Mutation Research 777 (2015) 54–61

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Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

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The cosmetic dye quinoline yellow causes DNA damage in vitro

Farah Maria Drumond Chequera,b,∗, Vinícius de Paula Venâncioa,Maíra Rocha de Souza Pradoa, Luiz Raimundo Campos da Silva e Cunha Juniorc,Thiago Mescoloto Lizierd, Maria Valnice Boldrin Zanonid, Rommel Rodríguez Burbanoc,Maria Lourdes Pires Bianchia, Lusânia Maria Greggi Antunesa

a Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,USP, Ribeirão Preto, SP 14040-903, Brazilb Departamento de Análises Clínicas e Toxicológicas, Faculdade Federal de Minas Gerais, UFMG, Belo Horizonte, MG 31270-901, Brazilc Laboratório de Citogenética Humana, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazild Instituto de Química. Departamento de Química Analítica, Universidade Estadual Paulista – UNESP, Quitandinha 14800-900, Araraquara/SP, Brazil

a r t i c l e i n f o

Article history:Received 29 June 2014Received in revised form 6 November 2014Accepted 11 November 2014Available online 18 November 2014

Keywords:GenotoxicityCosmetic dyeMicronucleusComet assayOxidation

a b s t r a c t

Quinoline yellow (QY) is a chinophthalon derivative used in cosmetic compositions for application tothe skin, lips, and/or body surface. However, regulatory data about the genotoxicity and/or mutageni-city of this compound are still controversial. Therefore, this work evaluated the genotoxicity of QY usingthe comet assay and the cytokinesis-block micronucleus cytome assay (CBMN-Cyt) in the metabolicallycompetent cell line HepG2, which closely mimics phase I metabolism. This research also identified theproducts formed after electrochemical oxidation of the QY dye, which simulates hepatic biotransforma-tion. The primary products generated after the oxidation process were analyzed by High PerformanceLiquid Chromatography coupled with a Diode Array Detector (HPLC/DAD), which detected the productionof 4,4′-diaminodiphenylmethane, 2-methoxy-5-methylaniline and 4,4′-oxydianiline. The results demon-strated that low (from 0.5 to 20 �g mL−1) QY concentrations were genotoxic in HepG2 cells on both assaysand those harmful compounds were detected after the oxidation process. Our findings suggest that thiscolorant could cause harmful effects to humans if it is metabolized or absorbed through the skin.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Synthetic dyes are used extensively in many industries,including the cosmetics, textile, pharmaceutical, food, plastics,photography and paper industries [1–4]. It is estimated that over10,000 different dyes and pigments are used industrially and thatover 7 × 105 tons of synthetic dyes are produced annually world-wide [4–6]. However, there is insufficient information about theirpotential health risks for humans and the environment [7,8]. Theavailable toxicological data about cosmetics dyes have showneffects that range from contact allergies to different types ofgenetic damages, including genotoxicity, mutagenicity and earlyage leukemia [9–13].

∗ Corresponding author at: Departamento de Análises Clínicas, Toxicológicas eBromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universi-dade de São Paulo, USP, Ribeirão Preto, SP 14040-903, Brazil. Tel.: +55 16 3602 4186;fax: +55 16 3602 4725.

E-mail address: farahchequer@gmail.com (F.M.D. Chequer).

The dye quinoline yellow (QY) is a chinophthalon derivativeused in cosmetic compositions for application to the skin, lips,and/or body surface [14]. This dye (also known as D&C Yellow no.11) was found to induce allergic contact dermatitis; in a humanmaximization test, 15 of 20 subjects became sensitized [15,16]. Theregulatory data regarding QY genotoxicity and/or mutagenicity arestill controversial [14,17]. Therefore, we studied the dye QY in thisresearch. The aim of this investigation was to evaluate the genotox-icity of QY using the alkaline comet assay and the cytokinesis-blockmicronucleus cytome assay (CBMN-Cyt) in the metabolically com-petent cell line HepG2, which closely mimics phase I metabolism.Micronuclei (MN) were also analyzed using the fluorescence in situhybridization (FISH) technique for further hazard characterization.

