Cytotoxic and genotoxic effects of two hair dyes used in the formulation of black color

Accepted Manuscript Cytotoxic and genotoxic effects of two hair dyes used in the formulation of black color Yaliana Tafurt Cardona, Paula Suares Rocha, Thaís Cristina, Casimiro Fernandes, Maria Aparecida Marin-Morales PII:

S0278-6915(15)30061-2

DOI:

10.1016/j.fct.2015.09.010

Reference:

FCT 8397

To appear in:

Food and Chemical Toxicology

Received Date: 9 July 2015 Revised Date:

19 August 2015

Accepted Date: 18 September 2015

Please cite this article as: Cardona, Y.T., Rocha, P.S., Cristina, T., Fernandes, C., Marin-Morales, M.A., Cytotoxic and genotoxic effects of two hair dyes used in the formulation of black color, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

OF BLACK COLOR

a

a

RI PT

CYTOTOXIC AND GENOTOXIC EFFECTS OF TWO HAIR DYES USED IN THE FORMULATION

a

Yaliana Tafurt Cardona , Paula Suares Rocha , Thaís Cristina Casimiro Fernandes , Maria a*

SC

Aparecida Marin-Morales

a

M AN U

UNESP- São Paulo State University “Júlio de Mesquita Filho” Institut of Biosciences, Department

of Biology, Rio Claro Campus, Av. 24-A, 1515, Bela Vista, Rio Claro, São Paulo, Brazil 13506-900 (*)

AC C

EP

TE D

[email protected] (corresponding author)

ACCEPTED MANUSCRIPT

CYTOTOXIC AND GENOTOXIC EFFECTS OF TWO HAIR DYES USED IN THE FORMULATION OF BLACK COLOR

RI PT

1. Introduction In the last decades, studies have shown that topical products can be absorbed, leading to systemic effects in the organisms exposed to them. Thereby, the possible toxic effects stemming from population exposure to cosmetic products have been reassessed (Nohynek et

SC

al., 2010). According to the International Agency for Research on Cancer (IARC), in vitro and in vivo studies (in exposed human populations) have shown that some hair dyes and many

(IARC,1993; IARC, 2010).

M AN U

chemicals used in the hair dyeing process can be considered mutagenic and carcinogenic

The permanent dyes represent approximately three quarters of the global dye use (Bolduc and Shapiro, 2001). At the end of the 1990s, approximately 33% of women over 18 years old and 10% of men over 40 years old in the United States used some kind of hair dye (La Vecchia and

TE D

Tavani, 1995; Ghosh and Sinha, 2008). Studies in the first decade of this century showed an increase in this percentage, with 42% of the women and 25% of the men making use of hair dyes (Rosenkranz et al., 2007). In Brazil, according to the National Institute of Metrology,

EP

Standardization and Industrial Quality (INMETRO), 26% of the adults use hair dye, most of which are women (INMETRO, 2014).

AC C

In North America and Europe, there are approximately two million professional hairdressers, barbers and beauticians who are routinely exposed to hair dyes (Gago-Dominguez et al., 2001; Czene et al., 2003). Epidemiological studies indicate that hairdressers occupationally exposed to hair dyes have a higher risk of developing bladder cancer, proportional to the exposure time (<10 years, risk increased by 0.5 times; 10 years or more, risk increased by 5.1 times), and of lymphoid malignancies (Holly et al., 1998; Zhang et al., 2004). Population studies also indicate 1

ACCEPTED MANUSCRIPT

that women using permanent hair dye at least once a month have a 2.1 times higher risk of bladder cancer in comparison with those who does not use it (Gago-Dominguez et al., 2001; Letašiová et al., 2012). Andrew et al. (2004) detected the presence of components of hair dye or

RI PT

their derivatives in the urine of users, indicating that the carcinogenic compounds can reach the target organ, the bladder. These results may be linked to the aromatic amines present in the hair dye (Yu et al., 2002). These compounds can be absorbed through the skin during the

SC

product application and induce this type of cancer which, according to in vitro and in vivo tests, are potentially mutagenic and carcinogenic (Ames et al., 1975; Bolt and Golka, 2007; Platzek,

M AN U

2010).

The black hair dye is composed of a mixture of several dyes, including the pure dyes Basic Red 51 (BR51) and Basic Brown 17 (BB17), which are temporary dyes of the azo group (R-N=N-R') (Figure 1), and the mixture Ebony, composed of other five hair dyes. Studies performed by the Scientific Committee on Consumer Safety – SCCSO (2011) indicated the mutagenic action of

TE D

BR51 and BB17 using the Ames test with Salmonella tiphymurim in the presence of metabolic activation (S9) (SCCS/1436/11; SCCS/1431/14).These azo dyes have been widely used by hair dye industries. During 2005, the total production of BR51 was 0.1 and 0.5 Tons, and of BR17, 1

EP

and 5 Tons (IARC, 2010).

