Abstract
Tartrazine is a food additive that belongs to a class of artificial dyes and contains an azo group. Studies about its genotoxic, cytotoxic and mutagenic effects are controversial and, in some cases, unsatisfactory. This work evaluated the potential in vitro cytotoxicity, genotoxicity and effects on DNA repair of human lymphocytes exposed to the dye. We assessed the cytotoxicity of tartrazine by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test and the response of DNA repair through comet assay (alkaline version). We used different concentrations of the dye, ranging from 0.25-64.0 mM. The results demonstrated that tartrazine has no cytotoxic effects. However, this dye had a significant genotoxic effect at all concentrations tested. Although most of the damage was amenable to repair, some damage remained higher than positive control after 24 h of repair. These data demonstrate that tartrazine may be harmful to health and its prolonged use could trigger carcinogenesis.
The food industry uses a wide range of additives that may be harmful to health, including dyes that belong to the azo class, which are extensively used in foods, drugs and cosmetics (1). The mutagenic, carcinogenic and toxic effects of azo dyes can be a result of direct action of the compound or arise from the formation of aryl amine derivatives generated during the reductive biotransformation of the azo bond. In mammals, water-soluble azo dyes are mainly metabolized by anaerobic intestinal microflora. Azo reductase in the liver can also catalyze reductive cleavage of azo bonds, specifically of the water-insoluble dyes (2, 3).
Tartrazine is a water-soluble azo dye with extremely low metabolism and oral absorption. Studies published in the literature show that less than 2% of the ingested tartrazine is actually absorbed. Most of the tartrazine is readily-metabolized in the colon by the intestinal flora, where it is reduced to sulfanilic acid and aminopyrazolone. Although mostly excreted in feces, these metabolites may be absorbed and excreted by urine as sulfanilic acid (4-6).
Tartrazine dye was evaluated by the Expert Committee on Food Additives (JECFA) and it was established that the acceptable daily intake (ADI) for such substance is 7.5 mg/kg/day. Based on toxicological, mutagenic, carcinogenic and teratogenic effects of tartrazine, Elhkim et al. (6) confirmed the initial hazard assessment conducted by the JECFA (6, 7).
A review performed by Elhkim et al. stated that tartrazine has no mutagenic or clastogenic potential, neither in vitro nor in vivo (6). Among the tools used to establish this were the Ames test, mutagenicity evaluation in yeast, unscheduled DNA synthesis assay, sister chromatid exchange (SCE) assay, chromosomal aberrations test and micronucleus assay (7-23).
Similarly, Poul et al. did not find mutagenic or cytotoxic effects of tartrazine (20, 200 or 1,000 mg/kg) when the dye was administered twice, at 24-h intervals, by oral gavage to mice (24). In their study, the in vivo gut micronucleus test was used for mutagenic effects and the cytotoxicity evaluation was performed by quantification of apoptotic and 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 hamster cells when exposed to a concentration of 2.5 mg/ml (25, 26). Patterson and Butler showed that tartrazine induces in vitro chromosomal aberrations in Muntiacus muntjac fibroblasts exposed to concentrations ranging from 5-20 mg/ml, when compared to the control (27). According to Giri et al. this dye can induce chromosomal aberrations and SCE in bone marrow cells of mouse and rat when they are submitted to chronic exposure to high doses of the dye (28).
Ishidate et al., in their study with Chinese hamster fibroblast cell line, also found a slight increase of polyploid cells after treatment with tartrazine (2.5 mg/ml) for 48 h (26).
Mpountoukas et al. evaluated the genotoxic, cytotoxic and cytostatic potential of tartrazine yellow dye (0.02 to 8 mM) in human peripheral blood cells over an exposure time of 72 h. They observed that the two higher concentrations of tartrazine (4 and 8 mM) had a significant toxic effect on the quality of chromosomes, so the differentiation stain between two chromatids was not clear, maybe because tartrazine was toxic at the condensation of chromosomes in mitosis. Furthermore, tartrazine at 4 and 8 mM led to a statistically significant decrease of mitotic index and a significant delay in cell division compared to the control (29).
