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

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Effects on DNA Repair in Human Lymphocytes Exposed to the Food Dye Tartrazine Yellow 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 cytotoxicity, 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 assay 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 cell 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 cell culture. 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.
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