Some hair dyes contain aromatic amines which may be toxic (Takkouche et al., 2005),

AC C

mutagenic (Ames et al., 1975) or carcinogenic (Sontag, 1981), representing a risk to human health (Sanjosé et al., 2006), due to their worldwide use. By the widespread use of hair dyes, the proven toxicity of these chemicals and the hazard associated with their continued use, it is essential to perform further investigations of these cosmetic products to reach new information that can assist the comprehension of possible damages that these dyes can promote on the health of exposed organisms. This warning has also been suggested by the Scientific Committee on Cosmetic and Non-Food Products Intended for Consumers (SCCNFP) that, 2

ACCEPTED MANUSCRIPT

based on the available scientific information, recommends the application of different strategies for the identification of possible carcinogenic risks to humans, associated to the presence of several compounds of different kinds of dyes, including the precursors, couplers,

RI PT

oxidants/intermediates and the products of combined reaction (SCNFP/0720/03). This way, the present study aimed at investigating the cytotoxic and genotoxic potential of the hair dyes Basic Red 51 (BR51) and Basic Brown 17 (BB17), used in the composition of the black hair dye, using

SC

permanent cells derived from human hepatoma (HepG2). Therefore, two acute cytotoxicity assays were performed, the Thiazolyl Blue Tetrazolium Bromide (MTT) assay and the Trypan

M AN U

blue test (TB), which evaluate the integrity of two different cellular organelles (mitochondria and cell membranes, respectively). Moreover, in order to investigate the genotoxic potential of these dyes, comet assay and micronucleus test were also performed. The single cell gel electrophoresis assay detects single-strand breaks (SSB), double–strand breaks (DSB), alkalilabile sites (ALS) and SSB associated with incomplete repair of excision sites (Speit and

TE D

Hartmann, 1995; Tice et al., 2000), while cytokinesis-block MN assay (CBMN) in the formation of micronucleus (MN), can occur for any lost or chromosomal breakage and dysfunction of the mitotic spindle, indicating important genomic instability events (Fenech, 2007; Kirsch-Volders et

in HepG2 cells.

EP

al., 2011). This is the first study investigating the cytotoxic and genotoxic potential of these dyes

AC C

2. Materials and methods

2.1. Test compounds – BR51 and BB17 The

compounds

used

in

this

study

were

the

hair

dyes

BR51

(2-[((4-

Dimethylamino)phenyl)azo])-1,3-dimethyl-1H-imidazolium chloride (CAS No. 77061-58-6) and BB17

(8-[(4-Amino-3-nitrophenyl)azo])azo]-7-hydroxy-N,N,N-trimethyl-2-naphthalenaminium

chloride (CAS No. 68391-32-2) (Figure 1), used in the formulation of black dye, according to the proportion of the commercial use, indicated by the manufacturer (Arianor Cherry Red 0.002% 3

ACCEPTED MANUSCRIPT

and Arianor Sienna Brown 0.04%). All substances were obtained in powder by the commercial

SC

RI PT

dye company Sensient Cosmetic Technologies Brasil, from São Paulo, Brazil.

Fig.1. Chemical structures of the dyes: (A) Basic Red 51 – BR51 (2-[((4-Dimethylamino)phenyl)azo])-1,3-

M AN U

dimethyl-1H-imidazolium chlorid (SCCS/1436/11); (B) Basic Brown 17 – BB17 (8-[(4-Amino-3nitrophenyl)azo]) azo]-7-hydroxy-N,N,N-trimethyl-2-naphthalenaminium (SCCS/1431/14).

2.2. Treatment solution

The powders of each dye were dissolved in sterilized bi-distilled water, in a concentration of 20mg/mL for BR51, and of 40mg/mL for BB17. Then, these solutions were again re-dissolved

TE D

in Minimal Essential Medium (MEM Gibco/Cultilab) and the following concentrations were tested for cell viability tests (MTT and Trypan Blue assays): BR51 2000 µg/mL, 200 µg/mL, 20 µg/mL, 2 µg/mL, 0.2 µg/mL and 0.02 µg/mL; BB17 62.5 µg/mL, 31.2 µg/mL, 15.6 µg/mL, 7.8 µg/mL, 3.9

EP

µg/mL and 1.95 µg/mL, being the highest concentrations, the same concentrations use in the commercial formula for the hair dye. Then, the viability tests indicate the higher concentration

AC C

inducing at least 80% of viability, to be use in the specific tests, where cytotoxic effects should be avoided.

2.3. Biological materials: human cell culture HepG2 cells, isolated from human hepatoma, were used as biological material for testing

the toxicity of the dyes. These cells are considered an important tool for the assessment of mutagens and pro-mutagens, since they can express different xenobiotic metabolizing enzymes (Westerink and Schoonen, 2007; Valentin-Severin et al., 2003). 4

ACCEPTED MANUSCRIPT

HepG2 cells were obtained from the American Type Culture Collection (ATCC No HB 8065, Rockville, MD) and were cultivated in 25 cm2 culture flasks with 5 mL of MEM (Gibco/Cultilab) supplemented with 10% fetal bovine serum (FBS) and 0.1% of antibiotic-antimycotic solution

RI PT

(penicillin 10,000 IU/mL / streptomycin 10 mg/mL, Cultilab). The flasks were maintained in a CO2 incubator (5%) until reaching approximately 80% confluence with a cell cycle of approximately 24 hours (Salvadori et al., 1993).

SC

2.4. Thiazolyl Blue Tetrazolium Bromide test (MTT)

The MTT test is a cytotoxicity assay that has been used to evaluate the survival,

M AN U

proliferation and activation of cells. This test is based on the ability of viable cells to convert tetrazolium salt (Thiazolyl Blue Tetrazolium Bromide, CAS No. 298-93-1, Sigma), an insoluble purple formazan salt in succinate dehydrogenase within the mitochondria. The formazan product is impermeable to the cell membranes of viable cells, accumulating inside (Fotakis and Timbrell, 2006).