Some studies regarding the mutagenic effect of tartrazine metabolites are also available in the literature. Chung et al. evaluated the mutagenicity of sulfanilic acid and aminopiralozone metabolites using the Ames test and did not find significant results (11). However, through this same assay, Henschler and Wild analyzed rat urine after feeding them with tartrazine. The excreta was sterilized by filtration, concentrated by lyophilization and subsequently diluted in sterile water. The results showed a dose-dependent mutagenic response in Salmonella typhimurium strains TA 98 (+S9) (17). Instead, also using the Ames test, but analyzing rat feces extract, Munzner and Wever showed no positive results (20).
Other research highlighted the mutagenicity of photoexcited tartrazine by using the Ames test (30) and Somatic Mutation And Recombination Test (SMART) (31).
Tartrazine has been widely used by the Brazilian pharmaceutical industry, being the most common food additive (30%) amongst azo dyes (32). It is noteworthy that in some cases, the food industry has used this compound at concentrations above the permitted level (33, 34).
Since many studies regarding the damaging potential of this dye have reported contradictory and, sometimes, unsatisfactory results, it is necessary to carry-out further research to investigate this dye more precisely. Thus, the present study assessed the in vitro cytotoxic and genotoxic activity of tartrazine and the DNA repair potential of cells that had undergone treatment with this compound.
Materials and Methods
Chemicals. Tartrazine (CAS 1934-21-0, purity ≥85%) and doxorubicin (CAS 25316-40-9, purity 98-102%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen (Carlsbad, CA, USA).
Samples and Experimental Design. In the current study, human lymphocytes were used as an experimental model. Peripheral blood (10 ml) from four healthy individuals (approximately 18-35 years of age, two females and two males) was collected in heparinized syringes. The donors gave written informed consent to participate in the study and history for all of them was in accordance with standard guidelines for genotoxicity tests (not being a smoker, with no recent infection, no chronic use of medication in the previous four months and no radiation exposure in the previous six months). For the cell viability assay (MTT), lymphocytes were isolated from whole blood using Histopaque (Sigma-Aldrich).
To assess genotoxicity by comet assay, we performed temporary culture of lymphocytes from decanted plasma. For this assay, we used cells without treatment as negative control (NC) and cells treated with doxorubicin at different concentrations (0.001 mM and 0.003 mM) as positive control (PC), in order to compare the genotoxicity level.
Comet assay was performed with two purposes: to assess the genotoxic effect of tartrazine after 3 hours of exposure, and to assess DNA repair potential 24 hours after removal of tartrazine. It is important to notice that separate lymphocyte cultures were prepared for each experiment.
Tartrazine was solubilized at different concentrations using sterile distilled water. For the MTT assay, we used 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 mM tartrazine and for the comet assay we included the concentration of 64.0 mM. All concentrations used were based on data available in the literature (26, 27, 29).
Lymphocyte cell culture. For the cytotoxicity assay, peripheral blood lymphocyte isolation was carried out according to Fenech (35). For the genotoxicity assay, blood was stored for 1 hour at 37°C for separation and decanting of plasma from whole blood. After visual separation of blood fractions, the leukocyte layer was resuspended with gentle stirring of the syringe. Then, 400 μl of resuspended plasma was added to 100 μl of whole blood in 15-ml falcon tubes (TPP, Zollstrasse, Trasadingen, Switzerland) containing 4.5 ml of RPMI-1640 medium (#52400-025; Gibco, Carlsbad, CA, USA), supplemented with 20% fetal bovine serum (FBS; #10106-169; Gibco), 4% phytohemagglutinin (#10576-05; Gibco), 1% penicillin-streptomycin (10000 units of penicillin and 10000 μg of streptomycin per ml, #15140-122; Gibco). The cells were seeded at 37°C for 20 hours before treatment in an incubator containing 5% CO2, as were the cells isolated by histopaque, which were seeded in 5 ml of the same medium.
MTT assay. Lymphocytes were grown in 96-well culture plates at a density of 0.64×105 cells per well and incubated for 24 h. After the initial period of incubation, cells were treated with different concentrations of tartrazine (0.25-32.0 mM) for 24 h. At the end of this period, 100 ml of MTT (5,000 mg/ml) was added to the cells for 3 hours. Then, the MTT was removed and 100 ml of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added for 1 h in order to dissolve the formazan obtained during the process. Afterward, the absorbance was then measured by spectrophotometry (λ=562 nm). Cell survival was calculated as the percentage absorbance compared with that of the control. All experiments were carried-out in duplicate.
