Mutation Research 777 (2015) 5461

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Mutation Research/Genetic Toxicology and

Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres

The cosmetic dye quinoline yellow causes DNA damage in vitro

Farah Maria Drumond Chequer a,b, , Vincius de Paula Venncio a ,
Mara Rocha de Souza Prado a , Luiz Raimundo Campos da Silva e Cunha Junior c ,
Thiago Mescoloto Lizier d , Maria Valnice Boldrin Zanoni d , Rommel Rodrguez Burbano c ,
Maria Lourdes Pires Bianchi a , Lusnia Maria Greggi Antunes a
a
Departamento de Anlises Clnicas, Toxicolgicas e Bromatolgicas, Faculdade de Cincias Farmacuticas de Ribeiro Preto, Universidade de So Paulo,
USP, Ribeiro Preto, SP 14040-903, Brazil
b
Departamento de Anlises Clnicas e Toxicolgicas, Faculdade Federal de Minas Gerais, UFMG, Belo Horizonte, MG 31270-901, Brazil
c
Laboratrio de Citogentica Humana, Instituto de Cincias Biolgicas, Universidade Federal do Par, Belm, PA, Brazil
d
Instituto de Qumica. Departamento de Qumica Analtica, 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 2014
Received in revised form 6 November 2014
Accepted 11 November 2014
Available online 18 November 2014
Keywords:
Genotoxicity
Cosmetic dye
Micronucleus
Comet assay
Oxidation

a b s t r a c t
Quinoline yellow (QY) is a chinophthalon derivative used in cosmetic compositions for application to
the skin, lips, and/or body surface. However, regulatory data about the genotoxicity and/or mutagenicity of this compound are still controversial. Therefore, this work evaluated the genotoxicity of QY using
the comet assay and the cytokinesis-block micronucleus cytome assay (CBMN-Cyt) in the metabolically
competent cell line HepG2, which closely mimics phase I metabolism. This research also identied the
products formed after electrochemical oxidation of the QY dye, which simulates hepatic biotransformation. The primary products generated after the oxidation process were analyzed by High Performance
Liquid Chromatography coupled with a Diode Array Detector (HPLC/DAD), which detected the production
of 4,4 -diaminodiphenylmethane, 2-methoxy-5-methylaniline and 4,4 -oxydianiline. The results demonstrated that low (from 0.5 to 20 g mL1 ) QY concentrations were genotoxic in HepG2 cells on both assays
and those harmful compounds were detected after the oxidation process. Our ndings suggest that this
colorant 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 [14]. It is estimated that over
10,000 different dyes and pigments are used industrially and that
over 7 105 tons of synthetic dyes are produced annually worldwide [46]. However, there is insufcient information about their
potential health risks for humans and the environment [7,8]. The
available toxicological data about cosmetics dyes have shown
effects that range from contact allergies to different types of
genetic damages, including genotoxicity, mutagenicity and early
age leukemia [913].

Corresponding author at: Departamento de Anlises Clnicas, Toxicolgicas e

Bromatolgicas, Faculdade de Cincias Farmacuticas de Ribeiro Preto, Universidade de So Paulo, USP, Ribeiro 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).
http://dx.doi.org/10.1016/j.mrgentox.2014.11.003
1383-5718/ 2014 Elsevier B.V. All rights reserved.

The dye quinoline yellow (QY) is a chinophthalon derivative

used 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 human
maximization test, 15 of 20 subjects became sensitized [15,16]. The
regulatory data regarding QY genotoxicity and/or mutagenicity are
still controversial [14,17]. Therefore, we studied the dye QY in this
research. The aim of this investigation was to evaluate the genotoxicity of QY using the alkaline comet assay and the cytokinesis-block
micronucleus cytome assay (CBMN-Cyt) in the metabolically competent cell line HepG2, which closely mimics phase I metabolism.
Micronuclei (MN) were also analyzed using the uorescence in situ
hybridization (FISH) technique for further hazard characterization.
In addition, it is known that aromatic amines can be produced
during oxidative and/or reductive processes [3,18], and the formation of these aromatic amine byproducts could be important for
understanding the chemical transformation of dyes. Therefore, the
present work also aimed to determine if 16 aromatic amines used
as standard models of amines classied by IARC [19] could be produced during the oxidative and/or reductive conditions. In addition,