TE D

The MTT test was performed in 96-well plates, according to the protocol established by Mosmann (1983), with some modifications. In each well, 2.34x104 cells were seeded, with a total volume of 100 µL of medium, supplemented with FBS, except for the wells related to the

EP

blank, which were composed by medium without FBS. The plates were incubated for 24 hours. After this period, the medium was removed and a new culture medium (without FBS) was added

AC C

to the treatments, with a final volume of 200 µL. Positive and negative controls were performed in order to validate the experiments, the positive control composed of Triton X-100 diluted in culture medium (without FBS) at a concentration of 1%, and the negative control (NC) performed with pure culture medium (without FBS) in separated wells. In the remaining wells, different concentrations of treatments were added. The plates were incubated for 4 hours at 37°C. After this period, medium was discarded and r eplaced with 150 µL of MTT diluted in PBS at a concentration of 1 × 10-6 mg/mL. 5

ACCEPTED MANUSCRIPT

After this period, the MTT solution was discarded and 100 µL of dimethyl sulfoxide (DMSO) were added to each well. The plates were read with a spectrophotometer microplate reader, using a filter of 540 nm (Multiskan apparatus FC – ThermoScientific). Statistical analyses were

RI PT

performed by the SPSS statistic software for Windows, version 15 (SPSS Inc., Chicago, IL, USA) with ANOVA, followed by Tukey’s comparison test (p<0.05). 2.5. Trypan blue test (TB)

SC

The Trypan blue test assesses damage to the membrane. Cells with disrupted plasma membrane stained with Trypan blue present a blue color, whereas cells with intact plasma

M AN U

membrane are not stained with Trypan blue (Zwolak, 2014). A total of 2.34 × 104 cells per well were seeded in 96-well plates, with a total volume of 100 µL of medium supplemented with FBS. The plates were then incubated for a period of 24 hours. After this, cells were exposed to different concentrations of treatments for 4 hours, as already described for the MTT test. For this evaluation, 0.4% (w/v) Trypan blue (Gibco) were mixed with the cell suspension at 1:1.

TE D

Subsequently, cells were scored for each treatment. The unstained cells (viable) were considered alive and the blue-stained cells (inviable) were considered dead or membranedamaged. Finally, the percentage of viable cells was calculated, using the following formula:

EP

viable cell count (white) / total number of cells (white and blue) × 100% (Zwolak, 2014). 2.6. Comet assay

AC C

The comet assay was performed to evaluate the genotoxic effects of physical and chemical agents, as well as DNA repair kinetics. The comet assay was carried out according to the protocol established by Singh et al. (1988) and Tice et al. (2000) with some modifications. Test were performed in triplicate. A total of 5×105 cells were seeded in each flask and incubated for 24 hours at 37 °C, in a CO 2 incubator (5%). After this period, cells were exposed for 4 hours to the treatments. The positive control (PC) was performed with Methyl Methanesulfonate (MMS, Sigma-Aldrich, CAS No. 66-27-3) MMS (4 x 10-4 M) diluted in MEM and the negative 6

ACCEPTED MANUSCRIPT

control (NC), was performed with MEM with 1% of sterile water. After this period, cells were trypsinized and collected for obtaining a suspension, and subsequently subjected to a cell viability assay with trypan blue (Gibco). For this evaluation, 5 mL of the cell suspension were

RI PT

mixed with 5 mL of trypan blue, with 100 cells scored for each treatment. After cell viability analysis, 20 µL of cell suspension were mixed with 120 µL of low melting point agarose 0.5% at 37 °C and placed on slides with standard 1% agarose and a coverslip. After solidification, the

SC

coverslip was removed and cells were exposed to a lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris and 8g of NaOH pH 10) in the dark, at 4 °C, for at least 1 hour. After lysis, the slides

M AN U

were transferred to electrophoresis and covered with alkaline buffer (pH<13) (300 mM NaOH and 1 mM EDTA solution), where the slides remained for 20 minutes, for denaturation of the DNA. After this period, they were subjected to electrophoresis at 39 V, 300 mA (~ 0.8 V/cm) for 20 min and then neutralized in Tris buffer (0.4 M Trizma HCl pH 7.5), fixed in absolute ethanol for 10 minutes and stored at 4 °C until the time of the analysis. The slide staining was

TE D

performed by adding 50 µL of GelRed® solution and they were analyzed immediately in an epifluorescence microscope Leica, with a magnification of 400 ×, and a filter B-34 (excitation: i = 420 nm-490 nm barrier: I = 520 nm). One hundred nucleoids were analyzed per slide, totaling

EP

600 per treatment.

Finally, the images were analyzed with the CASP program (Comet Assay Software Project), a

AC C

free software available on the Internet, which is used to quantify the DNA damage by variables such as: Olive Tail Moment (OTM = distance between the center of gravity of DNA in the tail and the center of gravity of DNA in the head in x-direction)/% percent tail DNA) and Comet Length (CL = Length of the entire comet from the left border of the head area to end of tail) (Końca et al., 2003, Olive and Durand, 2005; Garcia et al., 2007).

7

ACCEPTED MANUSCRIPT

Afterwards, the induction factor (IF) were calculated by the comparison of median values of OTM from exposed cells to median values of OTM from corresponding negative controls (Kosmehl et al., 2004).

RI PT

2.7. Cytokinesis-block MN assay (CBMN) The micronucleus assay (MN) allows rapid detection of clastogenic and aneugenic damage caused in the genetic material of organisms exposed to toxic substances, such as

SC

chromosome breakage and loss of whole chromosomes. These losses of genetic material are easily visualized in the daughter cells as a structure similar to the main core, however, in a

M AN U

reduced size (Bolt et al., 2011).

