Mutagenesis Advance Access originally published online on April 20, 2005
Mutagenesis 2005 20(3):229-233; doi:10.1093/mutage/gei030
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Evaluation of photo-mutagenicity and photo-cytotoxicity of food coloring agents
Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530, Japan
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Abstract |
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Pigments extracted from natural products are widely used for food coloration in Japan. An investigation concerning the photo-mutagenicity and photo-carcinogenicity of frequently used colorants in Japan was performed. Colorants examined were from Laccifer lacca (lac-color), Coccus cacti (cochineal-color), Carthamus tinctorius (carthamus yellow), Gardenia augusta (gardenia yellow and gardenia blue), Monascus anka and Monascus purpureus (monascus red), the skin of Vitis vinifera and Vitis labrusca (grape-skin color), Tamarindus indica (tamarind brown) and Beta vulgaris (beet red). No significant increase in bacterial mutation was found when Salmonella typhimurium TA98, TA100 and TA102 were simultaneously treated with colorants and subjected to UVA irradiation for 30 min. When colorant solutions were subjected to UVA irradiation for 4 h, irradiated solutions containing lac-color became slightly mutagenic toward S.typhimurium TA98 without metabolic activation. A decrease in cell survival resulted when WTK-1 cells were subjected to UVA irradiation for 60 min in the presence of purpurin at 1 mg/ml. Delayed cytotoxicity was also observed following 24 h incubation in fresh medium of samples that were subjected to UVA irradiation for 60 min in the presence of colorant (carthamus yellow, grape-skin color, gardenia blue, cochineal-color, monascus red or purpurin).
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The photo-genotoxic potential of drugs and cosmetic products has recently been the focus of intense research (1
). UV-absorbing compounds, such as sunscreen preparations and antibacterial agents have been examined for photomutagenic activity (2–5). 8-Methoxypsoralen, chlorpromazine and antibacterial agents, clinafloxacin and related quinolone, were established as photomutagens. Several pigments, which consist of UV and visible light-absorbing compounds, are derived from natural substances and are widely used in Japan as food colorants (6). Lac-color is a red-orange pigment derived from Laccifer lacca Kerr, cochineal-color is obtained from Coccus cacti L., carthamus yellow from safflower (Carthamus tinctorius L.), gardenia yellow and gardenia blue from gardenia (Gardenia augusta Merr), monascus red from Monascus anka and Mornascus purpureus, beet red from beet (Beta vulgaris L.), grape-skin color from grape (Vitis vinifera L. and Vitis labrusca L.) and tamarind brown from Tamarindus indica. L. Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a purple pigment that is present in the root of Japanese madder (Rubia tinctorum L.) (7). Monascus red showed no mutagenicity toward Salmonella typhimurium TA98 or TA100 up to 5 mg/plate in the Ames test (8). Purpurin is non-mutagenic in the majority of Ames tester strains (7,9), and is only weakly mutagenic in TA1537 (9). Other colorants mentioned above were reported as non-mutagenic in the Ames test in the absence of irradiation (10). Additionally, monascus pigments, purpurin, chlorophyllin and curcumin were reported to inhibit the mutagenicity of heterocyclic amines and other carcinogens (8,11–13).
Solar UV light is a causal factor in the case of skin cancer in humans, and UVA represents the predominant source of radiant energy in sunlight (14). UVA can reach the subcutaneous area of the skin containing blood vessels. We suspected that ingested and absorbed colorants in blood vessels might function as photo-sensitizers of sunlight UVA. We also suspected that photo-activated colorants or photo-generated products from irradiated colorants might act as mutagenic and cytotoxic agents. To date, the photo-mutagenic potential of colorants has not been assessed. Photo-genotoxicity testing and hazard identification of these colorants in the presence of UVA irradiation may constitute a prudent step toward the evaluation of food safety.
This report is mainly concerned with photo-mutagenicity tests involving bacterial assays and photo-cytotoxicity tests with human-derived cultured cells in the presence of the aforementioned colorants and UVA irradiation.
