CELLULAR & MOLECULAR BIOLOGY LETTERS
Volume 9, (2004) pp 107 – 122 http://www.cmbl.org.pl Received 26 August 2003 Accepted 27 January 2004
THE EVALUATION OF THE GENOTOXICITY OF TWO COMMONLY USED FOOD COLORS: QUINOLINE YELLOW (E 104) AND BRILLIANT BLACK BN (E 151) VIOLETTA K. MACIOSZEK* and ANDRZEJ K. KONONOWICZ** Department of Cytogenetics and Plant Molecular Biology, University of Łódź, S. Banacha 12/16, 90-237 Łódź, Poland Abstract: Additives, especially colors, are in widespread use in the food industry. With the exception of the quinolines, food colors are relatively weak mutagens and are certified as safe additives despite reports that some people have allergic reactions to them. The number of food additives is still on the increase, and research on their potential mutagenic/carcinogenic activity in vivo is very expensive. Using two different cellular model systems, human lymphocytes in vitro and Vicia faba root tip meristems of in vivo, we evaluated the potential cytological and genotoxic effects of two dyes: Quinoline Yellow (E 104) and Brilliant Black BN (E 151). Two relatively new, very sensitive and rapid tests – the micronucleus and Comet assays – were used in this study. The data provided in this paper showed the genotoxic effects of the two analyzed food colors, and confirmed the diagnostic value of the MN and Comet assays for screening potentially genotoxic substances. Key Words: Vicia faba, Comet Assay, DNA Damage, Food Colors, Genotoxicity, Human Lymphocytes, Micronucleus Assay
*Current address: Institute of Plant Biochemistry, Department of Stress and Developmental Biology, Weinberg 3, 06 120 Halle, Germany; e-mail:
[email protected] **Corresponding author: e-mail:
[email protected]. Abbreviations used: BB - Brilliant Black BN; BN - binucleated lymphocytes; DAPI 4’,6-diamidino-2-phenylindole; DEB - Diepoxybutane (1,3-butadiene diepoxide); EB ethidium bromide; LMP agarose - low melting point agarose; MH - maleic acid hydrazide; NDI - nuclear division index; NMP agarose - normal melting point agarose; QY - Quinoline Yellow.
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INTRODUCTION Additives, both natural and artificial (synthetic), are commonly used in the food industry. Each country has established its own legislation governing the procedures for the approval, application and acceptable content/dosage of food additives. In Poland, a detailed framework for the authorization of all food additives is provided by the Directive of the Ministry of Health and Social Care, March 31st, 1993, in “Authorized additives and technical contaminants in food and condiments” [1]. One of the major groups of food additives considered in the Polish legislature is colors. Colors and color additives are defined as dyes, pigments or substances that can impart color (including color stabilizers, color fixatives, and color retention agents) when added or applied to food, drugs, or cosmetics, and to certain medical devices such as contact lenses. Color additives are used in food for many reasons, including to offset color loss due to storage or processing of foods, to correct natural variations in food color, and finally, to enhance the appearance of certain foods to meet consumer demands and expectations; e.g. natural carotene (E 160a) or annatto (E 160b) are added to butter for this last reason. In the United States, EU member countries, and several other developed countries (e.g. Japan, Australia and Canada) colors permitted for use in foods are classified as certified or exempt from certification, but all must meet certain legal criteria for specifications and purity [2]. However, many of the colors and color adjuncts, which are permitted for use in European countries, have not been granted certification in the United States and Australia. Quinoline Yellow and Brilliant Black BN (Fig. 1) are synthetic dyes that are certified as safe and are permitted for use as food additives in Poland.
Fig. 1. Structural formula of Brilliant Black BN (A) and Quinoline Yellow (B).