In addition, it is known that aromatic amines can be producedduring oxidative and/or reductive processes [3,18], and the forma-tion of these aromatic amine byproducts could be important forunderstanding the chemical transformation of dyes. Therefore, thepresent work also aimed to determine if 16 aromatic amines usedas standard models of amines classified by IARC [19] could be pro-duced during the oxidative and/or reductive conditions. In addition,

http://dx.doi.org/10.1016/j.mrgentox.2014.11.0031383-5718/© 2014 Elsevier B.V. All rights reserved.

F.M.D. Chequer et al. / Mutation Research 777 (2015) 54–61 55

this present study also investigated the oxidation and reductionbyproducts of QY dye by using electrochemistry to mimic thesereactions.

2. Materials and methods

2.1. Chemical compounds

Quinoline yellow (QY; D&C Yellow no. 11; CAS: 8003-22-3; CI: 47,000; purity>95%) (Fig. 1), acridine orange (CAS: 10127-02-3), cytochalasin B (CAS: 14930-96-2) and trypan blue (CAS: 72-57-1) were obtained from Sigma–Aldrich (St Louis,MO, USA). Dimethylsulfoxide (DMSO; CAS: 67-68-5) was purchased from Merck(Darmstadt, Germany). Doxorubicin (DXR; CAS: 23214-92-8) was obtained fromLaboratório Bérgamo (Taboão da Serra, Brazil). GelRedTM (CAS: 7732-18-5) waspurchased from Biotium (Hayward, CA, USA). Dulbecco’s Modified Eagle Medium(DMEM), fetal bovine serum (FBS) and a penicillin–streptomycin solution were pur-chased from Gibco (Carlsbad, CA, USA). Normal- and low-melting point agaroses(CAS: 9012-36-6) were obtained from Invitrogen (Carlsbad, CA, USA), and all otherchemicals were analytical-grade products of the highest purity available.

2.2. Cell line and chemical concentration selection

HepG2 cells, a hepatocellular carcinoma cell line, were obtained from the Amer-ican Type Culture Collection (HB-8065, ATCC, Rockville, MD, USA) and culturedin DMEM containing 10% heat-inactivated FBS and 1% antibiotic solution (peni-cillin/streptomycin). The cells were maintained in an incubator (Forma Series II,Thermo Electron Corporation, USA) at 37 ◦C in a humidified atmosphere of 5% CO2

and 95% air. The QY and doxorubicin concentrations used in this study were selectedusing the trypan blue dye exclusion method when the cells were harvested. Thehighest DMSO concentration used was 1.0%.

2.3. Genotoxicity assessment using the alkaline comet assay

The alkaline comet assay (i.e., single-cell gel electrophoresis assay) was per-formed according to the methods described by Singh et al. [20] and Tice et al. [21],with minor modifications. Briefly, 2 × 105 HepG2 cells were seeded in a 24-wellplate and incubated for 24 h. The cells were then treated with 0.5, 1.0, 2.0, 5.0, 10.0,15.0 or 20.0 �g mL−1 (final concentrations) of QY for 4 h. Positive control (doxoru-bicin 0.3 �g mL−1) and vehicle control (1.0% DMSO) samples were also included.The cells were mixed with 37 ◦C low-melting-point agarose and placed on normal-melting-point agarose-coated slides. The slides were then incubated in lysis solution(2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO and 1% Triton X-100) overnightat 4 ◦C. The lysed cells were then incubated in an electrophoresis solution (300 mMNaOH and 1 mM EDTA) for 40 min at 4 ◦C before being transferred to a horizontalelectrophoresis unit containing the same solution. The electrophoresis conditionswere 0.78 V/cm and 300 mA for 20 min at 4 ◦C. Finally, the slides were washed in aneutralization buffer (0.4 M Tris) for 20 min at 4 ◦C and fixed in ethanol for 5 min.The slides were stained with Gel RedTM (1:10,000) immediately before analysis andscored using a fluorescence microscope (Axiostar, Zeiss, Germany) equipped witha 515–560 nm excitation filter, a 590-nm barrier filter and an integrated digitalcamera. For each treatment, the tail moment (i.e., the product of the proportionof the tail’s intensity and the displacement of the tail’s center of mass relative tothe center of the head) and the tail intensity (i.e., the percentage of DNA in the tail)values for 100 nucleoids were evaluated at 400× magnification using the CometAssay IV software (Perceptive Instruments, Suffolk, UK). A total of three independentexperiments were performed.

