ANTICANCER RESEARCH 35: 1465-1474 (2015)

Effects on DNA Repair in Human Lymphocytes Exposed to the Food Dye Tartrazine Yellow

BRUNO MOREIRA SOARES1,2, TAÍSSA MAÍRA THOMAZ ARAÚJO1,2, JORGE AMANDO BATISTA RAMOS1, LAINE CELESTINO PINTO1,2, BRUNA MEIRELES KHAYAT2, MARCELO DE OLIVEIRA BAHIA1, RAQUEL CARVALHO MONTENEGRO1,2, ROMMEL MARIO RODRÍGUEZ BURBANO1,2 and ANDRÉ SALIM KHAYAT1,2

1Human Cytogenetics Laboratory and 2Oncology Research Federal University of Pará, Belém, Brazil

Abstract. Tartrazine is a food additive that belongs to a Tartrazine is a water-soluble azo dye with extremely low class of artificial dyes and contains an azo group. Studies metabolism and oral absorption. Studies published in the about its genotoxic, cytotoxic and mutagenic effects are literature show that less than 2% of the ingested tartrazine is controversial and, in some cases, unsatisfactory. This work actually absorbed. Most of the tartrazine is readily- evaluated the potential in vitro , genotoxicity and metabolized in the colon by the intestinal flora, where it is effects on DNA repair of human lymphocytes exposed to the reduced to sulfanilic acid and aminopyrazolone. Although dye. We assessed the cytotoxicity of tartrazine by 3-(4,5- mostly excreted in feces, these metabolites may be absorbed Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and excreted by urine as sulfanilic acid (4-6). test and the response of DNA repair through comet Tartrazine dye was evaluated by the Expert Committee on (alkaline version). We used different concentrations of the dye, Food Additives (JECFA) and it was established that ranging from 0.25-64.0 mM. The results demonstrated that the acceptable daily intake (ADI) for such substance is tartrazine has no cytotoxic effects. However, this dye had a 7.5 mg/kg/day. Based on toxicological, mutagenic, significant genotoxic effect at all concentrations tested. carcinogenic and teratogenic effects of tartrazine, Elhkim et Although most of the damage was amenable to repair, some al. (6) confirmed the initial hazard assessment conducted by damage remained higher than positive control after 24 h of the JECFA (6, 7). repair. These data demonstrate that tartrazine may be harmful A review performed by Elhkim et al. stated that to health and its prolonged use could trigger carcinogenesis. tartrazine has no mutagenic or clastogenic potential, neither in vitro nor in vivo (6). Among the tools used to establish The food industry uses a wide range of additives that may this were the Ames test, mutagenicity evaluation in yeast, be harmful to health, including dyes that belong to the azo unscheduled DNA synthesis assay, sister chromatid class, which are extensively used in foods, drugs and exchange (SCE) assay, chromosomal aberrations test and cosmetics (1). The mutagenic, carcinogenic and toxic effects micronucleus assay (7-23). of azo dyes can be a result of direct action of the compound Similarly, Poul et al. did not find mutagenic or cytotoxic or arise from the formation of aryl amine derivatives effects of tartrazine (20, 200 or 1,000 mg/kg) when the dye generated during the reductive biotransformation of the azo was administered twice, at 24-h intervals, by oral gavage to bond. In mammals, water-soluble azo dyes are mainly mice (24). In their study, the in vivo gut micronucleus test metabolized by anaerobic intestinal microflora. Azo was used for mutagenic effects and the cytotoxicity reductase in the liver can also catalyze reductive cleavage of evaluation was performed by quantification of apoptotic and azo bonds, specifically of the water-insoluble dyes (2, 3). mitotic cells (24). On the other hand, several studies showed that tartrazine does have a mutagenic potential. Ishidate et al. observed that this dye can induce chromosomal aberrations in Chinese Correspondence to: André Salim Khayat, Professor, Federal hamster cells when exposed to a concentration of 2.5 mg/ml University of Pará, Belém 66075-110, Pará, Brazil. Tel.: +55 (25, 26). Patterson and Butler showed that tartrazine induces 09132018425, Fax: +55 09132017601, e-mail: [email protected] in vitro chromosomal aberrations in Muntiacus muntjac Key Words: Food dye, tartrazine, cytotoxicity, genotoxicity, DNA fibroblasts exposed to concentrations ranging from repair. 5-20 mg/ml, when compared to the control (27). According

