscope (Axioskop2 mot plus; CarlZeiss MicroImaging, Inc.) using ×10 magnification.At least 50 cells are analyzed for DNA damage in eachtreatment using Komet6 software (Andor Technology,Belfast, Northern Ireland).

H2AX phosphorylation foci analysis

For assessment of DSB induced by HQ in A549 cells,the cells were seeded on cover slips sitting in six-wellplate and incubated for overnight. After treatment withHQ, cells were washed with PBS and fixed in ice-coldmethanol for 10 min and incubated in nuclear extrac-tion buffer (10 mM PIPES pH 6.8, 100 mM NaCl,300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5 %Triton X-100) for 15 min at room temperature. Non-specific binding was blocked by incubation with 5 %FCS/PBS/0.1 % Triton X-100 for 1 h at room temper-ature. Immunostaining for gamma H2AX (1:800;Abcam, Cambridge, United Kingdom) was followedby detection with species specific secondary AlexaFluor conjugates (1:1,000; Invitrogen, Carlsbad,

Cell Biol Toxicol

USA). Images were captured using a digital camera(Carl Zeiss) attached to a fluorescent microscopeAxioskop2 mot plus (Carl Zeiss). The numbers ofH2AX foci were quantified in 50–60 nuclei using thepublic domain software ImageJ version 1.42q (NIH,USA).

GSH assay

Total cellular glutathione (GSH+GSSG)was determinedenzymatically using a Glutathione Assay Kit (CS0260,Sigma). Briefly, A549 cells 1×107 cells grown on60 cm2 dishes were incubated with HQ at 2.5, 5.0, 10,20, and 40 μM and the medium was removed after 0.5,1, 6, and 24 h. Cells were washed three times with coldPBS and scraped into a microcentrifuge tube. An aliquot(100 μL) of 5 % 5-sulfosalicylic acid was added to thepacked cell pellet and vortexed. The cell suspension wasfrozen and thawed three times (liquid nitrogen was usedto freeze and a 37 °C water bath to thaw), and thencentrifuged at 13,000 rpm (10,392×g) for 10 min. Totalglutathione in the supernatant was determined kineticallybymeasuring the formation of 5-thio-2-nitrobenzoic acidfrom 5, 5′-dithionitrobenzoic acid in the presence ofNADPH and glutathione reductase fluorometrically at405 nm. The oxidized form (GSSG) was measured fol-lowing derivatization of GSH with 2-vinyl-pyridiumtrifluoromethane sulfonate to remove it from the reac-tion. Briefly, standard solutions of GSSG or aliquots ofsamples were mixed with 1 μL of 2-vinylpyridine per50 μL of sample volume. All solutions were adjusted topH 7.5 with triethanolamine. After incubation for 60 minat room temperature, the assay was performed as de-scribed for total glutathione. The amounts of total gluta-thione and GSSG were normalized to protein contentand expressed as nanomoles GSH per milligram of pro-tein. Intracellular GSH levels were measured by a mod-ified method (Tietze 1969). In brief, A549 cells after HQtreatment were washed three times with cold PBS, andsuspended in 1 mL of ice-chilled PBS containing 0.01%Triton X-100 and 0.6 % sulfosalicyclic acid. Cells werehomogenized and centrifuged at 4 °C for 5 min at2,500 rpm (384×g). An aliquot (20 μL) of supernatantwas added to 120 μL of 0.1 M phosphate buffer, 5 mMEDTA (pH 7.5), containing 100 μL of 5 mM GSSG-reductase-5-5,-dithiobis-2-nitrobenzoic acid and 0.5U ofglutathione reductase. NADPH (60 μL of 2.4 mM) wasadded and the rate of change in absorbance was mea-sured for 3 min at 412 nm. A standard curve using GSH

in the range of 0.33–1.35 nmol was prepared prior tomeasurement of the samples. The results were expressedin terms of nanomoles of GSH per micrograms protein.Intracellular GSSG levels were estimated by the methodof Griffith (Griffith 1980). Briefly, standard solutions ofGSSG or aliquots of samples were mixed with 2μL of 2-vinylpyridine per 100 μL of sample volume. All solu-tions were adjusted to pH 7.5 with triethanolamine. Afterincubation for 1 h at room temperature, the assay wasthen performed as described for total glutathione.

Detection of 8-Oxo-dG by immunofluorescence

Immunocytochemistry was carried out for 8-Oxo-dGmeasurement using anti-8-Oxo-dG antibody (1:250;Trevigen), according to the manufacturer’s instructionswith slight modifications. Briefly, cells on cover slipswere fixed with 100 % pre-chilled methanol for 5 minand immersed in 100 % prechilled acetone for 5 min.Cover slips were subsequently air-dried, treated with0.05 M HCl for 5 min on ice, and washed three timeswith PBS. RNA was digested by incubating the coverslips in 100 μg/mL RNase with 15 mM sodium citratefor 1 h at 37 °C. After RNA digestion, cover slips weresequentially washed in PBS, 35, 50, and 75 % ethanolfor 2 min each. DNA was denatured by incubating thecover slips with 0.15 M NaOH in 70 % ethanol for4 min. A series of washes was performed starting with70 % ethanol containing 4 %v/v formaldehyde and then50 % ethanol, 35 % ethanol, and finally PBS for 2 mineach. Proteins were digested with 5 μg/mL proteinase Kin TE, pH 7.5, for 10 min at 37 °C. After several PBSwashes, cover slips were incubated with anti-8-Oxo-dGantibody in PBT20 (1×PBS/1 % BSA/0.1 % Tween 20)for 1 h at room temperature. After several washes with0.1×PBS, 8-Oxo-dG was detected using an Alexa Fluor488 secondary antibody (1:500 in PBT20; Invitrogen).Nuclei were counterstained with DAPI (Invitrogen,Mulgrave, Australia). Images were captured using adigital camera (Carl Zeiss) attached to a fluorescentmicroscope Axioskop2 mot plus (Carl Zeiss). The stain-ing intensity of nuclei was quantified using the publicdomain software ImageJ version 1.42q (NIH, USA).