Comet assay (alkaline version). Cultures of lymphocytes from four individuals were treated with different concentrations of tartrazine (0.25-64.0 mM) for 3 h. Doxorubicin (0.001 and 0.003 mM) was used as positive control. After treatment, a 450 μl suspension of 1 ml of cells were transferred to 1.5 ml culture tubes submerged in ice (to inactivate repair enzymes). Cells were harvested by centrifugation for five minutes at 106 g and homogenized in 300 ml of a low-melting-point agarose (0.8%), spread onto microscope slides pre-coated with a normal melting point agarose (1.5%) and covered with a coverslip (24×60 mm). After 8 min at 4°C, the coverslip was removed and the slides were immersed in cold lysis solution (2.5 M NaCl; 100 mM EDTA; 10 mM Tris, 10% DMSO 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). Electrophoresis was performed for 20 min (34 V and 300 mA). Afterwards, the slides were neutralized by submersion in distilled water (4°C) for 5 min and fixed in 100% ethanol for 3 minutes. Staining of the slides was performed immediately before the analyses using ethidium bromide (20 mg/ml). Slides were prepared in duplicate and 100 cells were screened per sample (50 cells from each slide) using a fluorescence microscope (Olympus BX41, Tokyo, Japan) at ×40 magnification.
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.
The damage index (DI) was visually determined based on the size and intensity of the comet's tail. The following five categories (0-4) were used: level 0, no damage; level 1, little damage with a tail length shorter than the diameter of the nucleus; level 2, medium 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 times the diameter of the nucleus; and level 4, significant damage with a tail length greater than three times the diameter of the nucleus.
We also quantified the level of DNA damage by using a comparative method to verify which damage level was most prevalent intra- and inter-treatment.
In vitro comet assay for DNA repair. To assess the DNA repair potential, after treatment the cells were washed twice with 5 ml of phosphate buffer solution (PBS) 1X (#70011-044; Invitrogen) and seeded for 24 h in RPMI-1640 medium supplemented with 20% FBS and 1% penicillin-streptomycin. Therefore, the same methodology used to evaluate the DI was used to quantify the level of damage by intra- and inter-treatment.
Statistical analysis. The statistical analysis was performed by ANOVA, followed by Tukey test, through GRAPHPAD PRISM v.5.0 (GraphPad Software, San Diego, CA USA). p-Values of less than 0.05 were considered significant.
Results
Cytotoxicity evaluation. The addition of tartrazine yellow dye to lymphocyte cultures isolated by Ficoll and stimulated by phytohemagglutinin did not significantly (p>0.05) affect cell viability at any of the tested concentrations (0.25-32.0 mM). Thus, the MTT assay showed that tartrazine had no cytotoxic potential under the tested conditions (Figure 1).
Genotoxicity evaluation. After 3 h of exposure, tartrazine induced DNA damage in human lymphocytes at all concentrations (0.25-64.0 mM). The damage observed in all treatments, including the PC, were statistically significant (p<0.0001) compared to the NC. There was no significant difference between the two concentrations of PC (0.001 and 0.003 mM) in the damage induced, nor between them and the treatments with the dye, except at the concentration of 64.0 mM, which induced a higher incidence of DNA damage (Figure 2).
In the intra-treatment comparative analyses (Figure 3), we observed that tartrazine yellow dye induced damage more frequently at higher levels. Moreover, we also observed a dose-dependent increase in incidence of damage at level 4.
In the PC at 0.001 mM, damage at level 1 was the most frequent, followed by level 0, both statistically significant compared to level 3 (p=0.001 and p=0.0349, respectively). Damage at level 1 was also significant in relation to the other levels (p=0.0002). In the PC at 0.003 mM, damage at level 1 was the most frequent, but was statistically significant only in relation to level 3 (p=0.0146). In the NC, damage at level 0 was the most frequent, followed by level 1, both statistically significant in relation to the other levels of damage (p<0.0001).
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.