2.4. Genotoxicity assessment using the cytokinesis-block micronucleus cytome(CBMN-Cyt) assay

The genotoxicity of QY was also evaluated as described by Fenech [22], fol-lowing the method of Natarajan and Darroudi [23] with modifications. A total of5 × 105 HepG2 cells were incubated in 25-cm2 culture flasks for 24 h before beingtreated with 0.5, 1.0, 2.0, 5.0, 10.0, 15.0 or 20.0 �g mL−1 QY (final concentration),0.03 �g mL−1 doxorubicin or vehicle control (1.0% DMSO). A total of 44 h after thebeginning of the initial incubation (i.e., after 20 h of treatment), the cells werewashed with PBS, the culture medium was changed, and the cells were treated with

Fig. 1. Chemical structure of the dye quinoline yellow.

3.0 �g mL−1 cytochalasin B. The cells were then incubated for 28 h, harvested, treatedwith cold 1% (v/v) sodium citrate and fixed with formaldehyde and methanol–aceticacid (3:1). Immediately before the analysis, the slides were stained using 40 �g mL−1

of acridine orange, and the frequencies of binucleated cells with micronuclei (MNi)were determined at 1000× magnification. The frequency of nucleoplasmic bridges(NPBs, biomarkers of dicentric chromosomes resulting from telomere end fusions orDNA mis-repair) and nuclear buds (NBUDs, biomarkers for gene amplification andaltered gene dosage events) were also evaluated according to the methods describedby Fenech [24]. To evaluate the cytostatic effects, the Nuclear Division Index (NDI)was calculated. A total of 500 viable cells per experimental point were scored todetermine the percentage of cells with one, two, three and four nuclei, and the NDIwas calculated as follows [25]: NDI = (M1 + 2M2 + 3M3 + 4M4)/N, where M1–M4 rep-resent the numbers of cells with 1–4 nuclei, respectively, and N represents the totalnumber of cells scored.

The MNi, NPB and NBUD frequencies were evaluated in a total of 1000 binu-cleated cells. The NDI calculation was measured in 500 cells per treatment. Threeindependent experiments were performed.

2.5. Fluorescence in situ hybridization (FISH)

The QY concentrations with the highest MN frequencies were selected for FISHanalysis. Positive (0.03 �g mL−1 DXR) and negative (DMSO 1.0%) controls were alsoused. The FISH analysis of MN was performed using a biotin-labeled human pancen-tromeric probe (1695-B Pan Centromeric Probe Biotin Cambio Ltd., UK) accordingto the method described by Guimaraes et al. [26]. This probe was first tested onmetaphase chromosomes to assess centromere-specific labeling. FISH assays wereperformed on cells that were fixed with methanol/acetic acid on freshly preparedslides, according to the protocol described by Kapka et al. [27], with minor mod-ifications. The MNi were counterstained with DAPI/antifade. The MNi present inthe binucleated human HepG2 cells with intact cytoplasms were examined for thepresence of one or more centromeric spots and were classified as either centromerepositive (C + MN) or centromere negative (C − MN). A total of 1000 binucleated cellswere scored for each treatment.

2.6. Controlled potential electrolysis and cyclic voltammetry

All of the electrochemical measurements were carried out using a PotentiostatEG&G model 283 (PAR). The measurements were performed in a conventional 25.0-mL electrochemical cell into which the following three electrodes were inserted:a reference electrode of Ag/AgCl (KCl 3.0 mol L−1), a platinum wire as the auxiliaryelectrode and a glassy carbon working electrode (area of 3.14 mm2 for the cyclicvoltammetric measurements and 4.00 cm2 for the electrolysis experiments).