Chromosome aberration test

Chromosome aberration was assessed using anestablished protocol (Klein et al. 2001). A549 cells ofunder 15 passages were seeded at 2×105 cells/culture

Cell Biol Toxicol

dish (100×20 mm, BD Primaria™), with 8 mL ofDMEM medium. Cells were incubated for 16 to 20 hat 37 °C before replacement of medium with 5 % FBSand respective addition of 5, 10, and 20 μM (finalconcentration) of hydroquinone dissolved in DMSO.A final concentration of 500 ng/mL mitomycin(MMC) was used as the positive control. After 6 h ofexposure, the cells were washed and incubated in freshmedium with 5 % FBS for another 24 h. Cells weretreated with colchicine and cultured for further 3 h andwere harvested following centrifugation. After the hy-potonic treatment with 0.075 M KC1, cells were fixedwith a mixture of methanol and glacial acetic acid (3:1).Cells were placed onto pre-cleaned ice-cold slides,dried, and stained with 10 % Giemsa stain.Metaphases were located using Metafer MSearch(MetaSystems Germany) and sorted automaticallyaccording to the quality of the metaphase spread. Amaximum of 200 cells of best quality metaphase spreadof each treatment were captured and 100 cells of eachtreatment were scored. Since A549 cells have variablechromosome numbers, the parameter of total chromo-some numbers was not used. The chromosome typeaberrations such as breaks, dicentrics, rings, and chro-matid breaks were scored and analyzed for each treat-ment group.

Statistics analysis

Data was analyzed with statistical software(GraphPad Prism 5.0.). All values are expressedas mean ± SD. Nonparametric analysis of variance(Kruskal–Wallis method) and Mann–Whitney Utest were used to determine significant overallsignificant differences between groups and signifi-cant differences between treated and controlgroups. A value of p less than 0.05 was consideredto indicate significance.

Results

Cell survival assay

The results of cytotoxicity of A549 cells induced byHQ are presented in Fig. 1. A549 cells were sensitiveto micromolar concentrations of HQ. Exposure to HQfor 6 and 12 h, have similar effects on cell growth(IC50, 38 and 36 μM, respectively) whereas lowest

toxicity was evident after 1 h (IC50, 59 μM). Longerexposure to HQ to 24 h induced the most toxic effecton A549 cell (IC50, 33 μM). Based on these results weused a dose range of 0, 2.5, 5, 10, 20, and 40 μM forall subsequent experiments, as these doses with theexception of 40 μM, were below the IC50 valueobtained at all hours. Considering that 1 h treatmentof HQ induced low toxicity with IC50 of 59 μM, weincluded 40 μM as the highest concentration.

DNA damage measured by comet assay

DNA damage was measured in A549 cells treatedwith various doses of HQ for 1, 6, 12, and 24 h.Tail DNA content (%), Olive Tail Moment, and TailLength were used to evaluate the level of HQ-induced DNA damage in A549 cells measured bythe Comet assay under alkaline conditions (pH>13).DNA damage was detected at all concentrations ofHQ by 1 h and increased in a concentration-dependent manner (Table 1). With an increase inexposure time, levels of DNA damage decreased asdetermined by all three parameters in the Cometassay. The extent of this damage was more markedat lower concentration of HQ.

γH2AX foci formation induced by hydroquinone

DNA double strand breaks induced by HQ were ana-lyzed with time after exposure by measuring γH2AX

1.00 1.25 1.50 1.75 2.00 2.250

25

50

75

100

125 HQ-1 hHQ-6 hHQ-12 hHQ-24 h

log HQ (µM)

% o

f co

ntro

l

Fig. 1 Hydroquinone (HQ)-induced cytoxicity in A549 cells.Effects of HQ at different concentrations on A549 cell survivalusing the MTS assay. Exposure of cells to HQ for 1, 6, 12, and24 h gave LC50 of 59, 38, 36, and 32 μM, respectively. Eachresult represents the mean and standard deviation of three rep-licates and three independent experiments

Cell Biol Toxicol

foci formation. Average numbers of H2AX foci in 50nuclei were plotted over the HQ concentrations asshown in Fig. 2. HQ induced H2AX phosphoryla-tion in A549 cell in a dose–response pattern afterall time periods with strongest induction observedafter 1 h. Longer treatment of HQ generated fewerγH2AX foci.

Cellular GSSG and total GSH level

The intracellular reduced GSH and oxidized GSSGlevels in A549 cells were measured to examine theinvolvement of oxidative damage induced by HQ.A reduction in GSH level at 0.5 h was evident (p<0.05) at concentrations of 10, 20, and 40 μM com-pared with the control group (Fig. 3a). Recovery ofGSH to control levels was observed at 1 h forhigher concentrations (20 and 40 μM) and 6 h for

lower concentrations (2.5 to 10 μM). A significantincrease of GSH at 24 h was found at all concen-trations of HQ. The levels of GSSG were elevatedat all concentrations of HQ at 0.5, 1, and 6 h (p<0.05) following 40 μM exposure. Highest increasesin the levels of GSSG were observed after 6 h ofexposure of 10, 20, and 40 μM. No significantchanges in intracellular GSSG levels at 24 h expo-sure were apparent (Fig. 3b). GSH/GSSG ratioreduced significantly at 0.5 and 1 h and recoveredto control levels in 6 and 24 h as seen in Fig. 3c.