F.M.D. Chequer et al. / Mutation Research 777 (2015) 5461

this present study also investigated the oxidation and reduction

byproducts of QY dye by using electrochemistry to mimic these
reactions.
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: 14930-962) and trypan blue (CAS: 72-57-1) were obtained from SigmaAldrich (St Louis,
MO, USA). Dimethylsulfoxide (DMSO; CAS: 67-68-5) was purchased from Merck
(Darmstadt, Germany). Doxorubicin (DXR; CAS: 23214-92-8) was obtained from
Laboratrio Brgamo (Taboo da Serra, Brazil). GelRedTM (CAS: 7732-18-5) was
purchased from Biotium (Hayward, CA, USA). Dulbeccos Modied Eagle Medium
(DMEM), fetal bovine serum (FBS) and a penicillinstreptomycin solution were purchased from Gibco (Carlsbad, CA, USA). Normal- and low-melting point agaroses
(CAS: 9012-36-6) were obtained from Invitrogen (Carlsbad, CA, USA), and all other
chemicals 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 American Type Culture Collection (HB-8065, ATCC, Rockville, MD, USA) and cultured
in DMEM containing 10% heat-inactivated FBS and 1% antibiotic solution (penicillin/streptomycin). The cells were maintained in an incubator (Forma Series II,
Thermo Electron Corporation, USA) at 37 C in a humidied atmosphere of 5% CO2
and 95% air. The QY and doxorubicin concentrations used in this study were selected
using the trypan blue dye exclusion method when the cells were harvested. The
highest 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 performed according to the methods described by Singh et al. [20] and Tice et al. [21],
with minor modications. Briey, 2 105 HepG2 cells were seeded in a 24-well
plate 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 mL1 (nal concentrations) of QY for 4 h. Positive control (doxorubicin 0.3 g mL1 ) and vehicle control (1.0% DMSO) samples were also included.
The cells were mixed with 37 C low-melting-point agarose and placed on normalmelting-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) overnight
at 4 C. The lysed cells were then incubated in an electrophoresis solution (300 mM
NaOH and 1 mM EDTA) for 40 min at 4 C before being transferred to a horizontal
electrophoresis unit containing the same solution. The electrophoresis conditions
were 0.78 V/cm and 300 mA for 20 min at 4 C. Finally, the slides were washed in a
neutralization buffer (0.4 M Tris) for 20 min at 4 C and xed in ethanol for 5 min.
The slides were stained with Gel RedTM (1:10,000) immediately before analysis and
scored using a uorescence microscope (Axiostar, Zeiss, Germany) equipped with
a 515560 nm excitation lter, a 590-nm barrier lter and an integrated digital
camera. For each treatment, the tail moment (i.e., the product of the proportion
of the tails intensity and the displacement of the tails center of mass relative to
the 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 magnication using the Comet
Assay IV software (Perceptive Instruments, Suffolk, UK). A total of three independent
experiments 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], following the method of Natarajan and Darroudi [23] with modications. A total of
5 105 HepG2 cells were incubated in 25-cm2 culture asks for 24 h before being
treated with 0.5, 1.0, 2.0, 5.0, 10.0, 15.0 or 20.0 g mL1 QY (nal concentration),
0.03 g mL1 doxorubicin or vehicle control (1.0% DMSO). A total of 44 h after the
beginning of the initial incubation (i.e., after 20 h of treatment), the cells were
washed with PBS, the culture medium was changed, and the cells were treated with