In the treatment with tartrazine at 0.25 mM, damages at levels 0 and 1 were the most frequents, however, no differences were statistically significant, neither between them nor bewteen them and the others; at 0.50 mM, damage at level 0 was the most frequent but was significant only in relation to damage at level 3 (p=0.0304); at 1.0 mM, the most frequent damages were at levels 0, 1 and 4, and only level 0 was significantly more frequent in relation to levels 2 and 3 (p=0.0257); at 2.0 and 4.0 mM, damage was more frequent at levels 0 and 1 and they were statistically significant more frequent in relation to the other levels (p=0.0156 and p=0.0063, respectively); at 8.0 mM, damage levels 0, 1 and 4 were the most frequent, with no significant difference between them, but they were more significant in relation to the damage levels 2 and 3 (p<0.0001); at 16.0 mM, damage at level 0 was the most frequent (p=0.0023), followed by levels 1 and 4 (p=0.0039), and all were significantly more frequents in relation to level 3; at 32.0 and 64.0 mM, damage at level 4 was the most frequent, however, without statistical significance compared to the other levels.
In the inter-treatment comparative analyses (Figure 4), we observed a statistically significant decrease (p=0.0002) in the frequency of damage at level 0 in all groups treated with tartrazine, as well as those treated with doxorubicin at 0.001 mM (p=0.0006) and at 0.003 mM (p=0.0002), when compared to the NC. Although we observed an increase of damage at levels 1, 2 and 3 in relation to the NC, it was not statistically significant. Damage at level 4 showed a dose-dependent increase in frequency compared to the NC, however, it had significance only at the highest concentration (64.0 mM) (p=0.0130), which was also significant in relation to the PC at 0.001 mM (p=0.0366).
DNA repair potential evaluation. After 24 h without tartrazine to evaluate the damage repair potential, the average DI was also higher in all treatments compared to the NC, however, only at the concentrations of 0.25 and 16.0 mM the differences were statistically significant (p=0.0135) (Figure 5). In the intra-treatment comparative analyses, we found that damage at level 0 was significantly more frequent in all treatments compared to the other levels (p<0.0001). In the treatments ranging from 1.0-64.0 mM, we did not observe significant difference between damage at levels 1, 2, 3 and 4 (Figure 6).
In the treatment with tartrazine at 0.25 mM, damage at level 4 was the second most frequent, but was significant only in relation to damage level 3 (p=0.002); at 0.50 mM, damage at level 1 was the second most frequent and was also significant only in relation to damage at level 3 (p=0.0108).
In the PC at 0.001 mM and 0.003 mM, damage at level 1 was the second most frequent and it was statistically significant only when compared to damage at level 3 (p=0.0119 and p=0.0017, respectively). The frequency of damage at levels 2, 3 and 4 did not differ. In the NC, damage at levels 1 and 2 were the second and third most frequent, respectively, showing significant difference between them and between them and damage at levels 3 and 4 (p<0.0003 and p=0.0225, respectively).
Quantitative analysis of the levels of DNA damage observed in human lymphocytes exposed to different concentrations of tartrazine (0.25-64.0 mM). Data were obtained from the comet assay (alkaline version). The analysis was performed by comparing the different classes of lesion (0, 1, 2, 3 and 4) within each treatment. Columns which have different letters are significantly differents at p<0.0001. Statistical test: ANOVA, Tukey. Positive control (PC) 0.001 mM: level 0 vs. 3: p=0.0349; level 1 vs. 3: p=0.001; level 1 vs. 2 and 4: p=0.0002; PC 0.003 mM: level 1 vs. 3: p=0.0146; negative control (NC): level 0 vs. 1; level 0 and 1 vs. 2, 3 and 4: p<0.0001; tartrazine 0.50 mm: level 0 vs. 3: p=0.0304; tartrazine 1.0 mM: level 0 vs. 2 and 3: p=0.0257; tartrazine 2.0 mM: level 0 and 1 vs. 2, 3 and 4: p=0.0156; tartrazine 4.0 mM: level 0 and 1 vs. 2, 3 and 4 p=0.0063; tartrazine 8.0 mM: level 0, 1 and 4 vs. 2 and 3: p<0.0001; tartrazine 16.0 mM: level 1 vs. 3: p=0.0023); level 4 vs. 3: p=0.0039.
Quantitative analysis of groups by level of damage observed in human lymphocytes exposed to different concentrations of tartrazine (0.25-64.0 mM). Data were obtained from the comet assay (alkaline version). The analysis was performed by comparing the same type of level under the different treatments. Columns which have different letters are significantly differents at p=0.0002. Statistical test: ANOVA, Tukey. Damage level 0: d vs. a (p=0.0002), d vs. b (p=0.0006). Damage level 4: c vs. a (p=0.0130), c vs. b (p=0.0366).