The MN assays was performed according to the protocol established by Natarajan and Darroudi (1991), with some modifications. A total of 5×105 cells were seeded in 25 cm2 culture flasks. The flasks were incubated for 24 hours at 37°C and maintained in a CO2 incubator (5%). After this period, cells were exposed to different concentrations of treatments for 4 hours, as already

TE D

described for the comet assay. After treatment, the culture medium was changed and 50 µL of cytochalasin B (final concentration of 3 ng/mL) were added for 28 hours, in order to obtain binucleated cells. After this period, cells were treated with hypotonic solution (sodium citrate

EP

1%) and fixed with formaldehyde (40%) and ethanol-acetic acid (3:1). The slides were stained with Giemsa 5% for 8 minutes. Two thousand binucleated cells were scored per replica,

AC C

totalizing six thousand cells per treatment. The numbers of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were determined.

2.8. Statistical analysis

The statistical analysis was performed using SPSS for Windows, version 15 (SPSS Inc., Chicago, IL, USA). Additionally, significance analysis of the test results was performed by means of an ANOVA-on-Ranks-variance parametric statistical test, followed by Dunn’s 8

ACCEPTED MANUSCRIPT

comparison test (p<0.05) for calculating statistical differences between groups, using Sigmaplot TM

. The significance analysis of MN test results was performed by ANOVA (one way), a

parametric statistical test, followed by Tukey’s comparison test (p<0.05).

RI PT

3. Results 3.1. Assessment of the cytotoxic potential of the hair dyes BR51 and BB17

According to the results, higher concentrations of the studied dyes (2000 µg/mL, 200 µg/mL and

SC

20 µg/mL of the BR 51 dye, 62.5 µg/mL and 31.2 µg/mL of the dye BB17) induced cytotoxic effects. The lowest concentrations of the tested dyes did not induce cytotoxic effects after 4-

M AN U

hours exposure (cell viability above 80%), and these concentrations were used as the highest concentrations in the comet assay and MN test, avoiding cell mortality and allowing the assessment of specific effects. Table 1 and Fig. 2 present the results obtained with TB and MTT assays.

Treatments Basic Red 51 NC

Viability MTT (%) Mean±SD 100 ± 0.067 12.4 ± 0.005*

2000 µg/mL

12.4 ± 0.009*

200µg/mL

12.2 ± 0.047*

20 µg/mL 2 µg/mL

Viability TB (%) Mean±SD 98 ± 2.00

Treatments Basic Brown 17 NC

Viability MTT(%) Mean±SD 100 ± 0.036

Viability TB (%) Mean±SD 97 ± 2.00

7 ± 3.00*

PC

21.6 ± 0.028*

7 ± 3.00*

40 ± 1.52*

62.5 µg/mL

72.2 ± 0.048*

73 ± 4.16*

65 ± 5.0*

31.2 µg/mL

75.1 ± 0.045*

81 ± 2.5*

29.7 ± 0.039*

70 ± 2.08*

15.6 µg/mL

83.8 ± 0.056

96 ± 2.00

97.6 ± 0.096

94 ± 2.5

7.8 µg/mL

81.5 ± 0.046

97 ± 1.52

AC C

EP

PC

TE D

Table 1: Cell viability based on the colorimetric MTT assay and Trypan blue test in HepG2 exposed to different concentrations of the dyes BR51 and BB17.

0.2 µg/mL

98.7 ± 0.096

94 ± 2.5

3.9 µg/mL

99.4 ± 0.081

97 ± 1.15

0.02µg/mL

98.2 ± 0.056

95 ± 3.5

1.9 µg/mL

83.8 ± 0.030

96 ± 2.08

SD = standardd deviation; NC=negative control; PC=positive control(Triton X-100 1%); Values followed by *are statistically significant in relation to the negative control (ANOVA/Tukey, p < 0.05).

9

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig.2. Cell viability (%) based on the colorimetric MTT assay and Trypan blue (TB) test in HepG2 (A) Exposed to different concentrations of the dye BR51. (B) Exposed to different concentrations of dye BB17. NC=negative control; PC=positive control

3.2.1. Assay comet

TE D

3.2. Assessment of the genotoxic potential of the hair dyes BR51 and BB17

From the results obtained with the MTT test (cell viability above 80%), concentrations of

EP

2 µg/mL, 0.2 µg/mL and 0.02 µg/mL for BR51 and 15.6 µg/mL, 7.8 µg/mL and 3.9 µg/mL for BB17 dye were selected for the comet assay. According to the results, BR51 and BB17

AC C

presented genotoxic potential for all dye concentrations (Figs.3 and 4).

10

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

M AN U

Fig.3. Genotoxic effects obtained with comet assay, using HepG2 cells. Each box plot presents the mean OTM (Olive Tail Moment) of three different concentrations of the (A) dye BR51 and (B) dye BB17. Additionally, induction factors (IF) are indicated above each box plot. NC=negative control; PC=positive control; * significant genotoxic effects according to Dunn’s test (p<0.05).

EP

Fig.4. Examples of HepG2 cells, in comet assay. (A) cell from Negative Control, without DNA damage. (B) Treated cell, with intermediate DNA damage. (C) Treated cell, with high DNA damage.

3.2.2. Cytokinesis-block MN assay (CBMN)

AC C

The same concentrations used for the comet assay were used in the MN test. Results are presented in Table 2.

According to the MN test results, all tested concentrations of the two dyes induced significant chromosomal damage in relation to the NC, indicating high potential genotoxicity to HepG2 cells. For the other analyzed parameters (nuclear buds, nucleoplasmic bridges), the results were not statistically significant when compared to the NC.