HQ induced intracellular 8-Oxo-dG formation

Cellular 8-Oxo-dG formation by HQ was examinedusing immunofluorescence staining and the resultspresented as average fluorescent intensity per cell asshown in Fig. 4a dose–response relationship between

Table 1 HQ-induced DNAdamage in A549 cells measuredby Comet assay

*p<0.05; **p<0.01

Treatment time (h) Concentration(μM)

Tail DNA (%) Olive tail moment Tail length

1 0 8.47±1.61 3.01±0.58 39.00±4.24

2.5 17.75±1.25** 8.71±1.18** 63.91±2.24**

5 24.20±1.78** 12.21±1.18** 84.41±7.58**

10 32.56±3.95** 17.62±2.10** 99.47±18.50**

20 38.59±5.98** 19.72±5.66** 89.43±5.68**

40 43.90±7.93** 24.31±5.97** 104.08±17.56**

6 0 8.32±1.2 3.42±0.38 28.71±1.79

2.5 19.14±3.01** 10.18±1.96** 61.85±12.18**

5 31.03±1.90** 18.91±3.64** 81.23±19.66**

10 38.35±3.33** 25.88±2.26** 116.00±6.8**

20 41.14±6.98** 25.40±2.41** 104.75±11.78**

40 51.93±3.75** 37.68±2.79** 121.86±13.90**

12 0 10.60±0.98 3.95±0.66 37.81±4.35

2.5 13.71±2.31 6.28±0.77** 71.60±4.74**

5 15.76±3.15 6.79±1.84** 57.89±17.76**

10 19.55±3.85** 9.23±2.41** 68.07±21.91**

20 21.81±2.17** 11.77±1.27** 65.80±5.1**

40 32.39±5.68** 16.95±1.79** 87.49±12.29**

24 0 9.65±2.27 2.54±0.43 32.17±2.83

2.5 11.38±3.51 4.71±1.04 39.47±3.24

5 11.67±2.31 4.55±1.05 41.11±2.69*

10 14.53±2.39* 5.49±0.83** 46.64±7.47**

20 16.21±2.02** 6.64±1.28** 58.65±9.66**

40 21.80±4.78** 10.37±1.36** 62.54±6.01**

Cell Biol Toxicol

formation of 8-Oxo-dG and HQ concentrations wasfound for the exposure time intervals of 1 and 6 h.

Lower levels of 8-Oxo-dG were evident at longer timepoints (12 and 24 h).

Control 5 µM 10 µM 20 µM 40 µM

Dapi

Anti- H2AX

Merged

a

b

Fig. 2 Double-strand DNA breaks evaluated by measuringthe formation of H2AX phosphorylation (γH2AX) in A549cells treated with HQ at different concentrations for 6 h. aRepresentative photographs of H2AX foci formation inA549 cells. Immunofluorescent staining for γH2AX wasfollowed by detection with specific secondary Alexa Fluorconjugates. Red γH2AX, blue nuclear stained with Dapi. b

DNA double-strand DNA breaks are represented asγH2AX foci number per cell. A549 cells were treated withcontrol and different concentrations of HQ for 1, 6, 12,and 24 h. The numbers of γH2AX foci were quantified in50–60 nuclei using the public domain software ImageJversion 1.42q (NIH, USA). *p<0.05, **p<0.01

Cell Biol Toxicol

Fig. 3 Effects of HQ on cellularGSH and GSSG levels of A549cells. a Effect of HQ on total GSHlevels in A549 cell treated withdifferent concentrations of HQ for0.5, 1, 6, and 24 h. b GSSG levelsin A549 cells treated with differentconcentrations of HQ for 0.5, 1, 6,and 24 h. GSSG levels were mea-sured using glutathione assay kit(CS0260, Sigma). c GSH/GSSGratio in A549 cells. (*p<0.05,**p<0.01)

Cell Biol Toxicol

Chromosome aberrations induced by hydroquinone

The results of chromosome aberration in A549 cellstreated with HQ for 6 h are shown in Table 2. Positivecontrol, MMC at 500 ng/mL induced significant for-mation of chromatid breaks. The most significant

aberrations induced by HQ at 5 μM were structuralchanges as chromosome breaks and rings which in-creased at higher concentrations of HQ. Other aberra-tions such as chromatid breaks were also found withno significant differences observed between the con-trol and treated cells.

a

b

Control 5 µM 10 µM 20 µM 40 µM

Dapi

Anti-8-oxo-dg

Merged

Fig. 4 HQ-induced oxidative DNA damage in A549 cells. aRepresentative photographs of 8-oxo-dG formation in A549cells treated with HQ for 6 h. Immunofluorescence detectionwas carried out for 8-oxo-dG measurement using anti-8-oxo-dGmonoclonal antibody followed with specific secondary Alexa

Fluor conjugates. Green 8-oxo-dG, blue nuclear stained withDapi. b Fluorescent staining intensity of 50 nuclei for eachtreatment was quantified using the public domain softwareImageJ version 1.42q (NIH, USA). *p<0.05, **p<0.01

Cell Biol Toxicol

Discussion

HQ is produced naturally and anthropogenically andhuman may be exposed to HQ through ingestion,inhalation and dermal absorption. HQ has been stud-ied for its mutagenicity and genotoxicity in various invitro models especially in hematopoietic and lympho-cyte cells (Andreoli et al. 1999; Hiraku and Kawanishi1996; Levay et al. 1991; Levay et al. 1993; Peng et al.2012). On the other hand, HQ is found in large quan-tities in cigarette smoke and some industrial sources towhich human may be exposed through inhalation.However, whether the genotoxic effects of HQ whichhave been demonstrated in blood cells can occur inlung cells need to be investigated.