0250-7005/2015 $2.00+.40 1465 ANTICANCER RESEARCH 35: 1465-1474 (2015) to Giri et al. this dye can induce chromosomal aberrations Samples and Experimental Design. In the current study, human and SCE in bone marrow cells of mouse and rat when lymphocytes were used as an experimental model. Peripheral blood they are submitted to chronic exposure to high doses of (10 ml) from four healthy individuals (approximately 18-35 years of age, two females and two males) was collected in heparinized the dye (28). syringes. The donors gave written informed consent to participate Ishidate et al., in their study with Chinese hamster fibroblast in the study and history for all of them was in accordance with line, also found a slight increase of polyploid cells after standard guidelines for genotoxicity tests (not being a smoker, with treatment with tartrazine (2.5 mg/ml) for 48 h (26). no recent infection, no chronic use of medication in the previous Mpountoukas et al. evaluated the genotoxic, cytotoxic and four months and no radiation exposure in the previous six months). cytostatic potential of tartrazine yellow dye (0.02 to 8 mM) in For the cell viability assay (MTT), lymphocytes were isolated from human peripheral blood cells over an exposure time of 72 h. whole blood using Histopaque (Sigma-Aldrich). To assess genotoxicity by comet assay, we performed temporary They observed that the two higher concentrations of tartrazine culture of lymphocytes from decanted plasma. For this assay, we (4 and 8 mM) had a significant toxic effect on the quality of used cells without treatment as negative control (NC) and cells chromosomes, so the differentiation stain between two treated with doxorubicin at different concentrations (0.001 mM and chromatids was not clear, maybe because tartrazine was toxic 0.003 mM) as positive control (PC), in order to compare the at the condensation of chromosomes in mitosis. Furthermore, genotoxicity level. tartrazine at 4 and 8 mM led to a statistically significant Comet assay was performed with two purposes: to assess the decrease of mitotic index and a significant delay in cell genotoxic effect of tartrazine after 3 hours of exposure, and to assess DNA repair potential 24 hours after removal of tartrazine. It is division compared to the control (29). important to notice that separate lymphocyte cultures were prepared Some studies regarding the mutagenic effect of tartrazine for each experiment. metabolites are also available in the literature. Chung et al. Tartrazine was solubilized at different concentrations using sterile evaluated the mutagenicity of sulfanilic acid and distilled water. For the MTT assay, we used 0.25, 0.5, 1.0, 2.0, 4.0, aminopiralozone metabolites using the Ames test and did not 8.0, 16.0 and 32.0 mM tartrazine and for the comet assay we find significant results (11). However, through this same included the concentration of 64.0 mM. All concentrations used assay, Henschler and Wild analyzed rat urine after feeding were based on data available in the literature (26, 27, 29). them with tartrazine. The excreta was sterilized by filtration, concentrated by lyophilization and subsequently diluted in Lymphocyte . For the cytotoxicity assay, peripheral blood sterile water. The results showed a dose-dependent lymphocyte isolation was carried out according to Fenech (35). For the genotoxicity assay, blood was stored for 1 hour at 37˚C for mutagenic response in Salmonella typhimurium strains TA separation and decanting of plasma from whole blood. After visual 98 (+S9) (17). Instead, also using the Ames test, but separation of blood fractions, the leukocyte layer was resuspended analyzing rat feces extract, Munzner and Wever showed no with gentle stirring of the syringe. Then, 400 μl of resuspended positive results (20). plasma was added to 100 μl of whole blood in 15-ml falcon tubes Other research highlighted the mutagenicity of (TPP, Zollstrasse, Trasadingen, Switzerland) containing 4.5 ml of photoexcited tartrazine by using the Ames test (30) and RPMI-1640 medium (#52400-025; Gibco, Carlsbad, CA, USA), Somatic Mutation And Recombination Test (SMART) (31). supplemented with 20% fetal bovine serum (FBS; #10106-169; Gibco), 4% phytohemagglutinin (#10576-05; Gibco), 1% penicillin- Tartrazine has been widely used by the Brazilian streptomycin (10000 units of penicillin and 10000 μg of pharmaceutical industry, being the most common food streptomycin per ml, #15140-122; Gibco). The cells were seeded at additive (30%) amongst azo dyes (32). It is noteworthy that 37˚C for 20 hours before treatment in an incubator containing 5% in some cases, the food industry has used this compound at CO2, as were the cells isolated by histopaque, which were seeded concentrations above the permitted level (33, 34). in 5 ml of the same medium. Since many studies regarding the damaging potential of this dye have reported contradictory and, sometimes, MTT assay. Lymphocytes were grown in 96-well culture plates at a 5 unsatisfactory results, it is necessary to carry-out further density of 0.64×10 cells per well and incubated for 24 h. After the initial period of incubation, cells were treated with different research to investigate this dye more precisely. Thus, the concentrations of tartrazine (0.25-32.0 mM) for 24 h. At the end of present study assessed the in vitro cytotoxic and genotoxic this period, 100 ml of MTT (5,000 mg/ml) was added to the cells for activity of tartrazine and the DNA repair potential of cells 3 hours. Then, the MTT was removed and 100 ml of dimethyl that had undergone treatment with this compound. sulfoxide (DMSO) (Sigma-Aldrich) was added for 1 h in order to dissolve the formazan obtained during the process. Afterward, the Materials and Methods absorbance was then measured by spectrophotometry (λ=562 nm). Cell survival was calculated as the percentage absorbance compared Chemicals. Tartrazine (CAS 1934-21-0, purity ≥85%) and with that of the control. All experiments were carried-out in duplicate. doxorubicin (CAS 25316-40-9, purity 98-102%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol- Comet assay (alkaline version). Cultures of lymphocytes from four 2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from individuals were treated with different concentrations of tartrazine Invitrogen (Carlsbad, CA, USA). (0.25-64.0 mM) for 3 h. Doxorubicin (0.001 and 0.003 mM) was