11

ACCEPTED MANUSCRIPT

Table 2: Cytokinesis-block micronucleus test performed with HepG2 cells exposed to different concentrations of dyes BR51 and BB17. MN Mean±SD 36.0 ± 5.29

NBUDs Mean±SD 8.3 ± 4.16

NPBs Mean±SD 1.67 ± 2.08

Treatments Basic Brown 17 NC

MN Mean±SD 36.0 ± 5.29

NBUDs Mean±SD 8.33 ± 4.16

NPBs Mean±SD 1.67 ± 2.08

PC

71.6 ± 7.02*

28.0 ± 15.7

11.6 ± 6.65*

PC

71.6 ± 7.24*

28.0 ± 15.7

11.6 ± 6.65*

2 µg/mL

95.6 ± 8.50*

11.3 ± 4.04

4.0 ± 1.00

15.6 µg/mL

81.3 ± 11.01*

7.0 ± 1.0

5.67 ± 4.72

0.2 µg/mL

102.0 ± 8.88*

7.3 ± 2.30

0.67 ± 0.57

7.8 µg/mL

76.3 ± 12.66*

6.0 ± 1.0

1.0 ± 1.0

0.02µg/mL

104.0 ± 7.00*

5.67 ± 1.15

2.0 ± 1.73

3.9 µg/mL

70.0 ± 5.29*

7.0 ± 4.35

2.0 ± 0.0

RI PT

Treatments Basic Red 51 NC

SC

SD = standard deviation; NC=negative control; PC=positive control; MN=micronucleus; NBUDs=nuclear buds; NPBs=nucleoplasmic bridges. Values followed by *are statistically significant in relation to the negative control (ANOVA/Tukey, p < 0.05).

M AN U

4. Discussion

In spite of the fact that hair dyes are released worldwide for human use, studies have shown that these compounds may induce toxic effects in both, people who have their hair dyed and those applying the dyes (Ahn and Lee, 2002).

A Scientific Committee on Consumer Safety (SCCS), taking over the management of potential

TE D

risks to human health, established a maximum use concentration for hair dyes BR51 (2000 mg/mL) and BB17 (1000 mg/mL), with are similar to that used in the composition of the black hair dye (SCCS/1436/11; SCCS/1431/14). However, the present results showed that the

EP

concentrations of 2 µg/mL for BR51 and 15.6 µg/mL for BB17 were already extremely toxic to HepG2 cells, inducing cell death or promoting a low activity of the mitochondrial enzyme

AC C

succinate dehydrogenase, indicating the alarming cytotoxic potential of these samples. The results obtained in this study, indicated a high cytotoxic potential of the dyes BR51 and BB17, for HepG2, even in concentrations much lower than those used in the hair dye commercial formula. This high cytotoxic effect could be probably related to drastic changes in mitochondrial function, including reduction of its mass and transmembrane potential, which leads to an increase in the levels of hydrogen peroxide (H2O2) in the cells (Di Pietro et al., 2011; Villarini et al. 2014). 12

ACCEPTED MANUSCRIPT

According to studies conducted by Zanoni et al. (2014) the dye BR51 is a stressor for epithelial cells, capable of inducing an increase in apoptosis and a delay in the cell cycle. This effect may be a consequence of activation of various genes involved in cell cycle regulation, directly

RI PT

connected to the induction of cell death. Thus, the damaged cell can activate intrinsic apoptosis pathways through intracellular stimuli, such as DNA damage (Hengartner, 2000).

Regarding specific effects, the obtained results presented a high genotoxic potential of the

SC

samples, recorded with both, comet and MN assays, in HepG2. These two assays are routinely used to identify genotoxic compounds (Hartmann et al., 2001). The genotoxic effects observed

M AN U

in this study for BR51 and BB17 corroborate results obtained by other authors, investigating dyes with similar characteristics (Przybojewska et al., 1989; Wollin and Gorlitz, 2004). The commercial dye CI Disperse Blue 291, which is also an azo dye, also induced genotoxicity and mutagenicity in HepG2 cells (Tsuboy et al., 2007).

From the obtained results, it is possible to infer that the genotoxicity of the tested dyes can be

TE D

attributed, at least in part, to the presence of the azo structure, which is recognized as mutagenic and carcinogenic (Ben Mansour et al., 2007). According to Wollin and Gorlitz. (2004) the azo dyes are genotoxic substances of direct action, which can induce chromosomal

EP

aberrations, MN and abnormalities in germ cells. Therefore, the genotoxic effect of the dyes BR51 and BB17 recorded in HepG2 cells may have been influenced by one or more chemical

AC C

components present in the formulation. According to Kilinc and Kilinc (2005) azo derived dyes also generated NO2 and –OH radicals, which are able to damage DNA and other cellular structures. The nitroaromatic compounds may be reduced by nitroreductases to aryl nitroso, N-arylhydroxilamines or intermediate radicals (Kilinc and Kilinc, 2005), inducing modifications to DNA as a result of an electrophilic attack of activated nitrogen (Haack et al., 2001). Umbuzeiro et al. (2005) stated that the NO2 radical of the dye can be metabolized by xanthine oxidase, producing hydroxylamines. Naiman et al. 13

ACCEPTED MANUSCRIPT

(2012) affirmed that the hydroxylamines can be more toxic than the original compounds and are recognized for their ability to form DNA adducts. The radicals of these compounds, such as N(CH2CH3)2, can be metabolized by cytochrome P450 enzymes (CYP450) to form genotoxic

RI PT

intermediates (Guaratini and Zanoni, 2000; Tsuboy et al., 2007) that interact with the DNA molecule, inducing primary damage (as demonstrated by the comet assay) and damage in the chromosomal level (as demonstrated by the MN test) (Tafazoli et al., 1998; Møller and Wallin,

SC

2000). In mammalian enzyme systems, the CYP450 in liver can also reduce azo dyes, and such reductions can generate aromatic amines, which can be carcinogenic (DeVito, S.C, 1993).