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

55

3.0 g mL1 cytochalasin B. The cells were then incubated for 28 h, harvested, treated
with cold 1% (v/v) sodium citrate and xed with formaldehyde and methanolacetic
acid (3:1). Immediately before the analysis, the slides were stained using 40 g mL1
of acridine orange, and the frequencies of binucleated cells with micronuclei (MNi)
were determined at 1000 magnication. The frequency of nucleoplasmic bridges
(NPBs, biomarkers of dicentric chromosomes resulting from telomere end fusions or
DNA mis-repair) and nuclear buds (NBUDs, biomarkers for gene amplication and
altered gene dosage events) were also evaluated according to the methods described
by 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 to
determine the percentage of cells with one, two, three and four nuclei, and the NDI
was calculated as follows [25]: NDI = (M1 + 2M2 + 3M3 + 4M4)/N, where M1M4 represent the numbers of cells with 14 nuclei, respectively, and N represents the total
number of cells scored.
The MNi, NPB and NBUD frequencies were evaluated in a total of 1000 binucleated cells. The NDI calculation was measured in 500 cells per treatment. Three
independent experiments were performed.
2.5. Fluorescence in situ hybridization (FISH)
The QY concentrations with the highest MN frequencies were selected for FISH
analysis. Positive (0.03 g mL1 DXR) and negative (DMSO 1.0%) controls were also
used. The FISH analysis of MN was performed using a biotin-labeled human pancentromeric probe (1695-B Pan Centromeric Probe Biotin Cambio Ltd., UK) according
to the method described by Guimaraes et al. [26]. This probe was rst tested on
metaphase chromosomes to assess centromere-specic labeling. FISH assays were
performed on cells that were xed with methanol/acetic acid on freshly prepared
slides, according to the protocol described by Kapka et al. [27], with minor modications. The MNi were counterstained with DAPI/antifade. The MNi present in
the binucleated human HepG2 cells with intact cytoplasms were examined for the
presence of one or more centromeric spots and were classied as either centromere
positive (C + MN) or centromere negative (C MN). A total of 1000 binucleated cells
were scored for each treatment.
2.6. Controlled potential electrolysis and cyclic voltammetry
All of the electrochemical measurements were carried out using a Potentiostat
EG&G model 283 (PAR). The measurements were performed in a conventional 25.0mL electrochemical cell into which the following three electrodes were inserted:
a reference electrode of Ag/AgCl (KCl 3.0 mol L1 ), a platinum wire as the auxiliary
electrode and a glassy carbon working electrode (area of 3.14 mm2 for the cyclic
voltammetric measurements and 4.00 cm2 for the electrolysis experiments).
The voltammetric measurements were obtained by transferring 25 mL of the
stock solution of the original QY dye (1.0 mg mL1 in DMSO/0.01 mol L1 tetrabutylammonium tetrauoroborate solution (TBABF4 ) into the cell. The solution was
purged with nitrogen for 15 min, and the voltammetric curves were recorded. This
step was performed to determine the oxidation and reduction potentials of the dye
and thus be able to apply the same potentials in the controlled potential electrolysis
experiment.
For the controlled potential electrolysis experiments, the dye degradation products were submitted to increasingly oxidative conditions to reach a constant value
of current; the data were recorded to generate current vs time curves. The solutions of the QY dye were prepared at 1.0 mg mL1 in 0.01 mol L1 DMSO/TBABF4 .
Oxidation and reduction were conducted using +1.5 and 1.5 V, respectively, and
the reactions were monitored every 30 min during the 4.0 h analysis. To monitor
the change in coloration and the degradation of the dye, the generated products
were monitored by UV/Vis spectrometry and HPLC/DAD. All of the chromatographic
measurements were carried out after a pre-ltration of the sample using a MILLEX
Millipore (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 generated 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 Shimadzu
CLC-ODS (C18) guard column (1 cm 4.6 mm 5 m, 100 A). HPLC-DAD was used to
separate and identify standard solutions containing 4,4 -diaminodiphenylmethane
(Fluka, 97%); 4-chloroaniline (Fluka, 99%); 2-methoxy-5-methylaniline (Aldrich,
99%); 3,3 -dimethylbenzidine (SigmaAldrich, 97%); 2,4-diaminotoluidine (Fluka,
98%); 2-chloro-4-nitroaniline (Fluka, 98%); 4,4 -oxydianiline (Aldrich, 98%); aniline (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 a
MeOH/water + 30 mM of BMIm[NTf2 ] 70:30 mobile phase, 0.8 mL min1 ow 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-DAD
was used to identify the primary products generated after electrochemical degradation of QY.