The voltammetric measurements were obtained by transferring 25 mL of thestock solution of the original QY dye (1.0 mg mL−1 in DMSO/0.01 mol L−1 tetra-butylammonium tetrafluoroborate solution (TBABF4) into the cell. The solution waspurged with nitrogen for 15 min, and the voltammetric curves were recorded. Thisstep was performed to determine the oxidation and reduction potentials of the dyeand thus be able to apply the same potentials in the controlled potential electrolysisexperiment.

For the controlled potential electrolysis experiments, the dye degradation prod-ucts were submitted to increasingly oxidative conditions to reach a constant valueof current; the data were recorded to generate current vs time curves. The solu-tions of the QY dye were prepared at 1.0 mg mL−1 in 0.01 mol L−1 DMSO/TBABF4.Oxidation and reduction were conducted using +1.5 and −1.5 V, respectively, andthe reactions were monitored every 30 min during the 4.0 h analysis. To monitorthe change in coloration and the degradation of the dye, the generated productswere monitored by UV/Vis spectrometry and HPLC/DAD. All of the chromatographicmeasurements were carried out after a pre-filtration of the sample using a MILLEXMillipore (0.45 �m) system.

2.7. Chromatographic analysis

High Performance Liquid Chromatography coupled with a Diode Array Detector(HPLC/DAD) was used to monitor the QY dye as well as the products gener-ated after the controlled potential electrolysis using a Shimadzu CLC-ODS (C18)reversed-phase column (25 cm × 4.6 mm × 5 �m, 100 A) connected to a ShimadzuCLC-ODS (C18) guard column (1 cm × 4.6 mm × 5 �m, 100 A). HPLC-DAD was used toseparate and identify standard solutions containing 4,4′-diaminodiphenylmethane(Fluka, 97%); 4-chloroaniline (Fluka, 99%); 2-methoxy-5-methylaniline (Aldrich,99%); 3,3′-dimethylbenzidine (Sigma–Aldrich, 97%); 2,4-diaminotoluidine (Fluka,98%); 2-chloro-4-nitroaniline (Fluka, 98%); 4,4′-oxydianiline (Aldrich, 98%); ani-line (Sigma, 99%); 3,3′-dichlorobenzidine (Supelco, 99%); benzidine (Fluka, 98%);4-aminobiphenyl (Sigma, 90%); o-dianisidine (Sigma, 98%); o-anisidine (Aldrich,99%); o-toluidine (Aldrich, 98%); 4,4′-methyleno-bis-(2-chloroaniline) (Aldrich,85%); and 2-naphthylamine (Sigma, 98%). HPLC-DAD was performed using aMeOH/water + 30 mM of BMIm[NTf2] 70:30 mobile phase, 0.8 mL min−1 flow rate,column temperature of 45 ◦C, Phenomenex column Luna C18 (250 × 4, 6 mm, 5 �m)and �analysis = 230 nm [28]. Using the previously optimized conditions, HPLC-DADwas used to identify the primary products generated after electrochemical degra-dation of QY.

56 F.M.D. Chequer et al. / Mutation Research 777 (2015) 54–61

Fig. 2. The effects of 0.5, 1.0, 2.0, 5.0, 10.0, 15.0 or 20.0 �g mL−1 of quinoline yellow (QY) for 4 h on the tail intensity (A) and tail moment (B) of HepG2 cells evaluated bythe comet assay. The values shown represent the mean ± SD, and the data are based on three independent experiments. Vehicle control, 1.0% dimethylsulfoxide; positivecontrol, 0.3 �g mL−1 of doxorubicin. *: Significantly different from vehicle control group.

Table 1Assessment of the mutagenic effects of quinoline yellow (QY) on HepG2 cells using the cytokinesis-block micronucleus cytome assay (CBMN-Cyt).

