DNA damage induced by tartrazine in rat whole blood using comet assay (single cell gel electrophoresis).
The colour additive, Tartrazine, FD&C Yellow No. 5, C.I. No. 19140, is principally the trisodium salt of 3-carboxy-5-hydroxy-1-(p-sulphophenyl)-4-(p sulphophenylazo) pyrazole. Tartrazine is an orange-coloured, water soluble powder widely used in food products, drugs, cosmetics and pharmaceuticals, in a way to improve the esthetic quality of a food product. It has the chemical structure illustrated in Fig.1.
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Moreover, this food colorant is used in cooking in many developing countries as a substitute for saffron . The Acceptable Daily Intake (ADI) for humans is 0-7.5 mg kg-1 body weight . Tartrazine is reduced in the organism to an aromatic amine which is highly sensitizing. Its main metabolite identified to date is sulfanylic acid . It has been implicated as the food additive which is most often responsible for allergic reactions in specific human populations [4, 5, 6]. Some countries such as Sweden, Switzerland and Norway have withdrawn Tartrazine on the ground of its anaphylactic potential. The study of the carcinogenetic and mutagenetic effects of Tartrazine was established by some authors which gives variable results [7,8,9,10,11,12].
The alkaline Comet Assay or single cell gel electrophoresis (SCGE) assay is a rapid, sensitive and relatively simple method for DNA double--and single-strand breaks, alkali-labile sites and delayed repair site detection, in eukaryotic individual cells [13,14,15]. This technique is capable of detecting a wide variety of DNA damage and lesions such as DNA single strand breaks, double strand breaks, base damage.
The Comet Assay is based on the ability of negatively charged loops/fragments of DNA to be drawn through an agarose gel in response to an electric field. The extent of DNA migration depends directly on the DNA damage present in the cells. When viewed under microscope, a cell has the appearance of a comet with a head (the nuclear region) and a tail containing DNA fragments or strands migration in the direction of the anode .
As the food colorants are widely used in our food and little is known about their genotoxicological effects, the present study was designed to investigate in vitro the genotoxic effects on the whole blood cells Wistar rats induced by Tartrazine.
Materials and Methods
2.1 Blood sample collection:
The comet assay was performed in triplicate on 10 [micro]l of fresh blood collected via the abdominal aorta of Wistar rats as control samples in an experiment to test the effect of Tartrazine.
2.2 Preparation of cells:
The blood samples were checked for cell viability by use of the trypan bleu exclusion technique. Blood samples were centrifuged ay room temperature at 300 x g for 5 min. the cell pellet was washed twice and re-suspended in Ca2+- and Mg2+free phosphate-buffred saline (PBS) to obtain about 10,000 cells in 10 [micro]l .
All chemicals used were of reagent to higher grade. Tartrazine (CAS 1934-21-0, Purity 86.7%) was purchased from Alfa Aesar (Germany).
2.4 Comet assay:
The comet assay was performed under pH >13 alkaline conditions according to Singh et al.  and the guidelines for using this assay in genetic toxicology . A volume of 10 [micro]l of the peripheral blood cell suspension, was mixed with 100 [micro]l of 0.5% low melting point agarose (37[degrees]C), layered on pre-coated slides with normal melting point agarose, covered with a coverslip and placed at 4[degrees]C, for 5 min, in order to solidify the agarose. The coverslip was gently removed and the slides were submersed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100) at 4 [degrees] C for 1 h. After lysis, the slides were briefly washed in PBS to remove the excess of lysis solution, and placed on horizontal electrophoresis unit filled with fresh electrophoresis alkaline buffer (300 Mm NaOH and 1 mM EDTA, pH > 13) for 20 min at 4[degrees]C. Electrophoresis was conducted at 4 [degrees] C for 20 min at 25 V and 300 mA. The slides were neutralized in a buffer (0.4 M Tris at pH 7.5).
After being dried at ambient temperature, the slides were stored until analysis. Each slide was stained with 30 [micro]l ethidium bromide (20 [micro]l/ml) and immediately analyzed. All the steps were conducted in the dark or dimmed light. The comets observations were made at 400 x magnification using a fluorescence microscope (Leica DM1000 LED) using an automated image analysis system (software CASP was downloaded from http://www.casp.of.pl) .
2.5 Quantification of the Comet assay:
To quantify the induced DNA damage, 100 cells per probe were examined for the Olive Tail Moment (OTM) reflecting the percentage of DNA in the tail of the comet multiplied by the median migration distance , the percentage of DNA in the Tail (%DT), Tail Moment (TM) and the Tail Length (TL) .
2.6 Statistical analysis:
Data are presented in tables or figures as the mean [+ or -] SEM. The statistical significance of the differences between control and experimental groups was evaluated by Student's t-test using GraphPad Instat 3.06. Results:
We assessed the genotoxicity of Tartrazine in rat whole blood using the comet assay as a highly effective tool for the biomonitoring of DNA integrity. The comet assay is a valuable and sensitive tool for detecting genetic damage in individual cells .
The migration of DNA from the cells blood treated with different concentrations of Tartrazine for each group is shown in Figs. (Fig.2). Furthermore, beginning at 3 mM, there was also evidence of an increase in dose-dependence. However, no significant effect on DNA migration was found at concentrations below 3 mM of Tartrazine (2.5, 2, 1.5,1, 0.5 mM).
In all concentrations of Tartrazine, the Tail Length was significantly higher than in controls (P < 0.05; Fig.3) and the difference between all groups was significant (P < 0.05). Tail DNA % in blood cells of all treated groups was higher than in controls (P < 0.05; Fig.4), and the difference between all groups was significant (P < 0.05). Tail moment and Olive Tail Moment also increased with the length of treatment, with significant differences between all groups (P < 0.05); (Fig.5-6). Our results indicated that, the levels of DNA degradation (Comet Tail Length) were dose-dependent.