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Quinoline Yellow (QY, E 104), which in Europe is used in jellies, caramels, processed seafood (e.g. caviar), lipsticks, hair products, colognes, liquors, and a wide range of medications, is banned in the USA, Australia and Norway. It has been proven that QY may cause dermatitis and allergic reactions in some individuals [3, 4]. Brilliant Black BN (BB, E 151) is used in jellies, brown sauces and blackcurrant cake mixes. This dye is banned in the United States, Australia and Western Europe (Denmark, Belgium, France, Germany, Switzerland, Sweden, Austria and Norway) based on reports of people suffering from allergic reactions to it [5]. The micronucleus (MN) and Comet assays are among the relatively new assays recommended for use in investigating potentially genotoxic substances, including foods and color additives. The former permits the detection of chromosomal breakage and is today recognized as a routine, preliminary, and relatively rapid method for assessing the potential mutagenicity of analyzed substances in a wide variety of model organisms [6, 7, 8]. MNs originate from chromosome fragments or whole chromosomes excluded from the main nucleus during cell division; therefore, the formation of micronuclei in analyzed cell populations indicates a clastogenic (causing structural chromosome aberrations) or aneugenic (disturbances of the mitotic spindle causing chromosome number aberrations) activity of the analyzed mutagen [9]. The Comet or the single cell gel electrophoresis assay is a sensitive, reliable, and rapid method for assessing DNA damage in individual eukaryotic cells. It permits the detection of DNA damage as subtle as 13] version of the comet assay was used. It is capable of detecting nuclear DNA single- and double-strand breaks, alkali labile sites in nuclear DNA, and delayed or incomplete excision repair sites in the DNA of individual cells [11]. Furthermore, under certain circumstances, the Comet assay can also detect DNA-DNA and DNA-protein cross-linking, which appears as a relatively reduced distance of DNA migration compared with concurrent controls [12]. The purpose of this study was to evaluate the potential genotoxic effects of two color additives, Quinoline Yellow and Brilliant Black BN, via the Comet and micronucleus assays, using human lymphocytes isolated from peripheral blood and a plant-based test – the root tip meristems of Vicia faba subsp. minor. In this paper, we provide data, which shows the genotoxic/mutagenic effects of Brilliant Black BN and Quinoline Yellow on human and plant cells. Moreover, the presented data confirms the usefulness and reliability of the MN and Comet assays for screening potential mutagens. MATERIALS AND METHODS Chemicals RPMI 1640 medium, NMP and LMP agarose, EDTA, cytochalasine B, DAPI and ethidium bromide, acridine orange and maleic acid hydrazide (MH, Cas no. 123-33-1) were purchased from SIGMA, Germany. Giemsa stain (GURR
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microscopy materials) was purchased from BDH Chemicals Ltd., England. Diepoxybutane (DEB, Cas no. 1464-53-5) and both the tested dyes, Brilliant Black BN and Quinoline Yellow, were purchased from ALDRICH. Brilliant Black BN (C.I. 28440, Cas no 2519-30-4, mw 867.69, synonyms: Black PN, C.I Food Black 1, BB) is an azo dye with the chemical formula 4acetamido-5-hydroxy-6-[7-sulfonato-4-(4-sulfonatophenylazo)-1-naphtylazo] naphthalene 1,7-disulfonate. Brilliant Black BN (BB) is described as the tetra sodium salt, but the calcium and the potassium salt are also available. The approved maximum amount of BB to be used in food is 100 mg l-1 (or kg-1) of the product weight (100 μg ml-1). Quinoline Yellow (C.I. 47005, Cas no. 8004-92-0, mw 477.38, synonyms: Quinoline Yellow-WS, C.I. Food Yellow 13, QY) is a quinophtalone dye prepared by sulfonating 2-(2-quinolyl) indian-1,3-dione. The dye essentially consists of sodium salts of a mixture of disulfonates (principally), monosulfonates and trisulfonates of the above compound. The maximum amount of QY permitted to be used in food is 200 mg l-1 (or kg-1) of the product weight (200 μg ml-1); there is no quantitative limit for liquors. In all our experiments, lymphocytes and 4-day-old seedlings of Vicia faba ssp. minor were treated with either Quinoline Yellow or Brilliant Black BN solutions at three concentrations: 8.67, 86.7, and 867 µg ml-1. The concentrations of the tested chemicals were based on the molecular weight of BB and the approved amount of each dye in food. Materials Human peripheral blood samples were obtained from the Blood Bank and Chemotherapy Center in Łódź. Seeds of Vicia faba (L.) subsp. minor were purchased from the Pałucka Plant Breeding Station (DANKO Group) in Sobiejuchy. Experimental procedures Human peripheral blood lymphocytes Heparinized peripheral venous blood samples were collected from healthy, nonsmoking donors and processed for the MN and Comet assays. A negative control (growth medium) was included in each experiment. DEB – a metabolite of 3,5butadiene, and a mutagenic and carcinogenic agent at the concentration 0.09 μg ml-1 – was used as a positive control to obtain a reference for the effects induced by the tested chemicals. Cytokinesis-block micronucleus assay (CBMN) Whole blood samples (72 h culture) were incubated in RPMI 1640 medium supplemented with 20% bovine serum, LF-7 (substance stimulating lymphocytes division), penicillin (100 U ml-1) and streptomycin (0.1 mg ml-1) at 37°C. Lymphocytes were treated with the tested chemicals for 24 h, centrifuged (1500 rpm, 5 min) and resuspended in fresh medium. Cytochalasine B (6 μg ml-1) was added 44 h after LF-7 stimulation, according to the modified method of Fenech