In this study, we used human lung adenocarcinomaalveolar basal epithelial cells (A549) as a cellular modelto investigate the cytotoxic and genotoxic effects of HQin the lung and the possibility of oxidative stress in-volvement. We found HQ to be highly toxic to A549cells. The lowest toxicity was observed after 1 h expo-sure (IC50, 59μM) and the highest at 24 h (IC50, 33μM;Fig. 1). These results are similar to other studies report-ed. For example, Syrian hamster embryo (SHE) cellsexposed to 30 μM HQ for 48 h decreased the cellsurvival to 61.2 % of that in untreated cells (Tsutsui etal. 1997). Similar toxicity of HQ was reported usingHL60, Jurkat, and U937 cells (Kim et al. 2009; Lee et al.2007). However, other cell lines such as Hela, RAW,and CHO K5 cells show much more resistance to HQ(Galvan et al. 2008; Kiffe et al. 2003), suggesting aselective cytotoxicity of HQ to certain cell types.Based on the cytotoxic data of HQ, we chose dosagerange 0, 2.5, 5, 10, 20, and 40 μM for assessing thegenotoxic effects of HQ.

HQ has been reported to be able to induce DNAadducts and damage in human HL-60, mouse bonemarrow macrophages, human bone marrow, lympho-cytes, and HepG2 cells (Andreoli et al. 1999; Hirakuand Kawanishi 1996; Levay et al. 1991; Levay et al.1993; Tietze 1969). To investigate the genotoxic effectof HQ on lung cells we examinedDNA damagewith theComet assay and the appearance of DNA double strandbreaks by γH2AX foci analysis. DNA damage causedby HQ in A549 cells was detectable as early as 1 h at alow dose (2.5 μM) as indicated by results from theComet assay (Table 1). The extent of DNA damagewas dose-dependent. Interestingly, during the earliertime exposures (1–6 h), HQ-induced DNA damageincreased at all doses, but declined thereafter at laterexposure time points (12–24 h; Table 1). H2AX is asubtype of histone H2A and plays a critical role in DNAdouble strand breaks (DSBs) (Podhorecka et al. 2010).In response to DNA damage, H2AX is quickly phos-phorylated on serine 139 and recruited to the sites ofDNA damage to form the foci (Rogakou et al. 1998).Recently, the formation of phosphorylated H2AX focihas been used as a genotoxicity biomarker in toxicolog-ical studies (Watters et al. 2009) since the number of focicorrelates to the amount of DSBs occurred (Löbrich etal. 2010). The number of foci can bemeasuredmanuallywith fluorescent microscope and image software orusing flow cytometry. The intensity of the foci whichmay be varied by different cell lines or antibodies caninfluence the foci counting and has to be set at certainthreshold for analysis (MacPhail et al. 2003). We foundan increase in the number of γH2AX foci during earlyexposure (1 h) was followed by a pronounced decline attimes thereafter (Fig. 2b). These results suggest thatDNA damage occurred very rapidly following exposure

Table 2 Chromosome aberrations in A549 cells treated with hydroquinone (HQ) for 6 h

Treatment Chromosome type aberrations Chromatid type Chromosome type Total chromosome

Breaks Dicentrics Rings Aberrations Aberrations Aberrations

MMC 1.09±0.31 0.09±0.09 0.00±0.00 1.18±0.00 1.82±0.40 3.00±0.55

0 0.80±0.13 0.17±0.04 0.04±0.02 1.01±0.02 0.59±0.11 1.60±0.19

2.5 1.20±0.16 0.09±0.03 0.07±0.03 1.36±0.03 0.70±0.11 2.06±0.22

5 1.58±0.25** 0.32±0.05 0.06±0.03 1.93±0.24** 0.41±0.06 2.34±0.24**

10 2.05±0.22** 0.30±0.12 0.09±0.03 2.44±0.03** 0.78±0.09 3.22±0.25**

20 6.14±0.29** 0.17±0.04 0.03±0.03 6.34±0.89** 0.27±0.06 6.34±0.89**

**p<0.01

Cell Biol Toxicol

to a wide-range of HQ concentrations. However by 12 hDNA damage was reduced suggesting a possible induc-tion of a protective cellular response which includesDNA repair mechanisms to rectify and protect DNAfrom further damage. Reduced DNA damaged observedat concentration of 40 μM for 6 h and longer may bepartially due to the reduced living cells because of thecytoxicity of HQ occurred at this concentration. Sincethe highest treatment concentration (40 μM) is higherthan IC50 at 6, 12, and 24 h, the decreased damagesobserved at this time points may be related to a lowersurvival rate.