Treatment(�g mL−1)

CBMN-Cyt NDI FISH

Total no. in 1000 BN cells C + MN(%)/C − MN(%)

MNi NPBs NBUDs

Vehicle control 19 ± 3 2 ± 2 5 ± 3 1.6 ± 0.1 57.1/42.9Positive control 99 ± 14* 16 ± 5 10 ± 7 1.6 ± 0.1 60.5/39.50.5 QY 53 ± 1* 3 ± 4 3 ± 3 1.5 ± 0.1 n/a1.0 QY 46 ± 8* 2 ± 3 3 ± 2 1.5 ± 0.1 n/a2.0 QY 50 ± 15* 2 ± 3 4 ± 3 1.5 ± 0.1 n/a5.0 QY 63 ± 13* 1 ± 1 6 ± 4 1.6 ± 0.1 n/a10.0 QY 65 ± 14* 2 ± 3 6 ± 2 1.5 ± 0.1 n/a15.0 QY 69 ± 7* 2 ± 2 7 ± 2 1.5 ± 0.1 57.9/42.120.0 QY 72 ± 4* 3 ± 2 4 ± 1 1.4 ± 0.1 57.2/42.8

Values shown are the mean ± SD; BN, binucleated cell; MNi, micronuclei; NPBs, nucleoplasmic bridges; NBUDs, nuclear buds; NDI, nuclear division index; C + MN, centromerepositive (i.e., MN containing one or more whole chromosome signals); C − MN, centromere negative (i.e., MN containing acentric chromosome fragment signals). The datashown are based on three independent experiments. Vehicle control, 1.0% dimethylsulfoxide; positive control, 0.03 �g mL−1 doxorubicin. *: Significantly different from thecontrol group (p < 0.05).

The conditions used for monitoring the degradation of the dye consisted of aMeOH/water 80:20 mobile phase, 1.0 mL min−1 flow rate, column temperature of45 ◦C, Phenomenex Luna C18 column (250 × 4, 6 mm, 5 �m) and �analysis = 450 nm.The analysis time was 10 min, and all of the analyses were conducted in triplicate.

All of these methodologies were conducted based on chromatographic param-eters such as retention time (tR), retention constant factor (k), selectivity (˛),resolution between peaks (Rs) and theoretical plate number (N). Standard curvesand a quantitative analysis of the target amines were obtained using a linear regres-sion of the peak area vs. concentration. Further comparisons were performed usingthe standard addition method in which aliquots of the working standard dissolved inmethanol were spiked into the samples. The procedure was conducted in triplicatefor each sample.

2.8. Statistical analysis

All of the data shown are expressed as the mean value ± SD of three independentexperiments. The results were analyzed using one-way ANOVA with post hoc Dun-nett’s tests (at a significance level p < 0.05) in GraphPad Prism 5 (GraphPad Software,USA).

3. Results

All of the QY concentrations used in this study resulted in atleast 80% cell viability prior to cell harvesting, as determined bythe trypan-blue exclusion method, and concentrations greater than20 �g mL−1 were excluded due to the low cell viability.

The results of the comet assay are shown in Fig. 2. The sensi-tivity of this in vitro assay was demonstrated by the response to0.3 �g mL−1 DXR, which induced a statistically significant increasein tail moment and tail intensity compared with the vehicle con-trol group (i.e., cells treated with 1.0% DMSO). Moreover, comparedwith the vehicle control, QY was genotoxic to HepG2 cells at con-centrations ranging from 2.0 to 20 �g mL−1.

In the CBMN-Cyt assay, the increased MNi frequencies (Table 1)indicated that this dye promoted genotoxic effects at each of theconcentrations tested (0.5–20 �g mL−1). The frequencies of NPBand NBUD and the NDI calculation (also shown in Table 1) werenot significantly different between the experimental and controlgroups. The FISH assay also showed that although the MNi frequen-cies were higher in the treated groups, there was no difference inthe C + MN/C − MN ratio between the treated groups and the nega-tive control group (p < 0.05). No difference between aneugenic andclastogenic effects was observed across the treatments. DXR wasused as a positive control, and 0.03 �g mL−1 DXR increased the MNfrequency compared with the vehicle control group.