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 did not observe any significant differences in the damage at levels ranging from 0 to 3. However, we observed an increase of damage at level 4 at the concentration of 0.25 mM, but it was significant only when compared to the NC (p=0.0333).
Discussion
Synthetic colorants have been replacing those of natural origin, since it is easier to obtain a stable and uniform color, contributing to the increase of their use by industries over time (36). However, several studies have shown that artificial colorings, including those belonging to the azo class, may have toxic, mutagenic and genotoxic effects (26, 29, 37, 38). Therefore, the ability to accurately measure cytotoxicity and genotoxicity (through MTT and comet assays, for example) is a very valuable tool in identifying compounds that might pose certain health risks in humans (39, 40).
Cytotoxic effect of tartrazine. In the current study, tartrazine did not exhibit cytotoxic effects at any concentration tested (0.25-64.0 mM). Although we observed a slight decrease of cell viability, it was not significant.
Similarly, Koutsogeorgopoulou et al., also by using MTT assay, did not find cytotoxic effects of tartrazine on human lymphocytes (41). However, Mpountoukas et al., also using an in vitro assay with human lymphocytes, showed that tartrazine has cytotoxic effects at concentrations of 4 mM and 8 mM, since at these concentrations, tartrazine treatment led to a statistically significant decrease of mitotic index and a significant cell division delay compared to the control (29).
These discrepant results may be related to the inaccuracy of the MTT assay in detecting cells in an intermediary condition between a metabolically active state and death, which may have masked the cytotoxicity results (42).
Genotoxic effect of tartrazine. Our data showed that tartrazine induced significant DNA damage to human lymphocytes at all concentrations (0.25-64.0 mM) compared to the NC. The concentration of 64.0 mM induced a higher incidence of DNA damage in relation to those induced by doxorubicin, an extensively used positive control in in vitro genotoxicity and mutagenicity assays (35, 43-46).
Through DNA electrophoretic mobility experiments, tartrazine dye was shown to be capable of strong binding to linear double stranded DNA causing its degradation. Thus, the genotoxic effects observed in our study through the comet assay may be related to the association of tartrazine with the DNA of lymphocytes (29).
Using the comet assay, the genotoxicity of 39 chemicals currently in use as food additives, including five that belongs to the azo class, was studied in eight mice organs by Sasaki et al (37). Their results showed that of all the additives, the dyes (amaranth, allura red, new coccine, tartrazine, erythrosine, phloxine, and rose bengal) were the most potent genotoxins in gastrointestinal organs (37). The same study also stated that all dyes induced DNA damage in gastrointestinal organs at a low dose (10 or 100 mg/kg). Among them, amaranth, allura red, new coccine and tartrazine induced DNA damage in the colon at close to the acceptable daily intakes. However, it is noteworthy that in their study, they did not use a positive control to compare the genotoxicity level, creating uncertainty in the validity of these results (37).
Quantitative analysis of the damage levels observed in human lymphocytes after allowing DNA damage repair for 24 hours. Data were obtained from the comet assay (alkaline version). The analysis was performed by comparing the different levels of damage (0, 1, 2, 3 and 4) within each treatment. Columns which have different letters are significantly differents at p<0.0001. Statistical test: ANOVA, Tukey. Positive control (PC) 0.001 mM: bp=0.0119, dp<0.0001; PC 0.003 mM: bp=0.0017, dp<0.0001; negative control (NC): bp=0.0225, cp<0,0003, dp<0.0001; tartrazine 0.25 mM: bp=0.002, dp<0.0001; tartrazine 0.50 mM: bp=0.0108, dp<0.0001.
Quantitative analysis of groups divided by type of damage level observed in human lymphocytes after allowing DNA damage repair for 24 hours. Data were obtained from the comet assay (alkaline version). The analysis was performed by comparing the same damage level under the different treatments. Columns which have different letters are significantly differents at p<0.05. Statistical test: ANOVA, Tukey. Damage level 4: bp=0.0333.
Hassan et al. assessed the in vivo genotoxic effect of tartrazine in rats orally-treated with a daily dose of 7.5 and 15 mg/kg for seven weeks. Their results showed that the high concentration of tartrazine in diets induced a retardation in growth and caused DNA, liver and kidney damage. However, like Sasaki et al., they did not use a positive control to compare the genotoxicity level, so the validity of these findings could be questioned (37, 38).