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Materials and methods |
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Materials
Lac-color and cochineal-color were obtained from San-Ei Gen F.F.I., Inc. (Osaka, Japan). Carthamus yellow, gardenia yellow, gardenia blue, monascus red, beet red, grape-skin color and tamarind brown were gifts from Yaegaki Bio-Industry, Inc. (Himeji, Japan). Purpurin [81-54-9], a purple pigment purified from the root of R.tinctorum (madder), was purchased form Tokyo Kasei (Tokyo, Japan). The lac and cochineal colorants were dissolved in 1% Na2CO3; monascus red, beet red and purpurin were dissolved in 10% ethanol, while carthamus yellow, gardenia blue, gardenia yellow, grape-skin color and tamarind brown were dissolved in water at 100 or 200 mg/ml as stock solutions. The stock solutions were diluted with phosphate-buffered saline (PBS) immediately before use. Irradiation was performed in a chamber (BC-2200, remodeled for irradiation, Taitec, Japan) at 37°C with four black-light bulbs (FL15BL-B, Matsushita, Japan) that emit light between 300 and 400 nm. The emission was filtered using a soft glass to eliminate most of the UVB light (shorter than 320 nm). Light intensity was measured using a photo-intensity meter (Ultraviolet products, USA). S.typhimurium TA98, TA100 and TA102 (15
) were gifts from Dr Bruce N.Ames (University of California, USA). The S9 fraction was prepared from the liver of Sprague–Dawley rats (male, 6 weeks old) induced by phenobarbital and 5,6-benzoflavone. The human-derived lymphoblastoid cell line WTK-1 was kindly supplied by Dr Akihiro Wakata (Yamanouchi Pharmaceutical Co., Ltd, Tokyo, Japan). UV-visible absorption profiles of colorants were measured using a U-2001 spectrophotometer (HITACHI Ltd, Tokyo, Japan).
Procedures for Ames test under simultaneous irradiation with UVA
Overnight cultures (40 ml) of S.typhimurium TA98, TA100 or TA102 were centrifuged at 3000 r.p.m. (2000 g) at 4°C for 10 min, and bacterial precipitates were re-suspended in 12 ml of ice-cold PBS. A bacterial suspension (0.5 ml) and a solution of colorant (0.5 ml) were placed into wells of a microtiter tray (24 wells) and mixed. The final concentration of colorant was 0.001, 0.01, 0.1, 1 or 10 mg/ml. As a negative control, a bacterial suspension (0.5 ml) and a solvent (PBS, 0.5 ml) were placed into the well of the tray and mixed. Mixtures were irradiated at 25°C for 30 min with continuous shaking. The dose of UVA was 1.25 ± 0.21 J/cm2. Irradiated samples (0.3 ml) were taken and poured onto agar plates with 2 ml of soft agar. To generate positive controls for the investigation of mutagenicity, 2-nitrofluorene (50 nmol/0.1 ml PBS) and 4-nitroquinoline-1-oxide (50 nmol/0.1 ml PBS) were mixed with 0.1 ml of bacterial suspension and poured into plates containing soft agar (15). Positive controls for the photomutagenic aspect of this study were obtained by mixing 0.5 ml of 8-methoxypsolarene (0.0025 mM) with 0.1 ml of S.typhimurium TA102 suspension and then irradiating the mixture for 2 min (3). Irradiated samples (0.3 ml) were taken and poured onto agar plates with 2 ml of soft agar. Plates were incubated for 48 h at 37°C and revertant colonies were scoured. Experiments were performed in triplicate.
Procedure for the Ames test for irradiated colorants
A solution of colorant (0.001, 0.01, 0.1, 1 or 10 mg/ml) in PBS was placed into a well of a microtiter tray (24 wells) and irradiated with UVA for 4 h (10 ± 1.7 J/cm2) at 37°C with continuous mixing. Following irradiation, samples (0.1 ml each) were assayed using the preincubation method (16) of the Ames test (15), with or without metabolic activation. A solution of lac-color (10 mg/ml), cochineal-color (10 mg/ml), carthamus yellow (10 mg/ml) or tamarind brown (0.1 or 10 mg/ml) in PBS was irradiated for 0.5, 1, 2 or 4 h (0.636 mW/cm2) at 37°C, and samples (0.1 ml each) were assayed without metabolic activation. For the non-irradiated control, a solution of colorant (10 mg/ml) in PBS was assayed using the Ames test with or without metabolic activation. 2-Aminoanthracene, 2-nitrofluorene and 4-nitroquinoline-1-oxide were used as positive controls. Experiments were performed in duplicate or in quadruplicate and average values of the results were determined.