Fig. 3A shows representative chromatograms of the HPLC-DAD data obtained for a 20 �L solution containing 50 ppmof the standard aromatic amine of interest (mobile phase:methanol/water 70:30 (v/v) containing 30 mM of liquid ionic

F.M.D. Chequer et al. / Mutation Research 777 (2015) 54–61 57

Fig. 3. (A) Chromatograms of HPLC/DAD obtained using 20 �L of a standard solution of aromatic amines. Mobile phase: methanol/water 70:30 (v/v) + 30 mM of BMIm-NTf2,� = 230 nm, column C18, T = 40 ◦C, flow rate = 0.80 mL min−1. a: 4,4′-diaminodiphenylmethane; b: 4-chloroaniline; c: 2-methoxy-5-methylaniline; d: 3,3′-dimethylbenzidine;e: 2,4-diamintoluidine; f: 4,4′-oxydianiline; g: 2-chloro-4-nitroaniline; h: aniline; i: 3,3′-dichlorobenzidine; j: benzidine; k: 4-aminobiphenyl; l: o-dianisidine; m: o-anisidine;n: o-toluidine; o: 4,4′-methyleno-bis-(2-chloroaniline); p: 2-naphthylamine. (B) Chromatograms from HPLC/DAD of quinoline yellow (1.0 mg mL−1) before (original dye,black line) and after oxidation at +1.5 V vs Ag/AgCl (blue line) or reduction at −1.5 V vs Ag/AgCL (red line) for 4.0 h. Mobile phase MeOH/Water 80:20, flow rate 1.0 mL min−1;45 ◦C; Phenomenex Luna C18 column (250 × 4.6 mm, 5 �m), �analysis = 450 nm and injection volume = 10 �L. (C) The UV–Vis spectra of the standard of quinoline yellow.

BMIm-NTf2 (v/v), � = 230 nm, column C18, T = 40 ◦C, flowrate = 0.80 mL min−1). The respective chromatograms presentedwell defined peaks for a: 4,4′-diaminodiphenylmethane; b:4-chloroaniline (tR = 7.42 min); c: 2-methoxy-5-methylaniline(tR = 6.31 min); d: 3,3′-dimethylbenzidine (tR = 5.09 min); e: 2,4-toluidine (tR = 7.6 min); f: 4,4′-oxydianiline (tR = 8.60 min); g:2-chloro-4-nitroaniline (tR = 10.3 min); h: aniline (tR = 14.4 min);i: 3,3′-dichlorobenzidine (tR = 13.2 min); j: benzidine(tR =13.9 min); k: 4-aminobiphenyl (tR = 12.5 min); l: o-dianisidine(tR = 16 min); m: o-anisidine (tR = 17 min); n: o-toluidine(tR = 18.6 min); o: 4,4′-methyleno-bis-(2-chloroaniline) (tR =21.9 min); p: 2-naphthylamine (tR = 20.0 min). Analytical curveswere obtained for each standard at concentrations ranging from1 × 10−6 mol L−1 to 1 × 10−5 mol L−1 with a linear relationship, andthe detection limit was approximately 2–10 ppb.

The chromatographic profile and HPLC/DAD analysis of theproducts resulting from the oxidation and reduction controlledpotential of QY were monitored every 30 min during the 4.0 h anal-ysis. As shown in Fig. 3B, the band pattern for the QY dye decreased75% in the reduction process, while this band decreased 90% afterthe oxidation process.

An HPLC/DAD analysis was performed with the products of theQY dye obtained at the end of the controlled potential electrolysisexperiment to identify the oxidation and reduction products thatformed.

After undergoing oxidation, the resulting 1.0 mg mL−1 quino-line yellow solution was analyzed by comparing the solution with

the compounds that were predicted to form after the electroly-sis. Based on this analysis, the following products were formed:4,4′-diaminodiphenylmethane (Peak 1, tR = 4.34 min), 2-methoxy-5-methylaniline (Peak 2, tR = 6.33 min), and 4,4-oxydianiline (Peak3, tR = 8.60 min) (Fig. 4). This identification was confirmed by com-paring the retention time obtained by HPLC with DAD detectorand UV–Vis spectral data with standard solutions. The results areshown in Table 2. None of the compounds were formed after reduc-tion. The chemical structures of the compounds identified as the QYoxidation products are shown in Table 2. The electrochemical oxi-dation illustrate that under a potential of +1.0 V electron transferstep in the majority cases leads the oxidation of –N=, in the chemicalstructure (Fig. 1) forming a cation radical as intermediates [29]. Thesubsequent reactions are justified due continuous charge transfersteps during all the 4 h of oxidation that can generate many radi-cals species are intermediate during the reaction. The results showthat three stable compounds are detected by HPLC-DAD, whichprobable formation is resumed in Schemes 1–3, respectively.

