the cosmetic dye quinoline yellow causes dna dmage in vitro

7/26/2019 The Cosmetic Dye Quinoline Yellow Causes DNA Dmage in VITRO

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Mutation Research 777 (2015) 54–61

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

journal homepage: www.elsevier .com/ locate /gentox

Commun i ty address : www.elsevier .com/ locate /mutres

The cosmetic dye quinoline yellow causes DNA damage in vitro

Farah Maria Drumond Chequera,b,∗, Vinícius de Paula Venâncioa,Maíra Rocha de Souza Prado a, Luiz Raimundo Campos da Silva e Cunha Junior c,Thiago Mescoloto Lizier d, Maria Valnice Boldrin Zanoni d, Rommel Rodríguez Burbanoc,Maria Lourdes Pires Bianchia, Lusânia Maria Greggi Antunes a

a Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdadede Ciências Farmacêuticas de Ribeirão Preto,Universidadede SãoPaulo,

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 CitogenéticaHumana, 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 2014

Received in revised form 6 November 2014

Accepted 11 November 2014

Available online18 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 mutageni-

city 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 identified the

products 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 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 demon-

strated that low (from 0.5to 20g mL −1) QY concentrations were genotoxic in HepG2 cells on both assays

and those harmful compounds were detected after the oxidation process. Our findings 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 [1–4]. 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 world-

wide [4–6]. However, there is insufficient 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 [9–13].

∗ Corresponding author at: Departamento de Análises Clínicas, Toxicológicas e

Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universi-

dade deSão Paulo,USP,Ribeirão Preto, SP14040-903, Brazil.Tel.: +5516 36024186;

fax: +55 16 3602 4725.

E-mail address: [email protected] (F.M.D. Chequer).

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. Theaim of this investigation was to evaluate thegenotox-

icity of QY using the alkaline comet assay andthe cytokinesis-block

micronucleus cytome assay (CBMN-Cyt) in the metabolically com-

petent cell line HepG2, which closely mimics phase I metabolism.

Micronuclei (MN) were also analyzedusingthe fluorescence 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 forma-

tion 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 classified by IARC [19] could be pro-

duced during theoxidative and/or reductiveconditions.In addition,

http://dx.doi.org/10.1016/j.mrgentox.2014.11.003

1383-5718/© 2014 Elsevier B.V. All rightsreserved.



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 20.0g mL −1 of quinoline yellow (QY) for 4h 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, 0.3g mL −1

of doxorubicin. *: Significantly different from vehicle control group.

Table 1

Assessment of themutagenic effects of quinolineyellow (QY) on HepG2 cells using thecytokinesis-block micronucleus cytomeassay (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 57.1/42.9

Positive control 99 ± 14* 16 ± 5 10 ± 7 1.6 ± 0.1 60.5/39.5

0.5 QY 53 ± 1* 3 ± 4 3 ± 3 1.5 ± 0.1 n/a

1.0 QY 46 ± 8* 2 ± 3 3 ± 2 1.5 ± 0.1 n/a

2.0 QY 50 ± 15* 2 ± 3 4 ± 3 1.5 ± 0.1 n/a

5.0 QY 63 ± 13* 1 ± 1 6 ± 4 1.6 ± 0.1 n/a

10.0 QY 65 ± 14* 2 ± 3 6 ± 2 1.5 ± 0.1 n/a

15.0 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

Valuesshown arethemean±SD; BN, binucleatedcell; 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, 0.03g mL −1 doxorubicin. *: Significantly 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, 1.0mL min−1 flow rate, column temperature of

45 ◦C, Phenomenex Luna C18 column (250×4, 6 m m, 5m) and analysis = 450nm.

The analysis time was 10min, and all of theanalyseswere conductedin triplicate.

All of these methodologies were conducted based on chromatographic param-

e ter s s uch as r et ent ion t ime (t R ), retention constant factor (k), selectivity (˛),

resolution between peaks (Rs) and theoretical plate number (N ). Standard curves

anda quantitativeanalysisof thetarget amineswereobtainedusing a linearregres-

sion of thepeak area vs.concentration.Further comparisons were performedusing

the standardadditionmethodin whichaliquotsof theworkingstandard dissolvedin

methanol were spiked into thesamples.The procedurewas conductedin triplicatefor each sample.

2.8. Statistical analysis

Allof thedatashownare expressed asthe mean value±SD of three independent

experiments.The results were analyzed using one-way ANOVA with post hocDun-

nett’s tests (ata significancelevel p< 0.05)in GraphPadPrism 5 (GraphPadSoftware,

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 exclusionmethod,and concentrations greater than

20g 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 to

0.3g mL −1 DXR, which induced a statistically significant increase

in tail moment and tail intensity compared with the vehicle con-

trol group (i.e., cells treated with1.0% DMSO). Moreover, compared

with the vehicle control, QY was genotoxic to HepG2 cells at con-

centrations ranging from 2.0 to 20g 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 (0.5–20g mL −1). The frequencies of NPB

and NBUD and the NDI calculation (also shown in Table 1) were

not significantly different between the experimental and control

groups. TheFISH assay also showedthatalthough the MNifrequen-

cies were higher in the treated groups, there was no difference in

the C+ MN/C−MN ratio between the treated groups and the nega-

tive 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 0.03g mL −1 DXR increased the MN

frequency compared with the vehicle control group.