M AN U

Small amounts of dyes present in hair dyes can be absorbed percutaneously during normal use of hair dyes, enter the blood circulation and, through this route, reach the bladder (Platzek, 2010). This way, the N-hydroxylamines associated to hair dyes, can circulate freely or form adducts with hemoglobin and can be transformed in glucuronide conjugates, being excreted through the kidney. On the bladder lumen, these glucuronide conjugates will be hydrolyzed, due

TE D

to the acidic characteristic of this environment, and with or without further bioactivation by Nacetyltransferase 1 (NAT1), to form a highly electrophilic N-acetoxy derivative, that can induce DNA adducts. If DNA damage is misrepair, these adducts may lead to mutations in proto-

EP

oncogenes and/or tumor suppressor genes, and this is a critical step in the process of changing a normal cell to a malignant phenotype (Yu et al., 2002), which may lead to bladder cancer.

AC C

Exposure to some azo dyes may also lead to the development of splenic sarcomas, hepatocellular carcinoma, cellular abnormalities and chromosomal aberrations (Caritá and Marin-Morales, 2008). According to Møller and Wallin. (2000) liver and bladder may be the target organs for the metabolism of products derived from azo structure. Thus, the carcinogenic effects of azo dyes could be derived from the direct action of dyes on the cells, mainly due to the formation of metabolites resulting from the reduction of the azo bond, which are capable of

14

ACCEPTED MANUSCRIPT

interacting with the DNA molecule (Umbuzeiro et al., 2005; Ventura-Camargo and MarinMorales, 2013). The results obtained in this study demonstrated that the dyes BR51 and BBR17, commonly use

RI PT

in the composition of hair dyes, are extremely toxic for human cells exposed to concentrations used in the commercial formula, as well as presented high genotoxic potential to the same cells. These findings indicated an alarming hazard related to the exposure of human beings to hair

SC

dyes. 5. Conclusions

M AN U

The hair dyes BR51 and BBR17, used in the formulation of the black color, were capable of inducing high cytotoxic and genotoxic effects in human cells, in in vitro experiments. These dyes were toxic even in concentration much lower than those used commercially, which point out to the hazard related to the exposure of humans to these compounds, during hair coloration processes.

TE D

Acknowledgements

The authors would like to thank AUIP (Asociación Universitaria Iberoamericana de Postgrado), the support program for PhD students from abroad (PAEDEX/PROPG),

EP

Fundunesp/Replan (covenant n. 1100.0067969.11.4) and the Instituto de Biociências of the Universidade Estadual Paulista (UNESP), campus of Rio Claro, Brazil, for their financial

AC C

support. References

Ahn, H.J., Lee, W.S., 2002. An ultrastuctural study of hair fiber damage and restoration following treatment with permanent hair dye. Int. J. Dermatol. 41, 88–92. Ames, B.N., Kammen, H.O., Yamasaki, E., 1975. Hair dyes are mutagenic: identification of a variety of mutagenic ingredients. Proc. Natl. Acad. Sci. U.S.A. 72, 2423–2427. Andrew, A.S., Schned, A.R., Heaney, J.A., Karagas, M.R., 2004. Bladder cancer risk and personal hair dye use. Int. J. Cancer 109, 581–586. Ben Mansour, H., Corroler, D., Barillier, D., Ghedira, K., Chekir, L., Mosrati, R., 2007. Evaluation of genotoxicity and pro-oxidant effect of the azo dyes: Acids yellow 17, violet 7 and orange 52, and of their degradation products by Pseudomonas putida mt-2. Food and Chemical Toxicology 45, 1670–1677. Benning, V., Brault, D., Duvinage, C., Thybaud, V., Melcion, C., 1994. Validation of the in vivo CD1