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F.M.D. Chequer et al. / Mutation Research 777 (2015) 5461

Fig. 2. The effects of 0.5, 1.0, 2.0, 5.0, 10.0, 15.0 or 20.0 g mL1 of quinoline yellow (QY) for 4 h on the tail intensity (A) and tail moment (B) of HepG2 cells evaluated by
the comet assay. The values shown represent the mean SD, and the data are based on three independent experiments. Vehicle control, 1.0% dimethylsulfoxide; positive
control, 0.3 g mL1 of doxorubicin. *: Signicantly different from vehicle control group.
Table 1
Assessment of the mutagenic effects of quinoline yellow (QY) on HepG2 cells using the cytokinesis-block micronucleus cytome assay (CBMN-Cyt).
Treatment
(g mL1 )

CBMN-Cyt

NDI

FISH
C + MN(%)/C MN(%)

Total no. in 1000 BN cells

MNi
Vehicle control
Positive control
0.5 QY
1.0 QY
2.0 QY
5.0 QY
10.0 QY
15.0 QY
20.0 QY

19
99
53
46
50
63
65
69
72

NPBs
3
14*
1*
8*
15*
13*
14*
7*
4*

2
16
3
2
2
1
2
2
3

NBUDs
2
5
4
3
3
1
3
2
2

5
10
3
3
4
6
6
7
4

3
7
3
2
3
4
2
2
1

1.6
1.6
1.5
1.5
1.5
1.6
1.5
1.5
1.4

0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

57.1/42.9
60.5/39.5
n/a
n/a
n/a
n/a
n/a
57.9/42.1
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, centromere
positive (i.e., MN containing one or more whole chromosome signals); C MN, centromere negative (i.e., MN containing acentric chromosome fragment signals). The data
shown are based on three independent experiments. Vehicle control, 1.0% dimethylsulfoxide; positive control, 0.03 g mL1 doxorubicin. *: Signicantly different from the
control group (p < 0.05).
The conditions used for monitoring the degradation of the dye consisted of a
MeOH/water 80:20 mobile phase, 1.0 mL min1 ow rate, column temperature of

45 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 parameters such as retention time (tR ), retention constant factor (k), selectivity (),
resolution between peaks (Rs) and theoretical plate number (N). Standard curves
and a quantitative analysis of the target amines were obtained using a linear regression of the peak area vs. concentration. Further comparisons were performed using
the standard addition method in which aliquots of the working standard dissolved in
methanol were spiked into the samples. The procedure was conducted in triplicate
for each sample.
2.8. Statistical analysis
All of the data shown are expressed as the mean value SD of three independent
experiments. The results were analyzed using one-way ANOVA with post hoc Dunnetts tests (at a signicance level p < 0.05) in GraphPad Prism 5 (GraphPad Software,
USA).

3. Results
All of the QY concentrations used in this study resulted in at
least 80% cell viability prior to cell harvesting, as determined by
the trypan-blue exclusion method, and concentrations greater than
20 g mL1 were excluded due to the low cell viability.

The results of the comet assay are shown in Fig. 2. The sensitivity of this in vitro assay was demonstrated by the response to
0.3 g mL1 DXR, which induced a statistically signicant increase
in tail moment and tail intensity compared with the vehicle control group (i.e., cells treated with 1.0% DMSO). Moreover, compared
with the vehicle control, QY was genotoxic to HepG2 cells at concentrations ranging from 2.0 to 20 g mL1 .
In the CBMN-Cyt assay, the increased MNi frequencies (Table 1)
indicated that this dye promoted genotoxic effects at each of the
concentrations tested (0.520 g mL1 ). The frequencies of NPB
and NBUD and the NDI calculation (also shown in Table 1) were
not signicantly different between the experimental and control
groups. The FISH assay also showed that although the MNi frequencies were higher in the treated groups, there was no difference in
the C + MN/C MN ratio between the treated groups and the negative control group (p < 0.05). No difference between aneugenic and
clastogenic effects was observed across the treatments. DXR was
used as a positive control, and 0.03 g mL1 DXR increased the MN
frequency compared with the vehicle control group.
Fig. 3A shows representative chromatograms of the HPLCDAD data obtained for a 20 L solution containing 50 ppm
of 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) 5461

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, ow rate = 0.80 mL min1 . 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 mL1 ) 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, ow rate 1.0 mL min1 ;
45 C; Phenomenex Luna C18 column (250 4.6 mm, 5 m), analysis = 450 nm and injection volume = 10 L. (C) The UVVis spectra of the standard of quinoline yellow.