GSH is an abundant cellular non-protein thiol, whichserves as the major intracellular antioxidant defensewithin the cells to protect against oxidative damage.As a strong reducing agent, reduced glutathione (GSH)is involved in reactions with ROS to form oxidizedglutathione (GSSG) (Franco et al. 2007). Consumptionof GSH leads to alterations in the GSH/GSSG ratio aknown traditional marker of oxidative stress (Balendiranet al. 2004). Depletion of reduced GSH by HQ has beenpreviously reported (Luo et al. 2008; Rubio et al. 2011;Smith 1999). Indeed, we found exposure to HQ caused atransient (0.5 h) depletion of GSH at higher HQ doses(Fig. 3a) suggesting that HQ can generate intracellularROS very rapidly. Moreover, elevated levels of GSSGwere observed proceeding exposure to high doses dur-ing early time points (0.5–6 h) compared with the con-trol values (Fig. 3b) supporting the notion that HQcauses an increase in ROS. The ROS induction wasmore evidently reflected in the GSH/GSSG ratio asshown in Fig. 3c. However, depletion in GSH evidentat 0.5 h for 10 and 20 μM HQ exposure was notassociated with an increase in GSSG implying thatoxidation of GSH during early exposure may not havebeen totally due to an increase in ROS. Previously,conjugation of HQ with GSH has been reported(Snyder et al. 1988). Thus, depletion of GSH may inpart be due to its conjugation to HQ resulting in theformation of less reactive and toxic metabolites, withincreased solubility and excretion. Hence HQ appears toproduce a depletion of intracellular GSH, concomitantwith GSH-conjugate formation, without significant ele-vation of GSSG. Importantly, proceeding GSH deple-tion, elevated levels of GSH were apparent by 6 h ofexposure to higher concentrations of HQ and remainedso at times thereafter (Fig. 3a). A possible mechanismfor this elevation in GSH was an increase in glutamatecysteine ligase (GCL) activity, the rate-limiting enzyme

of GSH biosynthesis. It has been reported that GCLnormally functions at less than its maximal rate becauseof feedback inhibition by GSH (Richman and Meister1975). A transient decrease of cellular GSH levels canrelease feedback inhibition of GCL enzyme activity.However, as GSH levels were above those of control,it seems unlikely that release of feedback inhibition byGSH depletion prevented its own synthesis. Indeed, HQexposure to human lung epithelial cells (Beas-2B) wasshown to up-regulate glutamate cysteine ligase modifiersubunit and glutathione synthetase, enzymes involved inGSH synthesis (Rubio et al. 2011). Therefore, it seemsplausible that short-term effects of oxidant-generatingsystems by HQ may up-regulate genes coding for GSHsynthesis in the lungs, possibly providing a protective/adaptive mechanism against subsequent oxidativestress. 8-Oxo-dG, a DNA adduct induced by oxygenfree radicals, has been used widely as a marker foroxidative DNA damage (Loft et al. 1994; Loft et al.1992; Toraason et al. 1999). This DNA oxidation iscaused by the modification of guanine by hydroxylradicals which are produced during HQ oxidation(DeCaprio 1999; Kondrova et al. 2007). Measurementof 8-Oxo-dG confirmed the presence of oxidative dam-age induced by HQ in the A549 cells (Fig. 3). At allexposure doses of HQ, short exposure time (1 and 6 h)resulted in maximal formation of 8-Oxo-dG. In compar-ison, longer exposure (12 and 24 h) led to reduced levelsof 8-Oxo-dG. Such results imply the up-regulation ofDNA repair mechanisms are employed during laterexposure times and further support the results we ob-served from the Comet assay and H2AX phosphoryla-tion (Table 1 and Fig. 2). When comparing the DNAdamages and GSH/GSSG, we can see the reverse cor-relation between DNA damages and GSH/GSSG at 1 h,suggesting the involvement of ROS in these genotoxiceffects of HQ at early time (Fig. 5a). Interestingly,GSH/GSSG was found to be recovered to control levelat 6 h (Fig. 5b) and longer treatment time while the DNAdamages were still persistent at these time periods. Therecovery of GSH/GSSG was due to the higher level ofGSH as stated previously. Induction of GSHmay be oneof protective mechanisms against further lesions byROS. But on the other hand, increased GSHmay inducefurther ROS production since the GSH-HQ conjugateswere found to be able to generate ROS through oxida-tive cycling and auto-oxidation (Lau et al. 2010; Smithet al. 1989). Recent work demonstrated GSH depletionled to a decrease in genotoxicity of benzene (Bird et al.

Cell Biol Toxicol

Fig. 5 Comparison of the ROS production and ROS damages.a Comparison of GSH/GSSG, 8-oxo-dG, and γH2AX fociformation in A549 cells treated with HQ for 1 h. The effects

of these parameters were expressed as relative to control forcomparison. b Comparison of GSH/GSSG, 8-oxo-dG, andγH2AX foci formation in A549 cells treated with HQ for 6 h

Cell Biol Toxicol

2010; Wetmore et al. 2008). These findings indirectlysupport the role of the GSH-metabolite conjugates sincebenzene exerts its genotoxic effects through its reactivemetabolites including HQ. The observed DNA damagemay also the results from the accumulative damages thatwere reduced over the treatment time periods (12 and24 h) possibly due to the DNA repair processes.Moreover, mutations induced by HQ were further sub-stantiated by the chromosome aberration test at 6 hwherein exposure to 10 and 20 μM of HQ led to signif-icant induction of chromosome breaks (Table 2).Chromosome aberrations occur as a result of unrepairedand/or misrepaired DNA damages which may happen inG1 phase and G2 phase leading to form chromosometype aberrations and chromatid type aberrations respec-tively. (Natarajan and Palitti 2008; Pfeiffer et al. 2000).In this study, we observed an increase in chromosomebreaks but not chromatid breaks at higher concentrationof HQ. In view our experimental conditions underwhich cells were treated with HQ for 6 h followed byrecovery time for 24 h, treated cells harboring the chro-matid type aberrations would have time for further rep-lication since A549 cell have a doubling time of 23.5 h(He et al. 2001). Consequently, chromatid breaks wereconverted by duplication into derived chromosome typeaberrations (Savage 1976) which were observed atmetaphase. The higher induction of chromatid breaksby positive control, MMC, is possibly due to its S-dependent chromosome breaking property with charac-teristics of producing chromatid type breaks (Sognierand Hittelman 1986). To our knowledge, this is the firsttime HQ has been shown to induce chromosomal aber-rations in A549 cells which provides additional evi-dence for a HQ-induced clastogenic effect. In agreementwith our results, other studies using Chinese hamsterlung fibroblast cells (Silva et al. 2003) and SHE cells(Tsutsui et al. 1997) confirm that HQ can induce chro-mosome aberrations. Based on these observations it isprobable that genotoxicity by HQ is mediated by avariety of different types of lesions in DNA as detectedby the Comet assay, DNA double strand breaksevidenced by appearance of γH2AX foci, and the oc-currence of chromosomal aberrations.