15

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

mouse splenocyte micronucleus test. Mutagenesis 9, 199–204. Bolduc, C., Shapiro, J., 2001. Hair care products: waving, straightening, conditioning, and coloring. Clin. Dermatol. 19, 431–436. Bolt, H.M., Golka, K., 2007. The debate on carcinogenicity of permanent hair dyes: new insights. Crit. Rev. Toxicol. 37, 521–536. Bolt, H.M., Stewart, J.D., Hengstler, J.G., 2011. A comprehensive review about micronuclei: mechanisms of formation and practical aspects in genotoxicity testing. Arch Toxicol 85, 861–862. Caritá, R., Marin-Morales, M.A., 2008. Induction of chromosome aberrations in the Allium cepa test system caused by the exposure of seeds to industrial effluents contaminated with azo dyes. Chemosphere 72, 722–725. Czene, K., Tiikkaja, S., Hemminki, K., 2003. Cancer risks in hairdressers: assessment of carcinogenicity of hair dyes and gels. Int. J. Cancer 105, 108–112. DeVito, S.C, B. M.A., 1993. Predicting azo dye toxicity., in: Critical Reviews in Environmental Science and Technology. In: Logan, T.J. (Ed.), Pearl River, 249–324. Di Pietro, A., Visalli, G., Baluce, B., Micale, R.T., La Maestra, S., Spataro, P., De Flora, S., 2011. Multigenerational mitochondrial alterations in pneumocytes exposed to oil fly ash metals. International Journal of Hygiene and Environmental Health 214, 138–144. Fenech, M., 2007. Cytokinesis-block micronucleus cytome assay. Nat. Protocols 2, 1084–1104. Fotakis, G., Timbrell, J.A., 2006. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters 160, 171–177. Gago-Dominguez, M., Castelao, J.E., Yuan, J.-M., Yu, M.C., Ross, R.K., 2001. Use of permanent hair dyes and bladder-cancer risk. Int. J. Cancer 91, 575–579. Garcia, O., Romero, I., González, J.E., Mandina, T., 2007. Measurements of DNA damage on silver stained comets using free Internet software. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 627, 186–190. Ghosh, P., Sinha, A.K., 2008. Hair Colors: Classification, Chemistry and a Review of Chromatographic and Electrophoretic Methods for Analysis. Analytical Letters 41, 2291-2321. Guaratini, C.C.I., Zanoni, M.V.B., 2000. Textile dyes. Química Nova 23, 71–78. Haack, T., Erdinger, L., Boche, G., 2001. Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 491, 183–193. Hartmann, A., Elhajouji, A., Kiskinis, E., Poetter, F., Martus, H.-J., Fjällman, A., Frieauff, W., Suter, W., 2001. Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test. Food and Chemical Toxicology 39, 843–858. Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407, 770–776. Holly, E.A., Lele, C., Bracci, P.M., 1998. Hair-color products and risk for non-Hodgkin’s lymphoma: a population-based study in the San Francisco bay area. Am J Public Health 88, 1767–1773. IARC, 2010. IARC Monographs Working Group on the Evaluation of Carcinogenic Risks to Humans,Some aromatic amines, organic dyes, and related exposures. IARC Monogr Eval Carcinog Risks Hum 99, 1–658. IARC,1993. IARC Working group on the evaluation of carcinogenic risks to humans: occupational exposures of hairdressers and barbers and personal use of hair colourants; some hair dyes, cosmetic colourants, industrial dyestuffs and aromatic amines. Proceedings. IARC Monogr Eval Carcinog Risks Hum 57, 7–398. INMETRO, 2014. Instituto Nacional de Metrologia, Normalização e Qualidade Industrial. Informações ao Consumidor: Tinturas para Cabelo. (accessed November 2014). Kilinc, K., Kilinc, A., 2005. Mutagenic Actions of Nitrogen Oxides. Indoor and Built Environment 14, 503– 512. Kirsch-Volders, M., Plas, G., Elhajouji, A., Lukamowicz, M., Gonzalez, L., Loock, K.V., Decordier, I., 2011. The in vitro MN assay in 2011: origin and fate, biological significance, protocols, high throughput methodologies and toxicological relevance. Arch Toxicol 85, 873–899. Końca, K., Lankoff, A., Banasik, A., Lisowska, H., Kuszewski, T., Góźdź, S., Koza, Z., Wojcik, A.,

16

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

2003. A cross-platform public domain PC image-analysis program for the comet assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 534, 15–20. Kosmehl, T., Krebs, F., Manz, W., Erdinger, L., Braunbeck, T., Hollert, H., 2004. Comparative genotoxicity testing of rhine river sediment extracts using the comet assay with permanent fish cell lines (rtg-2 and rtl-w1) and the ames test*. J Soils & Sediments 4, 84–94. La Vecchia, C., Tavani, A., 1995. Epidemiological evidence on hair dyes and the risk of cancer in humans. Eur. J. Cancer Prev. 4, 31–43. Letašiová, S., Medveďová, A., Šovčíková, A., Dušinská, M., Volkovová, K., Mosoiu, C., Bartonová, A., 2012. Bladder cancer, a review of the environmental risk factors. Environ Health 11, S11. Møller, P., Wallin, H., 2000. Genotoxic hazards of azo pigments and other colorants related to 1phenylazo-2-hydroxynaphthalene. Mutat. Res 462, 13–30. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Naiman, K., Dračínský, M., Hodek, P., Martínková, M., Schmeiser, H.H., Frei, E., Stiborová, M., 2012. Formation, Persistence, and Identification of DNA Adducts Formed by the Carcinogenic Environmental Pollutant o-Anisidine in Rats. Toxicol. Sci. 127, 348–359. Natarajan, A.T., Darroudi, F., 1991. Use of human hepatoma cells for in vitro metabolic activation of chemical mutagens/carcinogens. Mutagenesis 6, 399–403. Nohynek, G.J., Antignac, E., Re, T., Toutain, H., 2010. Safety assessment of personal care products/cosmetics and their ingredients. Toxicology and Applied Pharmacology, Safety Assessment Safety Assessment 243, 239–259. Nohynek, G.J., Fautz, R., Benech-Kieffer, F., Toutain, H., 2004. Toxicity and human health risk of hair dyes. Food and Chemical Toxicology 42, 517–543. Olive, P.L., Durand, R.E., 2005. Heterogeneity in DNA damage using the comet assay. Cytometry 66A, 1–8. Platzek, T., 2010. Risk from exposure to arylamines from consumer products and hair dyes. Front Biosci (Elite Ed) 2, 1169–1183. Przybojewska, B., Barański, B., Spiechowicz, E., Szymczak, W., 1989. Mutagenic and genotoxic activity of chosen dyes and surface active compounds used in the textile industry. Pol J Occup Med 2, 171–185. Rosenkranz, H.S., Cunningham, S.L., Mermelstein, R., Cunningham, A.R., 2007. The challenge of testing chemicals for potential carcinogenicity using multiple short-term assays: An analysis of a proposed test battery for hair dyes. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 633, 55–66. Salvadori, D.M.F., Ribeiro, L.R., Natarajan, A.T., 1993. The anticlastogenicity of β-carotene evaluated on human hepatoma cells. Mutation Research Letters 303, 151–156. Sanjosé, S. de, Benavente, Y., Nieters, A., Foretova, L., Maynadié, M., Cocco, P.L., Staines, A., Vornanen, M., Boffetta, P., Becker, N., Alvaro, T., Brennan, P., 2006. Association between Personal Use of Hair Dyes and Lymphoid Neoplasms in Europe. Am. J. Epidemiol. 164, 47–55. SCCNFP/0720/03,2003. The Scientific Committee on Cosmetic Products and non-food products, intended for consumers. Updated recommended strategy for testing hair dyes for their potential genotoxicity/mutagenicity/carcinogenicity. SCCS/1436/11, 2011. Opinion of the Scientific Committee on Consumer Safety (SCCS) opinion on Basic Red 51.Colipa n° B116. SCCS/1431/14.2014. Opinion of the Scientific Committee on Consumer Safety (SCCS). Opinion on Basic Brown 17.Colipa n° B007. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–191. Sontag, J.M., 1981. Carcinogenicity of substituted-benzenediamines (phenylenediamines) in rats and mice. J. Natl. Cancer Inst. 66, 591–602. Speit, G., Hartmann, A., 1995. The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay). Mutagenesis 10, 555–559. Tafazoli, M., Baeten, A., Geerlings, P., Kirsch-Volders, M., 1998. In vitro mutagenicity and genotoxicity study of a number of short-chain chlorinated hydrocarbons using the micronudeus test and the alkaline single cell gel electrophoresis technique (Comet assay) in human