The genotoxic potential of a substance can trigger cellular disorders, such as genetic instability, mutations and cell death. DNA damage originating from exposure to a chemical agent can be transmitted during cell division, thus giving rise to a greater number of cells with abnormal DNA. Importantly, long-term exposure can lead to irreversible changes that contribute to oncogenesis (47, 48).
The present study showed that the exposure of human lymphocytes to tartrazine led to a dose-dependent increase of damage at level 4 when compared to the NC. Furthermore, there was also an increase in the average frequency of damage at levels 2 and 3, which in some treatments were higher than that of the NC, although not statistically significant. Thus, the ingestion of tartrazine dye directly exposes the cells of gastrointestinal tract to its genotoxic effect, which may contribute to the onset of carcinogenesis.
DNA repair potential. The comet assay (alkaline version) detects DNA lesions such as single-strand breaks, double-strand breaks, alkali-labile sites, abasic sites and crosslinks (35). Depending on the extent of damage, the cell response to DNA damage occurs through activation of transcriptional and post-transcriptional gene clusters related to cell-cycle arrest, DNA repair and apoptosis (49, 50). DNA damage is first detected by sensor proteins, which in turn activate transducers in the signal cascade. These transducers then mediate the activation or inhibition of downstream effectors which can arrest the cell cycle or cause apoptosis (51, 52).
As far as we are aware of, this is the first study that evaluated DNA repair potential after exposure to tartrazine. In the evaluation, we observed a marked decrease in DI when compared to the analysis before the allowed repair time. The PC at 0.001 and 0.003 mM led to a decrease of 61.35% and 56.83% in DI, respectively. In the treatments with the dye, the reductions ranged from 34.46-75.45% (49.68%±18.39%). Additionally, there was a predominance of damage at level 0 (>60% in all treatments) and a decrease in damage at higher levels. However, although most of the damage was amenable to repair, damage at level 4 was higher than that of the NC at all concentrations, but was significant only at 0.25 mM.
We also observed that the lowest concentration of tartrazine (0.25 mM) led to high levels of DI and these levels decreased until reaching a point when they increased again (Figure 2). In the comet assay for DNA repair evaluation, the second increase occurred up to the concentration of 16.0 mM, and then values started decreasing (Figure 5). This variation in the response to the different concentrations may be explained by the activation of gene cascades involved in DNA repair mechanisms, such that with increasing doses, different genes were activated, leading to different cellular responses to DNA damage (53, 54).
The effects of DNA damage and its repair mechanisms are intimately associated with the development of cancer, since the cell attempts to correct alterations in DNA sequence can insert errors in the structure of genes, thus favoring tumor development. Some chemical carcinogens act directly in DNA to cause changes, others need to be metabolized to trigger such effects (55). Mpountoukas et al. evaluated the binding of tartrazine dye to DNA using the methods of spectroscopic titration, DNA mobility shift in agarose gel electrophoresis and PCR amplification and showed that tartrazine is capable of binding to double-stranded DNA triggering a degrading effect (29).
It is clear that tartrazine has a direct effect on the DNA of human lymphocytes and although many of the lesions can be repaired, we stated that the damage at level 4 did not show an efficient repair. Thus, the intake of this dye exposes the cells of gastrointestinal epithelium, inducing an increase in DNA damage, as observed by Sasaki et al. (37) in their in vivo experiment. Additionally, successive accumulation of damage, triggered by habitual ingestion of food containing this dye, may lead to the emergence of mutations, which often are associated with the onset of diseases, such as cancer.
Conclusion
In the present study, we observed that tartrazine yellow dye did not have any cytotoxic effects when assessed by the MTT assay. However, this dye had a significant genotoxic effect at all concentrations tested compared to the NC. The fact that some damage was irreparable suggests that the indiscriminate use of tartrazine for a long period of time could trigger carcinogenesis, since the accumulation of successive DNA errors may affect genes related to cell-cycle control, such as tumor-suppressor genes and proto-oncogenes.
Acknowledgements
We acknowledge the Federal University of Pará (PROPESP and Fadesp) for the technical support and the National Council for Scientific and Technological Development (CNPq) and Coordination for Enhancement of Higher Education Personnel (CAPES) for fellowship support.
- Received November 12, 2014.
- Revision received November 28, 2014.
- Accepted December 4, 2014.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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