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[13]. After a total of 72 h incubation, the cultures were harvested and treated with hypotonic solution (0.075M KCl) to lyse red blood cells. For the fixation, cold, freshly prepared fixative, a mixture of methanol:acetic acid (3:1, v/v) was used. Lymphocytes were spread onto microscopic slides and stained with acridine orange (2 μg ml-1) or Giemsa dye. A nuclear division index (NDI) was calculated according to Eastmond and Tucker [14] as follows: NDI = (M1 + 2 x M2 +3 x M3 + 4 x M4)/N, where M1-M4 respectively represent the number of cells with one to four nuclei and N is the total number of cells scored. The frequency of MN induction was evaluated by scoring 4000 binucleated lymphocytes (1000 cells per slide) per sample. Comet assay The Comet assay was performed on isolated lymphocytes under alkaline conditions following the procedure of Horváthová at al. [15] with minor modifications. All the steps of the procedure were conducted in dim light. Lymphocytes were isolated in Histopaque-1077 gradient [16] and treated for 1 h with the control solution and tested chemicals at 37°C. Following isolation, a mixture of 50 μl of lymphocytes suspension and 150 μl of 0.75% LMP agarose was placed onto a microscopic slide coated with 1% NMP agarose solution. Prior to electrophoresis, the slides were subjected for 1 h to alkaline (pH 10) lysis (2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, 0.5% SLS, 1% Triton X-100), at 4°C, followed by equilibration for 30 min at 10°C in alkaline (pH>13.0) electrophoretic solution (300 mM NaOH, 1 mM EDTA). Electrophoresis was performed for 20 min at 25 V (0.5 V cm-1) and 300 mA. After electrophoresis, the slides were neutralized for 15 min with Tris buffer, pH 7.5, at room temperature, air-dried, and kept in a refrigerator until analysis. For microscopic analyses, the slides were immersed in distilled water for 20 min, stained with 100 μl of DAPI (1 μg ml-1), and incubated for 12-16 h in a humid chamber, at 4°C. Two hundreds cells per sample were routinely analyzed with a Labotop-2 epifluorescence microscope with an EFD-3 objective (magnif. X20), 365/10 nm excitation filter and 400 nm barrier filter. The KOMET 3.1. image analysis system (Kinetic Imaging, U.K.) was employed to measure the tail moment of the comet images. Comet cells were sorted into 4 classes (0-III) according to the degree of DNA damage. Vicia faba ssp. minor Seed germination and treatment conditions were according to the protocol recommended by the International Program of Chemical Safety (IPSC) [17]. After soaking for 12-18 h in tap water, seeds were germinated in sterile, wet Perlite. Four-day-old seedlings with 2.5-3 cm long primary roots were used for all our experiments. Seedlings grown on sterile distilled water were the control. To evaluate the effect of the tested chemicals, seedlings treated for 2 h with MH at a concentration of 11.2 μg ml-1 were used as a positive control. For analysis of the potential mutagenic effect of the tested colors by micronucleus and Comet
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assays, seedlings were exposed for 2 h in darkness to different concentrations of either BB or QY. Micronucleus assay Following treatment with either control marker chemicals or color additives and 20 and 26 h recovery time, seedlings were harvested and root tips were fixed at 4°C in Carnoy’s solution (ethanol:glacial acetic acid, 3:1, v/v). Root tip meristems were stained according to Feulgen’s procedure: 1 h incubation with 4 N HCl at room temperature followed by 40 min staining in Schiff’s reagent. Squash preparations were made using the dry-ice technique. Slides were rinsed with distilled water, air-dried, and analyzed via light microscopy. The mitotic index was calculated as a percentage of the dividing cells and the MN frequency was determined by scoring 4000 cells (1000 cells per slide) for each experiment. Comet assay Immediately after treatment with MH or the dye solutions, cell nuclei were isolated from root tip meristems according to Van’t Hof’s protocol [18], modified by Macioszek and Maszewski [19] to meet the Comet assay requirements. The quality of the isolated nuclei was assessed by fluorescence microscope analysis of nuclei preparations stained with acridine orange (2 μg ml-1). The Comet assay was performed on root tip meristems of V. faba following the procedure applied to lymphocytes, except for the duration of the following steps: 40 min equilibration in electrophoretic buffer and 15 min electrophoresis. For each experiment, 200 comets per sample were analyzed (50 comets scored per replicate slide). Statistical analysis The mean and standard deviation values were calculated for the micronucleus assay, and the standard error values for the Comet assay. The statistical significance of the differences and correlation with the control were determined by the Student’s t-test for two independent probes using Statistica 5.1. software. RESULTS Human peripheral blood lymphocytes Diepoxybutane (DEB), a well-known environmental mutagen, was used in the micronucleus and Comet assays as a positive control and a marker of mutagenicity for both assays. DEB is a bifunctional alkylating agent that binds to the nucleophilic sites of DNA and forms cross-binding sites between the adjacent guanidines [20]. It is well documented that DEB induces the formation of micronuclei [21], chromosomal aberrations [22], and sister chromatid exchanges [23] in human lymphocytes in vitro. The capacity of BB and QY to induce MN in human lymphocyte cultures is illustrated in Tab. 1 and Fig. 2. A-C. Although a significantly (p