BMIm-NTf2 (v/v),  = 230 nm, column C18, T = 40 C, ow

rate = 0.80 mL min1 ). The respective chromatograms presented
well dened 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: 2,4toluidine (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 curves
were obtained for each standard at concentrations ranging from
1 106 mol L1 to 1 105 mol L1 with a linear relationship, and
the detection limit was approximately 210 ppb.
The chromatographic prole and HPLC/DAD analysis of the
products resulting from the oxidation and reduction controlled
potential of QY were monitored every 30 min during the 4.0 h analysis. As shown in Fig. 3B, the band pattern for the QY dye decreased
75% in the reduction process, while this band decreased 90% after
the oxidation process.
An HPLC/DAD analysis was performed with the products of the
QY dye obtained at the end of the controlled potential electrolysis
experiment to identify the oxidation and reduction products that
formed.
After undergoing oxidation, the resulting 1.0 mg mL1 quinoline yellow solution was analyzed by comparing the solution with

the compounds that were predicted to form after the electrolysis. Based on this analysis, the following products were formed:
4,4 -diaminodiphenylmethane (Peak 1, tR = 4.34 min), 2-methoxy5-methylaniline (Peak 2, tR = 6.33 min), and 4,4-oxydianiline (Peak
3, tR = 8.60 min) (Fig. 4). This identication was conrmed by comparing the retention time obtained by HPLC with DAD detector
and UVVis spectral data with standard solutions. The results are
shown in Table 2. None of the compounds were formed after reduction. The chemical structures of the compounds identied as the QY
oxidation products are shown in Table 2. The electrochemical oxidation illustrate that under a potential of +1.0 V electron transfer
step in the majority cases leads the oxidation of N=, in the chemical
structure (Fig. 1) forming a cation radical as intermediates [29]. The
subsequent reactions are justied due continuous charge transfer
steps during all the 4 h of oxidation that can generate many radicals species are intermediate during the reaction. The results show
that three stable compounds are detected by HPLC-DAD, which
probable formation is resumed in Schemes 13, respectively.
4. Discussion
In this study, quinoline yellow dye was evaluated for its potential to interact with DNA structure, which could cause breakages
and permanent DNA damage that could lead to genomic instability [30,31]. Although micronucleated cells originated from loss of
chromosomes can be eliminated by apoptosis [32], DNA rearrangements and mutations that are acquired in micronuclei can be also

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F.M.D. Chequer et al. / Mutation Research 777 (2015) 5461

Fig. 4. (A) Chromatogram from HPLC/DAD of quinoline yellow (1.0 mg mL1 ) 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. Chromatographic
conditions: mobile phase MeOH/water + 30 mM of BMIm [NTf2 ] 70:30, ow rate 0.8 mL min1 ; column temperature = 45 C; Phenomenex Luna C18 column (250 4, 6 mm,
5 m), analysis = 230 nm and injection volume = 10 L. (B) The UVVis spectra of the oxidation products of quinoline yellow.

incorporated into the genomes of developing cancer cells, and these

micronuclei can persist for many generations [33].
The alkaline version of comet assay was used once this methodology can detect DNA single-strand breaks, alkali-labile sites,
DNADNA and DNAprotein cross-linking and single strand breaks
associated with incomplete excision repair sites [21]. CBMN-Cyt
was chosen as an important complementary technique because
in addition to the DNA damage evaluation, this methodology can
detect dicentric chromosomes and gene amplication [24], providing a broad chrosomosome instability screening. In metabolically
competent HepG2 cells, QY was genotoxic by both the comet assay
and CBMN-Cyt. FISH assays revealed that QY induces DNA damage
through both aneugenic and clastogenic processes.
HepG2 cells are often used in toxicological investigations and
gene expression studies because they express metabolic enzymes
that can oxidize or reduce xenobiotics, closely mimicking the
in vivo activity of hepatocytes [3437]. These cells have retained
the inducibility and activities of several phase I and phase II xenobiotic metabolizing enzymes, and have been shown to be suitable
for the detection of different classes of indirect-acting genotoxic
agents [38]. In addition, this cell line is considered useful for avoiding false negative results in the detection of genotoxic carcinogens
[35,39,40], such as synthetic dyes and other chemical compounds
that 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-regulated
responses to DNA damage at the level of gene transcription and
translation [42,43].