4. Discussion

In this study, quinoline yellow dye was evaluated for its poten-tial to interact with DNA structure, which could cause breakagesand permanent DNA damage that could lead to genomic instabil-ity [30,31]. Although micronucleated cells originated from loss ofchromosomes can be eliminated by apoptosis [32], DNA rearrange-ments and mutations that are acquired in micronuclei can be also

58 F.M.D. Chequer et al. / Mutation Research 777 (2015) 54–61

Fig. 4. (A) Chromatogram from HPLC/DAD of quinoline yellow (1.0 mg mL−1) before and after oxidation and reduction at controlled potential at +1.0 and −1.0 V vs Ag/AgCl,respectively. The black line corresponds to the standard (original QY dye); red line: after 4 h of oxidation; and blue line: 4 h after the reduction process. Chromatographicconditions: mobile phase MeOH/water + 30 mM of BMIm [NTf2] 70:30, flow rate 0.8 mL min−1; column temperature = 45◦ C; Phenomenex Luna C18 column (250 × 4, 6 mm,5 �m), �analysis = 230 nm and injection volume = 10 �L. (B) The UV–Vis spectra of the oxidation products of quinoline yellow.

incorporated into the genomes of developing cancer cells, and thesemicronuclei can persist for many generations [33].

The alkaline version of comet assay was used once this method-ology can detect DNA single-strand breaks, alkali-labile sites,DNA–DNA and DNA–protein cross-linking and single strand breaksassociated with incomplete excision repair sites [21]. CBMN-Cytwas chosen as an important complementary technique becausein addition to the DNA damage evaluation, this methodology candetect dicentric chromosomes and gene amplification [24], provid-ing a broad chrosomosome instability screening. In metabolicallycompetent HepG2 cells, QY was genotoxic by both the comet assayand CBMN-Cyt. FISH assays revealed that QY induces DNA damagethrough both aneugenic and clastogenic processes.

HepG2 cells are often used in toxicological investigations andgene expression studies because they express metabolic enzymesthat can oxidize or reduce xenobiotics, closely mimicking thein vivo activity of hepatocytes [34–37]. These cells have retainedthe inducibility and activities of several phase I and phase II xeno-biotic metabolizing enzymes, and have been shown to be suitablefor the detection of different classes of indirect-acting genotoxicagents [38]. In addition, this cell line is considered useful for avoid-ing false negative results in the detection of genotoxic carcinogens[35,39,40], such as synthetic dyes and other chemical compoundsthat can be oxidized or reduced to become either more or less toxic[18,41]. HepG2 cells also express wild-type tumor suppressor TP53,making them an appropriate model for studying P53-regulatedresponses to DNA damage at the level of gene transcription andtranslation [42,43].

Here, HepG2 cells were treated with concentrations of QYthat were determined based on its solubility and low ADI(0–0.5 mg kg−1) [44]. The concentrations tested were not capableof inhibiting the cell cycle (no significant differences in the NDIbetween the experimental and control groups were observed) andresulted in cell viability levels of greater than 80% based on thetrypan-blue exclusion method, ensuring the consistency of ourresults. In addition, the percentage of binucleated cells observedusing CBMN-Cyt was greater than 35% in all of the treatments.