Fig. 3A shows representative chromatograms of the HPLC-

DAD data obtained for a 20L solution containing 50ppm

of the standard aromatic amine of interest (mobile phase:

methanol/water 70:30 (v/v) containing 30mM 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 20L of a standard solutionof aromatic amines. Mobile phase:methanol/water70:30 (v/v)+ 30mM of BMIm-NTf 2,

=230nm, columnC18, T = 40 ◦C,flowrate=0.80mL 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 mgmL −1) before (original dye,

black line) andafteroxidation at +1.5 V vs Ag/AgCl(blueline)or reductionat −1.5V vs Ag/AgCL (red line) for4.0 h. MobilephaseMeOH/Water 80:20,flow rate 1.0mLmin−1;

45 ◦C; Phenomenex Luna C18 column (250×4.6mm, 5m), analysis =450 nm and injection volume= 10L. (C) TheUV–Vis spectra of the standard of quinolineyellow.

BMIm-NTf 2 (v/v), =230nm, column C18, T = 40 ◦C, flow

rate=0.80mL min−1). The respective chromatograms presented

well defined peaks for a: 4,4-diaminodiphenylmethane; b:

4-chloroaniline (t R = 7.42 min); c: 2-methoxy-5-methylaniline

(t R =6.31min); d: 3,3-dimethylbenzidine (t R = 5.09 min); e: 2,4-

toluidine (t R = 7.6min); f : 4,4-oxydianiline (t R =8.60min); g :

2-chloro-4-nitroaniline (t R = 10.3 min); h: aniline (t R = 14.4 min);

i: 3,3-dichlorobenzidine (t R =13.2min); j: benzidine

(t R =13.9 min); k: 4-aminobiphenyl (t R = 12.5 min); l: o-dianisidine

(t R =16min); m: o-anisidine (t R =17min); n: o-toluidine

(t R =18.6min); o: 4,4-methyleno-bis-(2-chloroaniline) (t R =

21.9min); p: 2-naphthylamine (t R = 20.0min). Analytical curves

were obtained for each standard at concentrations ranging from1×10−6 mol L −1 to 1×10−5 mol L −1 with a linear relationship, and

the detection limit was approximately 2–10ppb.

The chromatographic profile and HPLC/DAD analysis of the

products resulting from the oxidation and reduction controlled

potential of QY were monitored every 30min duringthe 4.0h anal-

ysis. 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 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, t R = 4.34min), 2-methoxy-

5-methylaniline (Peak 2, t R = 6.33 min), and 4,4-oxydianiline (Peak

3, t R = 8.60min) (Fig. 4). This identification was confirmed by com-

paring the retention time obtained by HPLC with DAD detector

and UV–Vis spectral data with standard solutions. The results are

shown in Table 2. None of thecompounds were formedafterreduc-

tion.The chemical structures of thecompounds identifiedas theQY

oxidation products are shown in Table 2. The electrochemical oxi-

dation illustrate that under a potential of +1.0V electron transfer

step inthe majoritycasesleads the oxidationof –N=, inthe chemical

structure (Fig.1) forming a cation radical as intermediates [29]. Thesubsequent reactions are justified due continuous charge transfer

steps during all the 4 h of oxidation that can generate many radi-

cals 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 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 breakages

and permanent DNA damage that could lead to genomic instabil-

ity [30,31]. Although micronucleated cells originated from loss of

chromosomes can be eliminatedby apoptosis [32], DNA rearrange-

ments and mutations that are acquired in micronuclei can be also




Fig. 4. (A) Chromatogram from HPLC/DAD of quinolineyellow (1.0 mg mL −1) before and after oxidationand reduction at controlled potential at +1.0 and−1.0V 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 [NTf 2] 70:30, flow rate 0.8mL min−1; column temperature= 45◦ C; Phenomenex Luna C18 column (250×4, 6mm,

5m), analysis =230 nm and injectionvolume= 10L. (B)The UV–Vis spectra of theoxidation products of quinoline yellow.

incorporated intothe genomes of developing cancer cells, andthese

micronuclei 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 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 amplification [24], provid-

ing 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 thein vivo activity of hepatocytes [34–37]. These cells have retained

the inducibility and activities of several phase I and phase II xeno-

biotic 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 avoid-

ing 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 becomeeither more or less toxic

[18,41]. HepG2cells also express wild-typetumor suppressorTP53,

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(0–0.5 mg kg−1) [44]. The concentrations tested were not capable

of inhibiting the cell cycle (no significant 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 cos-

metic products,and theresults of this research indicatedthat these

colorants werepotentially toxicto human lymphocytes in vitro andcould possibly bind directly to DNA [12]. However, research in syn-

thetic dyes are often controversial andsome results of in vitro tests

do not show the same effects in vivo assays. For instance, accord-

ing to Poul et al. [45], acute oral exposure to food dye additives

amaranth, tartrazine and sunset yellow as well as to the hepa-

tocarcinogen azo dye dimethylaminoazobenzene (DAB) did not

induce genotoxiceffect in the gut using micronucleus assayin 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 2

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

Compound HPLC/DAD

Structure CAS number t R (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




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 toxic

than the original compound [47–49]. 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 4-

biphenylamine, 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 compoundswere identified: 4,4-diaminodiphenylmethane (t R = 4.34min), 2-

methoxy-5-methylaniline (t R = 6.33 min) and 4,4-oxydianiline

(t R =8.60min). Zanoni et al. [3] studied the dye Sudan III and

the products formed after the oxidation process. Two compounds

identified coincided with the products found after electrolysis

of the dye QY: 4,4-diaminodiphenylmethane and 2-methoxy-5-

methylaniline. These compounds are classifiedby the International

Agency for Research on Cancer (IARC) in category 2B or possibly

carcinogenicto humans [19]. Thus, thegenerationof toxic aromatic

aminescan beharmfulto humanbeings,whereas the invitromodel

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 concentra-

tions were genotoxic in HepG2 cells. Additionally, we found that




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 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.

Conflict of interest

The authors declare that there are no conflict of interest.

Acknowledgements

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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the cosmetic dye quinoline yellow causes dna dmage in vitro

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