1466 Soares et al: Tartrazine Yellow Effects on Human Lymphocytes

Figure 1. Percentage of cell viability of human lymphocytes exposed to different concentrations of tartrazine. The result was calculated from spectrophotometric measurement of optical density at 570 nm, from which an average of each duplicate was obtained and then the percentage of cell viability calculated considering the negative control as having a viability of 100%. Statistical test: ANOVA, Tukey.

used as positive control. After treatment, a 450 μl suspension of 1 methodology used to evaluate the DI was used to quantify the level ml of cells were transferred to 1.5 ml culture tubes submerged in of damage by intra- and inter-treatment. ice (to inactivate repair enzymes). Cells were harvested by centrifugation for five minutes at 106 g and homogenized in 300 ml Statistical analysis. The statistical analysis was performed by of a low-melting-point agarose (0.8%), spread onto microscope ANOVA, followed by Tukey test, through GRAPHPAD PRISM slides pre-coated with a normal melting point agarose (1.5%) and v.5.0 (GraphPad Software, San Diego, CA USA). p-Values of less covered with a coverslip (24×60 mm). After 8 min at 4˚C, the than 0.05 were considered significant. coverslip was removed and the slides were immersed in cold lysis solution (2.5 M NaCl; 100 mM EDTA; 10 mM Tris, 10% DMSO Results and 1% Triton-X, pH 10) overnight. After lysis, the slides were placed in an electrophoresis chamber and covered with freshly made electrophoresis buffer (300 mM NaOH; 1 mM EDTA, pH>13). Cytotoxicity evaluation. The addition of tartrazine yellow dye Electrophoresis was performed for 20 min (34 V and 300 mA). to lymphocyte cultures isolated by Ficoll and stimulated by Afterwards, the slides were neutralized by submersion in distilled phytohemagglutinin did not significantly (p>0.05) affect cell water (4˚C) for 5 min and fixed in 100% ethanol for 3 minutes. viability at any of the tested concentrations (0.25-32.0 mM). Staining of the slides was performed immediately before the Thus, the MTT assay showed that tartrazine had no cytotoxic analyses using ethidium bromide (20 mg/ml). Slides were prepared in duplicate and 100 cells were screened per sample (50 cells from potential under the tested conditions (Figure 1). each slide) using a fluorescence microscope (Olympus BX41, Tokyo, Japan) at ×40 magnification. Genotoxicity evaluation. After 3 h of exposure, tartrazine The damage index (DI) was visually determined based on the size induced DNA damage in human lymphocytes at all and intensity of the comet’s tail. The following five categories (0-4) concentrations (0.25-64.0 mM). The damage observed in all were used: level 0, no damage; level 1, little damage with a tail treatments, including the PC, were statistically significant length shorter than the diameter of the nucleus; level 2, medium (p<0.0001) compared to the NC. There was no significant damage with a tail length one or two times the diameter of the nucleus; level 3, significant damage with a tail length one or two difference between the two concentrations of PC (0.001 and times the diameter of the nucleus; and level 4, significant damage 0.003 mM) in the damage induced, nor between them and with a tail length greater than three times the diameter of the nucleus. the treatments with the dye, except at the concentration of We also quantified the level of DNA damage by using a 64.0 mM, which induced a higher incidence of DNA damage comparative method to verify which damage level was most (Figure 2). prevalent intra- and inter-treatment. In the intra-treatment comparative analyses (Figure 3), we observed that tartrazine yellow dye induced damage more In vitro comet assay for DNA repair. To assess the DNA repair potential, after treatment the cells were washed twice with 5 ml of frequently at higher levels. Moreover, we also observed a phosphate buffer solution (PBS) 1X (#70011-044; Invitrogen) and dose-dependent increase in incidence of damage at level 4. seeded for 24 h in RPMI-1640 medium supplemented with 20% In the PC at 0.001 mM, damage at level 1 was the most FBS and 1% penicillin-streptomycin. Therefore, the same frequent, followed by level 0, both statistically significant