[FIGURE 2 OMITTED]
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The comet assay is known as a sensitive, rapid and economical detection of DNA fractures and can be considered a good biomarker of genotoxicity of food additives. Whole blood was chosen for her role as carrier of toxic pollutants [21, 22]. Because Tartrazine is from the group of azo dye food colorants, it is metabolized into aromatic amine by intestinal flora and the formed aromatic amines can generate reactive oxygen species (ROS), which are well known to cause DNA damage. The ROS such as superoxide anion, hydroxyl radical and H2O2 could be produced in the metabolism of nitrosamines and increase oxidative stress .
By referring to a few studies including the study of Sasaki and al. (2002), who studied the genotoxicity of 39 chemicals currently in use as food additives. They treated groups of four male ddY mice once orally with each additive at up to half its LD50 or the limit dose (2000 mg/kg bw) and performed Comet assays on glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow, 3 and 24 hours after treatment. Tartrazine induced dose-related DNA damage in the glandular stomach, colon, and/or urinary bladder. All 7 food dyes tested induced DNA damage in the gastrointestinal organs at low doses (10 or 100 mg/kg). Among them, Amaranth, Allura Red, New Coccine, and Tartrazine induced DNA damage in the colon. Tartrazine also induced DNA damage in the stomach at doses of 10 and 2000 mg/kg bw without a dose-effect relationship .
G. Hassan (2010) studied the genotoxic effects induced byTartrazine and chocolate as a food coloring synthetic agents. The rats were divided into five equal groups, each composed of 4 rats, as follows: The 1st group (G1) served as untreated control. The 2nd (G2) and 3rd (G3) groups were orally treated with a daily dose of Tartrazine 7.5 and 15 mg/kg b.wt., respectively. The 4th (G4) and 5th (G5) groups were orally treated with a daily dose of chocolate brown 0.15 and 0.3 mg/kg b.wt., respectively for 7 weeks. The results revealed that Tartrazine and chocolate brown caused DNA liver and kidney damage as detected by comet assay. Chromosome ring and end to end association were the most common abnormalities observed on bone marrow cells of treated rats .
Furthermore, beginning at 3 mM, there was also evidence of an increase in dose-dependence, probably due to the direct contact of Tartrazine with nuclear DNA. These results are compatible with Panagiotis Mpountoukas and al. (2010) who tested the food coloring agents, amaranth, erythrosine and Tartrazine at 0.02-8 mM in human peripheral blood cells in vitro, in order to investigate their genotoxic, cytotoxic and cytostatic potential. Tartrazine showed cytotoxicity at 1 and 2 mM. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these dyes bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. These results indicate that these food colorants had a toxic potential to human lymphocytes in vitro and it seems that they bind directly to DNA. .
Data pertaining to the genotoxicity or carcinogenicity of Tartrazine in various systems with positive results are available; Tartrazine has been shown to induce chromosomal aberrations in fibroblast cells of Muntiacus muntjac , on bone marrow cells of mice and rats  and on chromosomes of Allium cepa . In this study higher concentrations of Tartrazine (3-25 mM) have a significant effect on DNA migration, so when using concentrations near to the ADI, the result is not significant. It was proved that the administration of Tartrazine up to ADI does not create cytogenetic damages. But, at higher concentrations has reverse effects [29, 30].
We believe that there are two plausible explanations for this behavior, namely (1) the production of carcinogenic compounds upon reductive cleavage of the azo bond (N=N) of Tartrazine by intestinal flora, and (2) binding of the Tartrazine to DNA . Based on these results, we believe that Tartrazine used in food products, cosmetics and pharmaceuticals can induce genetic damage, depending on the dose used.
The present study indicates that Tartrazine is not only genotoxic in whole blood cells under 3mM; but also its effects become more risky at higher doses because it can induce oxidative stress by formation of free radicals. Today, food colorants additives are randomly used in many foods. Therefore, it is necessary that people should be made aware about the hazardous effects of consuming Tartrazine.
Conflict of interest statement:
The authors declare that there are no conflicts of interest.
This research is financially sponsored by the <<CUD Commission Universitaire pour le Developpement>>. Also, we are grateful to Hakkou abdelkader for his help.
A part of this work was conducted in <<laboratory of biochemistry, faculty of medicine, university Mohammed 1er Oujda-Morocco>>.
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Acceptable Daily Intake (ADI); Single Cell Gel Electrophoresis (SCGE); Phosphate Buffred Saline (PBS); Olive Tail Moment (OTM); Percentage of DNA in the Tail (%DT);Tail Length (TL); Tail Moment (TM); reactive oxygen species (ROS).
(1) Imane Himri, (1) Faiza Souna, (2) Mohammed Aziz, (1) Abdelkader Hakkou, (1) Ennouamane Saalaoui
(1) Universite Mohamed ler, Faculte des Sciences, Laboratoire de Biochimie,
(2) Universite Mohamed ler, Faculte Des Sciences, Laboratoire de Physiologie Et Ethnopharmacologie,
Imane Himri, Universite Mohamed Ier, Faculte des Sciences, Laboratoire de Biochimie, E-mail: firstname.lastname@example.org Phone:+212629323982
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Himri, Imane; Souna, Faiza; Aziz, Mohammed; Hakkou, Abdelkader; Saalaoui, Ennouamane|
|Publication:||Advances in Environmental Biology|
|Date:||Oct 1, 2012|
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