17

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

lymphocytes: a structure–activity relationship (QSAR) analysis of the genotoxic and cytotoxic potential. Mutagenesis 13, 115–126. Takkouche, B., Etminan, M., Montes-Martínez, A., 2005. Personal Use of Hair Dyes and Risk of Cancer. JAMA: The Journal of the American Medical Association 293, 2516 –2525. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J. ‐C, Sasaki, Y.F., 2000. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis 35, 206–221. Tsuboy, M.S., Angeli, J.P.F., Mantovani, M.S., Knasmüller, S., Umbuzeiro, G.A., Ribeiro, L.R., 2007. Genotoxic, mutagenic and cytotoxic effects of the commercial dye CI Disperse Blue 291 in the human hepatic cell line HepG2. Toxicol In Vitro 21, 1650–1655. Valentin-Severin, I., Le Hegarat, L., Lhuguenot, J.-C., Le Bon, A.-M., Chagnon, M.-C., 2003. Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 536, 79–90. Umbuzeiro, G., Freeman, H., Warren, S.H., Kummrow, F., Claxton, L.D., 2005. Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using different protocols of the Salmonella assay. Food and Chemical Toxicology 43, 49–56. Ventura-Camargo, B. de C., Marin-Morales, M.A., 2013. Azo Dyes: Characterization and Toxicity– A Review. Textiles and Light Industrial Science and Technology, Textiles and Light Industrial Science and Technology 2, 85-103. Villarini, M., Pagiotti, R., Dominici, L., Fatigoni, C., Vannini, S., Levorato, S., Moretti, M., 2014. Investigation of the Cytotoxic, Genotoxic, and Apoptosis-Inducing Effects of Estragole Isolated from Fennel (Foeniculum vulgare). J. Nat. Prod. 77, 773–778. Westerink, W.M.A., Schoonen, W.G.E.J., 2007. Phase II enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicology in Vitro 21, 1592–1602. Wollin, K.-M., Gorlitz, B.-D., 2004. Comparison of Genotoxicity of Textile Dyestuffs in Salmonella Mutagenicity Assay, In Vitro Micronucleus Assay, and Single Cell Gel/Comet Assay. Journal of Environmental Pathology, Toxicology and Oncology 23, 12. Yu, M.C., Skipper, P.L., Tannenbaum, S.R., Chan, K.K., Ross, R.K., 2002. Arylamine exposures and bladder cancer risk. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 506–507, 21–28. Zanoni, T.B., Tiago, M., Faião-Flores, F., de Moraes Barros, S.B., Bast, A., Hageman, G., de Oliveira, D.P., Maria-Engler, S.S., 2014. Basic Red 51, a permitted semi-permanent hair dye, is cytotoxic to human skin cells: Studies in monolayer and 3D skin model using human keratinocytes (HaCaT). Toxicology Letters 227, 139–149. Zhang, Y., Holford, T.R., Leaderer, B., Boyle, P., Zahm, S.H., Flynn, S., Tallini, G., Owens, P.H., Zheng, T., 2004. Hair-coloring Product Use and Risk of Non-Hodgkin’s Lymphoma: A Populationbased Case-Control Study in Connecticut. Am. J. Epidemiol. 159, 148–154. Zwolak, I., 2014. Comparison of three different cell viability assays for evaluation of vanadyl sulphate cytotoxicity in a Chinese hamster ovary K1 cell line. Toxicol Ind Health. 1-13.

18

ACCEPTED MANUSCRIPT  Highlights       1. 2.

AC C

EP

TE D

M AN U

SC

RI PT

3.  

 Hair  dyes  may  be  mutagenic  and/or  carcinogenic  to  humans   The  azo-­‐dyes  basic  red  51  and  basic  brown  17  induced  significant  cyto/genotoxicity  in  human   cells   Dyes  were  toxic  in  concentration  much  lower  than  those  used  commercially