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Figure 2. Genotoxic effect of tartrazine on human lymphocytes. Data are expressed as the mean damage index obtained in the comet assay (alkaline version). Columns which have different letters are significantly differents at p<0.0001. Statistical test: ANOVA, Tukey.

compared to level 3 (p=0.001 and p=0.0349, respectively). compared to the NC. Although we observed an increase of Damage at level 1 was also significant in relation to the other damage at levels 1, 2 and 3 in relation to the NC, it was not levels (p=0.0002). In the PC at 0.003 mM, damage at level 1 statistically significant. Damage at level 4 showed a dose- was the most frequent, but was statistically significant only dependent increase in frequency compared to the NC, in relation to level 3 (p=0.0146). In the NC, damage at level however, it had significance only at the highest 0 was the most frequent, followed by level 1, both concentration (64.0 mM) (p=0.0130), which was also statistically significant in relation to the other levels of significant in relation to the PC at 0.001 mM (p=0.0366). damage (p<0.0001). In the treatment with tartrazine at 0.25 mM, damages at DNA repair potential evaluation. After 24 h without levels 0 and 1 were the most frequents, however, no tartrazine to evaluate the damage repair potential, the average differences were statistically significant, neither between them DI was also higher in all treatments compared to the NC, nor bewteen them and the others; at 0.50 mM, damage at however, only at the concentrations of 0.25 and 16.0 mM the level 0 was the most frequent but was significant only in differences were statistically significant (p=0.0135) (Figure relation to damage at level 3 (p=0.0304); at 1.0 mM, the most 5). In the intra-treatment comparative analyses, we found frequent damages were at levels 0, 1 and 4, and only level 0 that damage at level 0 was significantly more frequent in all was significantly more frequent in relation to levels 2 and 3 treatments compared to the other levels (p<0.0001). In the (p=0.0257); at 2.0 and 4.0 mM, damage was more frequent at treatments ranging from 1.0-64.0 mM, we did not observe levels 0 and 1 and they were statistically significant more significant difference between damage at levels 1, 2, 3 and frequent in relation to the other levels (p=0.0156 and 4 (Figure 6). p=0.0063, respectively); at 8.0 mM, damage levels 0, 1 and 4 In the treatment with tartrazine at 0.25 mM, damage at were the most frequent, with no significant difference level 4 was the second most frequent, but was significant between them, but they were more significant in relation to only in relation to damage level 3 (p=0.002); at 0.50 mM, the damage levels 2 and 3 (p<0.0001); at 16.0 mM, damage damage at level 1 was the second most frequent and was also at level 0 was the most frequent (p=0.0023), followed by significant only in relation to damage at level 3 (p=0.0108). levels 1 and 4 (p=0.0039), and all were significantly more In the PC at 0.001 mM and 0.003 mM, damage at level 1 frequents in relation to level 3; at 32.0 and 64.0 mM, damage was the second most frequent and it was statistically at level 4 was the most frequent, however, without statistical significant only when compared to damage at level 3 significance compared to the other levels. (p=0.0119 and p=0.0017, respectively). The frequency of In the inter-treatment comparative analyses (Figure 4), damage at levels 2, 3 and 4 did not differ. In the NC, damage we observed a statistically significant decrease (p=0.0002) at levels 1 and 2 were the second and third most frequent, in the frequency of damage at level 0 in all groups treated respectively, showing significant difference between them with tartrazine, as well as those treated with doxorubicin at and between them and damage at levels 3 and 4 (p<0.0003 0.001 mM (p=0.0006) and at 0.003 mM (p=0.0002), when and p=0.0225, respectively).