Short-term exposure has important implications withregard to the health of workers in various industries, aswell as the general public. We found that short-termexposure to HQ resulted in DNAdamage and an increasein oxidative stress. Longer exposure to a single dose ofHQ appears to induce cellular protective/adaptive

responses an indicated by a decline in DNA damage.Moreover, the induction of GSH may be like a double-edged sword as partially protective/adaptive cellular re-sponses on one hand and on the other hand, a pathway ofROS generation by conjugation with HQ. Further studiesare required to elucidate the balance and relationshipbetween GSH level and oxidative consequences.Besides, the HQ can react with DNA to form DNAadducts which also play a role in HQ-inducedgenotoxicity (Gaskell et al. 2005a; Gaskell et al.2005b). How much contribution from oxidative stressand DNA adduct to the genotoxic effects of HQ warrantfurther studies. Given that urbanization is increasing andsubsequently impaired air pollution, there is a definiteneed for further investigations for the development ofappropriate health risk assessment strategies in relationto such potentially toxic compounds. New knowledgegained will advance our understanding in the potentialadverse effects of HQ and provide information for amore refined health risk assessment of this compoundby considering all exposure sources.

Acknowledgment The project was funded by CRC CARE(grant no. 1-3-03-07/08). Entox is a partnership betweenQueensland Health and the University of Queensland. Technicaladvice and access to Metafer MSearch instrument for chromo-some aberration assay provided by Mr. Ross Brookwell and BenLundie of Sullivan and Nicolaides Pathology, Brisbane, areacknowledged.

References

Andreoli C, Rossi S, Leopardi P, Crebelli R. DNA damage byhydroquinone in human white blood cells: analysis by alkalinesingle-cell gel electrophoresis. Mutat Res. 1999;438:37–45.

Balendiran GK, Dabur R, Fraser D. The role of glutathione incancer. Cell Biochem Funct. 2004;22:343–52.

Bertram KM, Baglole CJ, Phipps RP, Libby RT. Molecularregulation of cigarette smoke induced-oxidative stress inhuman retinal pigment epithelial cells: implications forage-related macular degeneration. Am J Physiol CellPhysiol. 2009;297:C1200–10.

Bird MG, Letinski DJ, Nicolich M, Chen M, Schnatter AR,Whitman FT. Influence of toluene co-exposure on the metabo-lism and genotoxicity of benzene in mice using continuous andintermittent exposures. Chem Biol Interact. 2010;184:233–9.

Bolton JL, TrushMA, Penning TM, Dryhurst G,Monks TJ. Role ofquinones in toxicology. Chem Res Toxicol. 2000;13:135–60.

Castell JV, Donato MT, Gomez-Lechon MJ. Metabolism andbioactivation of toxicants in the lung. The in vitro cellularapproach. Exp Toxicol Pathol. 2005;57 Suppl 1:189–204.

Cell Biol Toxicol

Cheah NP, Pennings JL, Vermeulen JP, van Schooten FJ,Opperhuizen A. In vitro effects of aldehydes present intobacco smoke on gene expression in human lung alveolarepithelial cells. Toxicol In Vitro. 2013;27:1072–81.

DeCaprio AP. The toxicology of hydroquinone-relevance tooccupational and environmental exposure. Crit RevToxicol. 1999;29:283–330.

Deisinger PJ, Hill TS, English JC. Human exposure to naturallyoccurring hydroquinone. J Toxicol Env Health. 1996;47:31–46.

Franco R, Schoneveld OJ, Pappa A, Panayiotidis MI. Thecentral role of glutathione in the pathophysiology of humandiseases. Arch Physiol Biochem. 2007;113:234–58.

Galvan N, Lim S, Zmugg S, Smith MT, Zhang L. Depletion ofWRN enhances DNA damage in HeLa cells exposed to thebenzene metabolite, hydroquinone. Mutat Res. 2008;649:54–61.

Gaskell M, McLuckie KI, Farmer PB. Comparison of the repairof DNA damage induced by the benzene metabolites hydro-quinone and p-benzoquinone: a role for hydroquinone inbenzene genotoxicity. Carcinogenesis. 2005a;26:673–80.

Gaskell M, McLuckie KI, Farmer PB. Genotoxicity of thebenzene metabolites para-benzoquinone and hydroqui-none. Chem-Biol Interact. 2005b;153–154:267–70.

Gopalakrishna R, Chen ZH, Gundimeda U. Tobacco smoketumor promoters, catechol and hydroquinone, induce oxi-dative regulation of protein kinase C and influence inva-sion and metastasis of lung carcinoma cells. Proc NatlAcad Sci U S A. 1994;91:12233–7.