Here, HepG2 cells were treated with concentrations of QY

that were determined based on its solubility and low ADI
(00.5 mg kg1 ) [44]. The concentrations tested were not capable
of inhibiting the cell cycle (no signicant differences in the NDI
between the experimental and control groups were observed) and
resulted in cell viability levels of greater than 80% based on the
trypan-blue exclusion method, ensuring the consistency of our
results. In addition, the percentage of binucleated cells observed
using CBMN-Cyt was greater than 35% in all of the treatments.
In addition to QY, other cosmetic dyes have also been found to
be potentially genotoxic in mammalian cells. Mpountoukas et al.
[12] evaluated the genotoxic, cytotoxic and cytostatic potential of
the synthetic dyes amaranth, erythrosine and tartrazine in human
peripheral blood cells in vitro. These dyes are used in food and cosmetic products, and the results of this research indicated that these
colorants were potentially toxic to human lymphocytes in vitro and
could possibly bind directly to DNA [12]. However, research in synthetic dyes are often controversial and some results of in vitro tests
do not show the same effects in vivo assays. For instance, according to Poul et al. [45], acute oral exposure to food dye additives
amaranth, tartrazine and sunset yellow as well as to the hepatocarcinogen azo dye dimethylaminoazobenzene (DAB) did not
induce 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 reactions may occur after the absorption of a xenobiotic, and the

Table 2
Oxidation 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) 5461

59

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

oxidation and reduction processes play important roles in this

process because the products generated can be even more toxic
than the original compound [4749]. After the biotransformation
of dyes, it is possible to generate aromatic amines, and some of
these products can be carcinogenic and can accumulate in the food
chain. For example, the biphenylamines, such as benzidine and 4biphenylamine, are present in the environment and constitute a
threat to human health and to the ecosystems in general [50,51].
Considering that QY dye showed genotoxic potential in HepG2
cells, it is important to study the possible products formed after
the metabolism of QY. There is little available data concerning the
products formed after the oxidation and reduction of dyes. Thus,
for the adequate risk assessment of a chemical used as a dye, it is
important to evaluate the toxicity of the compound itself and also
the degradation products [3].

After the oxidation of QY, the following chemical compounds

were identied: 4,4 -diaminodiphenylmethane (tR = 4.34 min), 2methoxy-5-methylaniline (tR = 6.33 min) and 4,4 -oxydianiline
(tR = 8.60 min). Zanoni et al. [3] studied the dye Sudan III and
the products formed after the oxidation process. Two compounds
identied coincided with the products found after electrolysis
of the dye QY: 4,4 -diaminodiphenylmethane and 2-methoxy-5methylaniline. These compounds are classied by the International
Agency for Research on Cancer (IARC) in category 2B or possibly
carcinogenic to humans [19]. Thus, the generation of toxic aromatic
amines can be harmful to human beings, whereas the in vitro model
used in this study simulates a reaction that could occur in vivo after
the ingestion of food or water containing these dyes [3].
In conclusion, our study demonstrated that low QY concentrations were genotoxic in HepG2 cells. Additionally, we found that

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F.M.D. Chequer et al. / Mutation Research 777 (2015) 5461

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 breakages (clastogenic) and loss (aneugenic), which may represent an
important health risk to consumers of products that use this dye.
This study demonstrated the genotoxic property of QY, and when
combined with the existing toxicological and risk-assessment data
of QY in literature, we conclude that this colorant can offer risk to
humans if it is metabolized or absorbed through the skin.
Conict of interest

[4]
[5]

[6]

[7]
[8]

The authors declare that there are no conict of interest.

[9]

Acknowledgements
[10]

This work was supported by FAPESP (2011/01755-0 and

2011/14115-9). The authors also wish to thank Regislaine Valeria
Burin, Ph.D. for her assistance with technical procedures.
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