1468 Soares et al: Tartrazine Yellow Effects on Human Lymphocytes (p=0.0366). azine (0.25-64.0 mM). Data were obtained from the comet obtained from azine (0.25-64.0 mM). Data were c vs. b ne (0.25-64.0 mM). Data were obtained from the comet assay obtained from ne (0.25-64.0 mM). Data were s which have different letters are significantly differents at differents significantly are letters different have s which tment. Columns which have different letters are significantly differents at differents significantly are letters different have tment. Columns which (p=0.0130), l 1 vs. 2 and 4: p=0.0002; PC 0.003 mM: level 1 vs. 3: p=0.0146; negative l 1 vs. 2 and 4: p=0.0002; PC 0.003 mM: level 0 mM: level 0 vs. 2 and 3: p=0.0257; tartrazine 2.0 mM: level 0 and 1 vs. 2, 2.0 mM: level 0 vs. 2 and 3: p=0.0257; tartrazine 0 mM: level <0.0001; tartrazine 16.0 mM: level 1 vs. 3: p=0.0023); level 4 vs. 3: p=0.0039. 1 vs. 3: p=0.0023); level 16.0 mM: level <0.0001; tartrazine c vs. a (p=0.0006). Damage level 4: level (p=0.0006). Damage d vs. b (p=0.0002), d vs. a Quantitative analysis of the levels of DNA damage observed in human lymphocytes exposed to different concentrations of tartrazi concentrations to different exposed in human lymphocytes observed damage of DNA analysis of the levels Quantitative . Quantitative analysis of groups by level of damage observed in human lymphocytes exposed to different concentrations of tartr concentrations to different exposed in human lymphocytes observed of damage by level analysis of groups . Quantitative (alkaline version). The analysis was performed by comparing the different classes of lesion (0, 1, 2, 3 and 4) within each trea classes of lesion (0, 1, 2, 3 and 4) within each performed by comparing the different The analysis was (alkaline version). 1 vs. 3: p=0.001; leve 0 vs. 3: p=0.0349; level (PC) 0.001 mM: level control Positive Tukey. ANOVA, p<0.0001. Statistical test: 1. 0 vs. 3: p=0.0304; tartrazine 0.50 mm: level 0 and 1 vs. 2, 3 4: p<0.0001; tartrazine 0 vs. 1; level (NC): level control 0, 1 and 4 vs. 2 3: p 8.0 mM: level 0 and 1 vs. 2, 3 4 p=0.0063; tartrazine 4.0 mM: level 3 and 4: p=0.0156; tartrazine Figure 3. p=0.0002. Statistical test: ANOVA, Tukey. Damage level 0: level Damage Tukey. ANOVA, p=0.0002. Statistical test: assay (alkaline version). The analysis was performed by comparing the same type of level under the different treatments. Column treatments. under the different performed by comparing the same type of level The analysis was assay (alkaline version). Figure 4

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Figure 5. Genotoxic effect of tartrazine on human lymphocytes after allowing DNA damage repair for 24 h. Data are expressed as the mean damage index obtained in the comet assay (alkaline version). Columns which have different letters are significantly different. Statistical difference was observed only at concentrations of 0.25 and 16.0 mM (p=0.0135). Statistical test: ANOVA, Tukey.