Griffith OW. Determination of Glutathione and GlutathioneDisulfide Using Glutathione-Reductase and 2-Vinylpyridine.Anal Biochem. 1980;106:207–12.

Gut I, Nedelcheva V, Soucek P, Stopka P, Tichavska B.Cytochromes P450 in benzene metabolism and involve-ment of their metabolites and reactive oxygen species intoxicity. Environ Health Perspect. 1996;104:1211–8.

Hard GC, Whysner J, English JC, Zang E, Williams GM.Relationship of hydroquinone-associated rat renal tumorswith spontaneous chronic progressive nephropathy.Toxicol Pathol. 1997;25:132–43.

He LF, Yang CPH, Horwitz SB. Mutations in beta-tubulin mapto domains involved in regulation of microtubule stabilityin epothilone-resistant cell lines. Mol Cancer Ther.2001;1:3–10.

Hiraku Y, Kawanishi S. Oxidative DNA damage and apoptosisinduced by benzene metabolites. Cancer Res. 1996;56:5172–8.

Horita M, Wang DH, Tsutsui K, Sano K, Masuoka N, Kira S.Involvement of oxidative stress in hydroquinone-inducedcytotoxicity in catalase-deficient Escherichia coli mutants.Free Radical Res. 2005;39:1035–41.

IARC. IARC Monographs on the Evaluation of CarcinogenicRisks to Humans, Vol. 71 (part two), Re-evaluation ofsome organic chemicals, hydrazine and hydrogen peroxide.Lyon: IARC; 1999. p. 691–719.

Ishii H et al. Fhit-deficient hematopoietic stem cells survivehydroquinone exposure carrying precancerous changes.Cancer Res. 2008;68:3662–70.

Kang KW, Lee SJ, Kim SG. Molecular mechanism of Nrf2activation by oxidative stress. Antioxid Redox Signal.2005;7:1664–73.

Kari FW, Bucher J, Eustis SL, Haseman JK, Huff JE. Toxicityand carcinogenicity of hydroquinone in F344/N rats andB6C3F1 mice. Food Chem Toxicol. 1992;30:737–47.

Kiffe M, Christen P, Arni P. Characterization of cytotoxic andgenotoxic effects of different compounds in CHO K5 cellswith the comet assay (single-cell gel electrophoresis assay).Mutat Res. 2003;537:151–68.

Kim YJ, Woo HD, Kim BM, Lee YJ, Kang SJ, Cho YH, et al.Risk assessment of hydroquinone: differential responses ofcell growth and lethality correlated to hydroquinone con-centration. J Toxicol Environ Health A. 2009;72:1272–8.

Klein CB, Broday L, Costa M. Assays for Detecting ChromosomalAberrations, Current protocols in Toxicology. John Wiley &Sons, Inc., New York University School of Medicine. 2001,pp 3.7.1–3.7.16.

Kondrova E, Stopka P, Sousek P. Cytochrome P450 destructionby benzene metabolites 1,4-benzoquinone and 1,4-hydro-quinone and the formation of hydroxyl radicals in minipigliver microsomes. Toxicol in Vitro. 2007;21:566–75.

Lau SS, Kuhlman CL, Bratton SB, Monks TJ. Role ofhydroquinone-thiol conjugates in benzene-mediated toxic-ity. Chem Biol Interact. 2010;184:212–7.

Leanderson P, Tagesson C. Cigarette smoke-induced DNA-damage: role of hydroquinone and catechol in the formationof the oxidative DNA-adduct, 8-hydroxydeoxyguanosine.Chem Biol Interact. 1990;75:71–81.

Lee JY, Lim JY, Lee YG, Shin WC, Chun T, Rhee MH, et al.Hydroquinone, a reactive metabolite of benzene, reducesmacrophage-mediated immune responses. Mol Cells.2007;23:198–206.

Levay G, Pongracz K, Bodell WJ. Detection of DNA adducts inHL-60 cells treated with hydroquinone and p-benzoqui-none by 32P-postlabeling. Carcinogenesis. 1991;12:1181–6.

Levay G, Ross D, Bodell WJ. Peroxidase activation of hydro-quinone results in the formation of DNA adducts in HL-60cells, mouse bone marrow macrophages and human bonemarrow. Carcinogenesis. 1993;14:2329–34.

Löbrich M, Shibata A, Beucher A, Fisher A, Ensminger M,Goodarzi AA, et al. GammaH2AX foci analysis for mon-itoring DNA double-strand break repair: strengths, limita-tions and optimization. Cell Cycle. 2010;9:662–9.

Loft S, Vistisen K, Ewertz M, Tjonneland A, Overvad K,Poulsen HE. Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence ofsmoking, gender and body mass index. Carcinogenesis.1992;13:2241–7.

Loft S, Astrup A, Buemann B, Poulsen HE. Oxidative DNAdamage correlates with oxygen consumption in humans.FASEB J. 1994;8:534–7.

Luo LH, Jiang LP, Geng CY, Cao J, Zhong LF. Hydroquinone-induced genotoxicity and oxidative DNA damage inHepG2 cells. Chem Biol Interact. 2008;173:1–8.

MacPhail SH, Banath JP, Yu TY, Chu EHM, Lambur H, OlivePL. Expression of phosphorylated histone H2AX in cul-tured cell lines following exposure to X-rays. Int J RadiatBiol. 2003;79:351–8.

McGregor D. Hydroquinone: an evaluation of the human risksfrom its carcinogenic and mutagenic properties. Crit RevToxicol. 2007;37:887–914.