Procedure for cell exposure to UVA in the presence of colorants
WTK-1 cells were grown in RPMI 1640 medium containing 10% horse serum at 37°C in 5% CO2 as described in the literature (17,18). Cells were collected and re-suspended in PBS immediately before use. The colorants, at final concentrations of 0, 0.01, 0.1 or 1 mg/ml, were mixed with cells (2 x 106 cells/ml) in PBS (3 ml) and allowed to stand for 15 min. Cytotoxicity can also be measured by counting cells before and after irradiation (19). Small samples (0.1 ml) were taken from the mixture, and living and dead cells were scored in quadruplicate using trypan-blue staining. Cell survival (percent) as shown in Table II was obtained as follows: Number of living cells x 100/Total number of living and dead cells. Mixtures were then exposed to UVA for 1 h (3.00 ± 0.77 J/cm2) at 37°C. Cell survival was then determined. Irradiated mixtures were centrifuged at 900 r.p.m. (180 g) for 5 min to separate cells from the colorant solution. Cells were re-suspended in medium and cultured at 37°C in 5% CO2. Cytotoxicity was measured by counting the cells 24 h following treatment.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The UV-visible absorption profiles of the colorants are shown in Figure 1. The A360 of lac-color, cochineal-color, carthamus yellow, tamarind brown, monascus red, the grape-skin color, gardenia yellow, gardenia blue, beet red and purpurin at 1 mg/ml in PBS were calculated as 25.9, 18.3, 2.13, 2.28, 2.92, 1.35, 0.29, 0.34, 0.169, 1.80, respectively. When S.typhimurium TA98, TA100 or TA102 were treated with colorants (up to 10 mg/ml) under simultaneous irradiation with UVA for 30 min, no significant increase in bacterial mutation was found compared with those treated with UVA in the absence of colorant (data not shown). This suggested that no mutagenic products were formed and that no mutagenic interaction occurred between the pigments and DNA through energy transfer or chemical reaction during irradiation. This also suggested that no short-lived photo-activated reactants caused mutation in the treated bacteria.
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The effect of UVA exposure for periods >30 min with respect to the formation of mutagenic substances and mutagenic photo-decomposed products derived from the colorants during irradiation was then determined. Following 4 h of UVA irradiation, an increase of revertant colonies was observed in the samples of lac-color, cochineal-color, carthamus yellow and tamarind brown toward S.typhimurium TA98 without metabolic activation, as shown by the underlined values in Table I. Slight decrease of background lawn was observed in the assay plates of tamarind brown (10 mg/ml, UVA 4 h). A dose-dependent pattern was found for lac-color in terms of photo-derived mutagenicity. An irradiation-time dependency was also observed in the sample of lac-color (Table I), but not observed in those of cochineal-color, carthamus yellow or tamarind brown (data not shown). No mutagenicity was found in colorant solutions in the absence of UVA (Table I). It suggested that photoproducts were formed during irradiation in the presence of lac-color, and that these induced frameshift mutations in the bacteria. The lack of an apparent correlation between dose (of colorants or UVA) and mutagenicity for cochineal-color, carthamus yellow and tamarind brown may indicate that the observed increase of revertant colonies was the result of an artifactual effect. Although the concentration of colorants used was much higher than that expected in vivo, further investigations concerning the photo-mutagenicity of the colorant would be worthwhile and could lead to the delineation of the mechanisms at play.
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Cochineal-color and monascus red at 1 mg/ml displayed cytotoxic activity against WTK-1 cells with or without UVA (Table II). Simultaneous UVA irradiation with 1 mg/ml of purpurin induced cytotoxicity against WTK-1 cells (Table II). An increased incidence of urinary bladder tumors was observed when male F344 rats were fed purpurin at 1% of the diet for 520 days (20
). The observed photo-toxicity of purpurin at the highest dose may indicate an additional risk for humans following exposure to purpurin as a food additive. Delayed cytotoxicity was also found following incubation for 24 h in fresh medium of samples that had been subjected to UVA irradiation for 1 h in the presence of carthamus yellow (0.01–1 mg/ml), grape-skin color (1 mg/ml), gardenia blue (0.01 mg/ml), purpurin (1 mg/ml), cochineal-color (0.01–1 mg/ml) or monascus red (0.1 mg/ml) (Table II). Apoptosis was observed in WTK-1 cells following ionizing radiation, which caused chromosome damage and triggered a delayed type of apoptosis (21,22). Treatment of cells with colorants and UVA may trigger delayed cytotoxicity. The lack of a dose-dependent response in terms of photo-cytotoxicity in the case of carthamus yellow and gardenia pigments may have resulted owing to product saturation generated from the dose of UVA used in these experiments. This investigation dealt with fundamental research concerning photo-mutagenicity and cellular responses to UV-induced toxic effects of colorants. Evaluation of the photo-mutagenicity and photo-cytotoxicity of food colorants may contribute to a greater understanding and assessment of these substances with respect to food safety. The photo-cytotoxicity test using human-derived cultured cells could prove to be a convenient method for the successful screening of photo-toxic chemicals.
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Acknowledgments |
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This work was supported by the San-Ei Gen Foundation for Food Chemical Research.
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Notes |
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* To whom correspondence should be addressed. Tel: +81 86 251 7947; Fax: +81 86 251 7926; Email: arimoto{at}cc.okayama-u.ac.jp
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References |
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Received on January 18, 2005; revised on March 6, 2005; accepted on March 22, 2005.
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