In the inter-treatment comparative analyses (Figure 7), we These discrepant results may be related to the inaccuracy did not observe any significant differences in the damage at of the MTT assay in detecting cells in an intermediary levels ranging from 0 to 3. However, we observed an increase condition between a metabolically active state and death, of damage at level 4 at the concentration of 0.25 mM, but it which may have masked the cytotoxicity results (42). was significant only when compared to the NC (p=0.0333). Genotoxic effect of tartrazine. Our data showed that Discussion tartrazine induced significant DNA damage to human lymphocytes at all concentrations (0.25-64.0 mM) compared Synthetic colorants have been replacing those of natural to the NC. The concentration of 64.0 mM induced a higher origin, since it is easier to obtain a stable and uniform color, incidence of DNA damage in relation to those induced by contributing to the increase of their use by industries over doxorubicin, an extensively used positive control in in vitro time (36). However, several studies have shown that artificial genotoxicity and mutagenicity assays (35, 43-46). colorings, including those belonging to the azo class, may Through DNA electrophoretic mobility experiments, have toxic, mutagenic and genotoxic effects (26, 29, 37, 38). tartrazine dye was shown to be capable of strong binding to Therefore, the ability to accurately measure cytotoxicity and linear double stranded DNA causing its degradation. Thus, genotoxicity (through MTT and comet assays, for example) the genotoxic effects observed in our study through the is a very valuable tool in identifying compounds that might comet assay may be related to the association of tartrazine pose certain health risks in humans (39, 40). with the DNA of lymphocytes (29). Using the comet assay, the genotoxicity of 39 chemicals Cytotoxic effect of tartrazine. In the current study, tartrazine currently in use as food additives, including five that belongs did not exhibit cytotoxic effects at any concentration tested to the azo class, was studied in eight mice organs by Sasaki et (0.25-64.0 mM). Although we observed a slight decrease of al (37). Their results showed that of all the additives, the dyes cell viability, it was not significant. (amaranth, allura red, new coccine, tartrazine, erythrosine, Similarly, Koutsogeorgopoulou et al., also by using MTT phloxine, and rose bengal) were the most potent genotoxins in assay, did not find cytotoxic effects of tartrazine on human gastrointestinal organs (37). The same study also stated that lymphocytes (41). However, Mpountoukas et al., also using all dyes induced DNA damage in gastrointestinal organs at a an in vitro assay with human lymphocytes, showed that low dose (10 or 100 mg/kg). Among them, amaranth, allura tartrazine has cytotoxic effects at concentrations of 4 mM red, new coccine and tartrazine induced DNA damage in the and 8 mM, since at these concentrations, tartrazine colon at close to the acceptable daily intakes. However, it is treatment led to a statistically significant decrease of noteworthy that in their study, they did not use a positive mitotic index and a significant cell division delay compared control to compare the genotoxicity level, creating uncertainty to the control (29). in the validity of these results (37).

1470 Soares et al: Tartrazine Yellow Effects on Human Lymphocytes p<0,0003, c p=0.0225, b re obtained from the comet assay (alkaline obtained from re for 24 hours. Data were obtained from the comet obtained from Data were for 24 hours. which have different letters are significantly differents at differents significantly are letters different have which p<0.0001; negative control (NC): control p<0.0001; negative d mns which have different letters are significantly differents at differents significantly are letters different have mns which p=0.0017, b p<0.0001; PC 0.003 mM: p<0.0001. d d p=0.0119, p=0.0108, b b p=0.0333. b p<0.0001; tartrazine 0.50 mM: p<0.0001; tartrazine d p=0.002, b Quantitative analysis of the damage levels observed in human lymphocytes after allowing DNA damage repair for 24 hours. Data we for 24 hours. repair damage DNA after allowing in human lymphocytes observed levels analysis of the damage Quantitative Quantitative analysis of groups divided by type of damage level observed in human lymphocytes after allowing DNA damage repair repair damage DNA after allowing in human lymphocytes observed level by type of damage divided analysis of groups Quantitative p<0.0001; tartrazine 0.25 mM: p<0.0001; tartrazine Figure 6. d version). The analysis was performed by comparing the different levels of damage (0, 1, 2, 3 and 4) within each treatment. Colu treatment. (0, 1, 2, 3 and 4) within each of damage levels performed by comparing the different The analysis was version). (PC) 0.001 mM: control Positive Tukey. ANOVA, p<0.0001. Statistical test: p<0.05. Statistical test: ANOVA, Tukey. Damage level 4: level Damage Tukey. ANOVA, p<0.05. Statistical test: Figure 7. assay (alkaline version). The analysis was performed by comparing the same damage level under the different treatments. Columns treatments. under the different level performed by comparing the same damage The analysis was assay (alkaline version).