Cell Biol Toxicol

McHale CM, Zhang L, Smith MT. Current understanding of themechanism of benzene-induced leukemia in humans: impli-cations for risk assessment. Carcinogenesis. 2012;33:240–52.

Natarajan AT, Palitti F. DNA repair and chromosomal alter-ations. Mutat Res. 2008;657:3–7.

Olumide YM, Akinkugbe AO, Altraide D, Mohammed T,Ahamefule N, Ayanlowo S, Onyekonwu C, Essen N.Complications of chronic use of skin lightening cosmetics.Int J Dermatol. 2008;47:344–53.

Peng D, JiaxingW, Chunhui H,Weiyi P, XiaominW. Study on thecytogenetic changes induced by benzene and hydroquinonein human lymphocytes. Hum Exp Toxicol. 2012;31:322–35.

Pfeiffer P, Goedecke W, Obe G. Mechanisms of DNA double-strand break repair and their potential to induce chromo-somal aberrations. Mutagenesis. 2000;15:289–302.

Podhorecka M, Skladanowski A, Bozko P. H2AX phosphory-lation: its role in dna damage response and cancer therapy.J Nucleic Acis. 2010. doi:10.4061/2010/920161.

Pons M, Marin-Castaño ME. Cigarette smoke-related hydroqui-none dysregulates MCP-1. VEGF and PEDF expression inretinal pigment epithelium in vitro and in vivo. Plos One.2011;6:e16722.

Richman PG, Meister A. Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibitionby glutathione. J Biol Chem. 1975;250:1422–6.

Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNAdouble-stranded breaks induce histone H2AX phosphoryla-tion on serine 139. J Biol Chem. 1998;273:5858–68.

Rubio V, Zhang J, Valverde M, Rojas E, Shi ZZ. Essential role ofNrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol in Vitro. 2011;25:521–9.

Savage JR. Classification and relationships of induced chromo-somal structual changes. J Med Genet. 1976;13:103–22.

Sharma A, Patil JA, Gramajo AL, Seigel GM, Kuppermann BD,Kenney CM. Effects of hydroquinone on retinal and vas-cular cells in vitro. Indian J Ophthalmol. 2012;60:189–93.

Shibata MA, Hirose M, Tanaka H, Asakawa E, Shirai T, Ito N.Induction of renal cell tumors in rats and mice, and en-hancement of hepatocellular tumor development in miceafter long-term hydroquinone treatment. Jpn J Cancer Res.1991;82:1211–9.

Shimada ALB, Lino-dos-Santos-Franco A, Bolonheis SM,Nakasato A, Damazo AS, Tavares-de-Lima W, et al. In vivohydroquinone exposure causes tracheal hyperresponsivenessdue to TNF secretion by epithelial cells. Toxicol Lett.2012;211:10–7.

Silva MDC, Gaspar J, Silva ID, Leao D, Rueff J. Mechanisms ofinduction of chromosomal aberrations by hydroquinone inV79 cells. Mutagenesis. 2003;18:491–6.

Smith MT. Benzene, NQO1, and genetic susceptibility to can-cer. Proc Natl Acad Sci U S A. 1999;96:7624–6.

Smith MT, Yager JW, Steinmetz KL, Eastmond DA. Peroxidase-dependent metabolism of benzene’s phenolic metabolites andits potential role in benzene toxicity and carcinogenicity.Environ Health Perspect. 1989;82:23–9.

Snyder R. Benzene and leukemia. Crit Rev Toxicol. 2002;32:155–210.

Snyder CA, Sellakumar AR, James DJ, Albert RE. The carci-nogenicity of discontinuous inhaled benzene exposures inCD-1 and C57Bl/6 mice. Arch Toxicol. 1988;62:331–5.

Sognier MA, Hittelman WN. Mitomycin-induced chromatidbreaks in HeLa cells: a consequence of incomplete DNAreplication. Cancer Res. 1986;46:4032–40.

Tietze F. Enzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione: ap-plications to mammalian blood and other tissues. AnalBiochem. 1969;27:502–22.

Toraason M et al. Oxidative stress and DNA damage in Fischerrats following acute exposure to trichloroethylene or per-chloroethylene. Toxicology. 1999;138:43–53.

Tsutsui T, Hayashi N, Maizumi H, Huff J, Barrett JC. Benzene-,catechol-, hydroquinone- and phenol-induced cell transfor-mation, gene mutations, chromosome aberrations, aneu-ploidy, sister chromatid exchanges and unscheduled DNAsynthesis in Syrian hamster embryo cells. Mutat Res.1997;373:113–23.

Wan J, Winn LM. Benzene’s metabolites alter c-MYB activityvia reactive oxygen species in HD3 cells. Toxicol ApplPharm. 2007;222:180–9.

Watters GP, Smart DJ, Harvey JS, Austin CA. H2AX phosphor-ylation as a genotoxicity endpoint. Mutat Res. 2009;679:50–8.

Wetmore BA et al. Genotoxicity of intermittent co-exposure tobenzene and toluene in male CD-1 mice. Chem BiolInteract. 2008;173:166–78.

Winn LM. Homologous recombination initiated by benzenemetabolites: a potential role of oxidative stress. ToxicolSci. 2003;72:143–9.

Yin SN, Hayes RB, Linet MS, Li GL, Dosemeci M, Travis LB,Zhang ZN, Li DG, Chow WH, Wacholer S, Blot WJ. Anexpanded cohort study of cancer among benzeneexposedworkers in China. Benzene Study Group. Environ HealthPerspect. 1996;104(suppl 6):1339–1341.

Cell Biol Toxicol