In addition to QY, other cosmetic dyes have also been found tobe potentially genotoxic in mammalian cells. Mpountoukas et al.[12] evaluated the genotoxic, cytotoxic and cytostatic potential ofthe synthetic dyes amaranth, erythrosine and tartrazine in humanperipheral blood cells in vitro. These dyes are used in food and cos-metic products, and the results of this research indicated that thesecolorants were potentially toxic to human lymphocytes in vitro andcould possibly bind directly to DNA [12]. However, research in syn-thetic dyes are often controversial and some results of in vitro testsdo not show the same effects in vivo assays. For instance, accord-ing to Poul et al. [45], acute oral exposure to food dye additivesamaranth, tartrazine and sunset yellow as well as to the hepa-tocarcinogen azo dye dimethylaminoazobenzene (DAB) did notinduce genotoxic effect in the gut using micronucleus assay in mice.However, the DNA damage induced by amaranth and tartrazine,previously noted in the in vivo comet assay in mouse colon [46],was not corroborate in the gut micronucleus assay in mice [45].

Additionally, it is known that several biotransformation reac-tions may occur after the absorption of a xenobiotic, and the

Table 2Oxidation products obtained from QY, as determined by HPLC/DAD.

Compound HPLC/DAD

Structure CAS number tR (min)

Peak 1: 4,4′-diaminodiphenylmetane 101-77-9 4.34

Peak 2: 2-methoxy-5-methylaniline 120-71-8 6.33

Peak 3: 4,4′-oxydianiline 101-80-4 8.60

F.M.D. Chequer et al. / Mutation Research 777 (2015) 54–61 59

Scheme 1. Electrochemical oxidation of quinoline yellow and subsequent formation of 4,4′-oxydianiline.

oxidation and reduction processes play important roles in thisprocess because the products generated can be even more toxicthan the original compound [47–49]. After the biotransformationof dyes, it is possible to generate aromatic amines, and some ofthese products can be carcinogenic and can accumulate in the foodchain. For example, the biphenylamines, such as benzidine and 4-biphenylamine, are present in the environment and constitute athreat to human health and to the ecosystems in general [50,51].Considering that QY dye showed genotoxic potential in HepG2cells, it is important to study the possible products formed afterthe metabolism of QY. There is little available data concerning theproducts formed after the oxidation and reduction of dyes. Thus,for the adequate risk assessment of a chemical used as a dye, it isimportant to evaluate the toxicity of the compound itself and alsothe degradation products [3].

After the oxidation of QY, the following chemical compoundswere identified: 4,4′-diaminodiphenylmethane (tR = 4.34 min), 2-methoxy-5-methylaniline (tR = 6.33 min) and 4,4′-oxydianiline(tR = 8.60 min). Zanoni et al. [3] studied the dye Sudan III andthe products formed after the oxidation process. Two compoundsidentified coincided with the products found after electrolysisof the dye QY: 4,4′-diaminodiphenylmethane and 2-methoxy-5-methylaniline. These compounds are classified by the InternationalAgency for Research on Cancer (IARC) in category 2B or possiblycarcinogenic to humans [19]. Thus, the generation of toxic aromaticamines can be harmful to human beings, whereas the in vitro modelused in this study simulates a reaction that could occur in vivo afterthe ingestion of food or water containing these dyes [3].

In conclusion, our study demonstrated that low QY concentra-tions were genotoxic in HepG2 cells. Additionally, we found that

60 F.M.D. Chequer et al. / Mutation Research 777 (2015) 54–61

Scheme 2. Electrochemical oxidation of quinoline yellow and subsequent formation of 2-methoxy-5-methylaniline.

Scheme 3. Electrochemical oxidation of quinoline yellow and subsequent formation of 4,4′-diaminodiphenylmethane.

QY interferes with DNA stability by causing chromosome break-ages (clastogenic) and loss (aneugenic), which may represent animportant health risk to consumers of products that use this dye.This study demonstrated the genotoxic property of QY, and whencombined with the existing toxicological and risk-assessment dataof QY in literature, we conclude that this colorant can offer risk tohumans if it is metabolized or absorbed through the skin.

Conflict of interest

The authors declare that there are no conflict of interest.

Acknowledgements

This work was supported by FAPESP (2011/01755-0 and2011/14115-9). The authors also wish to thank Regislaine ValeriaBurin, Ph.D. for her assistance with technical procedures.

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