1471 ANTICANCER RESEARCH 35: 1465-1474 (2015)

Hassan et al. assessed the in vivo genotoxic effect of levels decreased until reaching a point when they increased tartrazine in rats orally-treated with a daily dose of 7.5 and again (Figure 2). In the comet assay for DNA repair 15 mg/kg for seven weeks. Their results showed that the high evaluation, the second increase occurred up to the concentration of tartrazine in diets induced a retardation in concentration of 16.0 mM, and then values started growth and caused DNA, liver and kidney damage. However, decreasing (Figure 5). This variation in the response to the like Sasaki et al., they did not use a positive control to different concentrations may be explained by the activation compare the genotoxicity level, so the validity of these of gene cascades involved in DNA repair mechanisms, findings could be questioned (37, 38). such that with increasing doses, different genes were The genotoxic potential of a substance can trigger cellular activated, leading to different cellular responses to DNA disorders, such as genetic instability, mutations and cell damage (53, 54). death. DNA damage originating from exposure to a chemical The effects of DNA damage and its repair mechanisms are agent can be transmitted during cell division, thus giving rise intimately associated with the development of cancer, since to a greater number of cells with abnormal DNA. the cell attempts to correct alterations in DNA sequence can Importantly, long-term exposure can lead to irreversible insert errors in the structure of genes, thus favoring tumor changes that contribute to oncogenesis (47, 48). development. Some chemical carcinogens act directly in The present study showed that the exposure of human DNA to cause changes, others need to be metabolized to lymphocytes to tartrazine led to a dose-dependent increase trigger such effects (55). Mpountoukas et al. evaluated the of damage at level 4 when compared to the NC. binding of tartrazine dye to DNA using the methods of Furthermore, there was also an increase in the average spectroscopic titration, DNA mobility shift in agarose gel frequency of damage at levels 2 and 3, which in some electrophoresis and PCR amplification and showed that treatments were higher than that of the NC, although not tartrazine is capable of binding to double-stranded DNA statistically significant. Thus, the ingestion of tartrazine dye triggering a degrading effect (29). directly exposes the cells of gastrointestinal tract to its It is clear that tartrazine has a direct effect on the DNA of genotoxic effect, which may contribute to the onset of human lymphocytes and although many of the lesions can be carcinogenesis. repaired, we stated that the damage at level 4 did not show an efficient repair. Thus, the intake of this dye exposes the DNA repair potential. The comet assay (alkaline version) cells of gastrointestinal epithelium, inducing an increase in detects DNA lesions such as single-strand breaks, double- DNA damage, as observed by Sasaki et al. (37) in their in strand breaks, alkali-labile sites, abasic sites and crosslinks vivo experiment. Additionally, successive accumulation of (35). Depending on the extent of damage, the cell response damage, triggered by habitual ingestion of food containing to DNA damage occurs through activation of this dye, may lead to the emergence of mutations, which transcriptional and post-transcriptional gene clusters often are associated with the onset of diseases, such as related to cell-cycle arrest, DNA repair and (49, cancer. 50). DNA damage is first detected by sensor proteins, which in turn activate transducers in the signal cascade. Conclusion These transducers then mediate the activation or inhibition of downstream effectors which can arrest the cell cycle or In the present study, we observed that tartrazine yellow dye cause apoptosis (51, 52). did not have any cytotoxic effects when assessed by the As far as we are aware of, this is the first study that MTT assay. However, this dye had a significant genotoxic evaluated DNA repair potential after exposure to tartrazine. effect at all concentrations tested compared to the NC. The In the evaluation, we observed a marked decrease in DI fact that some damage was irreparable suggests that the when compared to the analysis before the allowed repair indiscriminate use of tartrazine for a long period of time time. The PC at 0.001 and 0.003 mM led to a decrease of could trigger carcinogenesis, since the accumulation of 61.35% and 56.83% in DI, respectively. In the treatments successive DNA errors may affect genes related to cell- with the dye, the reductions ranged from 34.46-75.45% cycle control, such as tumor-suppressor genes and proto- (49.68%±18.39%). Additionally, there was a predominance oncogenes. of damage at level 0 (>60% in all treatments) and a decrease in damage at higher levels. However, although most of the Acknowledgements damage was amenable to repair, damage at level 4 was We acknowledge the Federal University of Pará (PROPESP and higher than that of the NC at all concentrations, but was Fadesp) for the technical support and the National Council for significant only at 0.25 mM. Scientific and Technological Development (CNPq) and Coordination We also observed that the lowest concentration of for Enhancement of Higher Education Personnel (CAPES) for tartrazine (0.25 mM) led to high levels of DI and these fellowship support.

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