Effects of certain food dyes on chromosomes of Allium cepa

Mutation Research, 223 (1989) 313-319 Elsevier

313

MUTGEN 01446

Effects of certain food dyes on chromosomes of A llium cepa Arati R o y c h o u d h u r y and Ashok K u m a r Girl * Centre of Advanced Studies in Cell and Chromosome Research, Department of Botany, University of Calcutta, Calcutta-700019 (India) (Received 19 July 1988) (Revision received 13 January 1989) (Accepted 17 February 1989)

Keywords: Food dyes; Allium cepa; Chromosomal aberrations; Micronuclei

Summary

The effects of 4 permitted food dyes, i.e., fast green FCF, indigo carmine, orange G and tartrazine, and the non-permitted dye metanil yellow on chromosomes of Allium cepa are reported. A significant increase in polyploid cells was observed in all cases. High doses of these dyes induced chromosome breaks and micronucleus formation. Although all dyes produced mitotic aberrations, metanil yellow and fast green FCF showed comparatively stronger clastogenic activity.

Despite sufficient data on the mutagenic and genotoxic effects of different environmental agents (Girl, 1986; Giri et al., 1984a; Hsu, 1982; Sharma and Talukder, 1987; Sugimura et al., 1981) little is known about the impact of dyes, especially those used in food stuffs. Combes and Haveland-Smith (1982) in their review urged the need for a detailed evaluation of the mutagenic and genotoxic effects of different permitted and non-permitted food dyes. The global production of food dyes is around 7500 tons per year, while India produces 150 tons (2% of the world production). In India, tartrazine accounts for 40% of the production and combined with the second largest colour, sunset yellow, forms

Correspondence: Dr. A. Roychoudhury, Centre of Advanced Studies in Cell and Chromosome Research, Department of Botany, University of Calcutta, Calcutta-700019 (India). * Present address: College of Pharmacy, University of Michigan, Ann Arbor, MI 48109 (U.S.A.).

over 70% of the net production; around 2.2 tons of indigo carmine are manufactured annually (National Institute of Nutrition, 1988, personal communication). Only a few authors have attempted to determine the mutagenic and clastogenic nature of these dyes in micro-organisms and in mammalian systems. The available information on the effects of these dyes on genetic systems is not sufficient and is often highly contradictory (see Table 1). Very little information is available about the effects of these permitted dyes on plant genetic systems. Therefore the present experiment was undertaken to study the chromosomal aberrations induced by 4 commonly used food dyes, fast green FCF, indigo carmine, orange G and tartrazine, on chromosomes of Allium cepa. Metanil yellow, a non-permitted dye, was also included since it is illicitly added to foods on a large scale in India (Khanna et al., 1973). It has often been found that certain substances show better reproducibility in plant genetic system than in mammalian bone marrow cells though the

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TABLE 1 MUTAGEN1C, CARCINOGENIC AND CLASTOGENIC EFFECTS OF CERTAIN DYES ON DIFFERENT TEST SYSTEMS Dye

Test system

Fast green FCF

Salmonella typhimurium Rat embryo cells Tumour formation in rats Bone marrow cells of mice

+ + +

CHO cells in vitro

+

Indigo carmine

Positive effect

Salmonella typhimurium

Saccharomyces cerevisiae

Metanil yellow

Orange G

Tartrazine

Rat embryo cells Bone marrow cells of mice

+ +

CHO cells in vitro Human lymphocytes

+

References Brown et al., 1978 Price et al., 1978 Nelson and Hagan, 1953; Hansen et al., 1966 Giri et al., 1986a, Gift et al., 1987 Au and Hsu, 1979 Auletta et al., 1977; Garner and Nutrnan, 1977; Brown et al., 1978 Sankaranarayanan and Murthy, 1979 Price et al., 1978 Giri et al., 1986a, Giri et al., 1987 Au and Hsu, 1979 Zhurkov, 1975 Szybalski, 1958 Muzzal and Cook, 1979 Massey and Raghuvanshi, 1986 Sinha and Sinha, 1986 Sinha and Sinha, 1986 Sinha and Sinha, 1986

Escherichia coli Salmonella typhimurium Root-tip cells of Hordeum vulgare Root-tip cells of Allium cepa Grasshopper spermatocytes Bone marrow cells of rat

+ + + +

Bone marrow cells of mice

+

Dominant lethal test in mice Human leukocytes

+ +

Giri et al., 1984b; Girl and Banerjee, 1986; Gift et al., 1986a, 1986b Prasad, 1986 Vaidaya and Godbole, 1979

+

Garner and Nutman, 1977 Landa et al., 1965 Au and Hsu, 1979

Salmonella typhimurium Drosophila melanogaster CHO cells in vitro Salmonella typhimurium Saccharomyces cerevisiae Rat embryo ceils V79 ceils in vitro Bone marrow cells of rat/mice

+ +

Human lymphocyte Human leukocyte

+ +

l a t t e r are a l s o a f f e c t e d . T h e e m p h a s i s a t p r e s e n t is principally o n a b a t t e r y of test s y s t e m s in w h i c h plant

Negative effect

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Auletta et al., 1977; Brown et al., 1978 Sankaranarayanan and Murthy, 1978 Price et al., 1978 Bhowmik and Gift, 1987 Girl and Das, 1987; Vaidaya and Godbole, 1978 Zhurkov, 1975 Vaidaya and Godbole, 1978

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Material and methods Chemicals

Fast green F C F (C.I. No. 42053), indigo carmine (C.I. No. 73015), metanil yellow (C.I. No. 13065), orange G (C.I. No. 16230), tartrazine (C.I. No. 19140), and ethyl methanesulfonate were dissolved in distilled water. All chemicals were obtained from Sigma Chemical Co., St. Louis, MO. Treatment and recovery schedules as suggested by Grant (1982) were used. Young and healthy bulbs of uniform size (20-25 g) of Allium cepa were selected and rooting was done in distilled water. When the roots had grown to 3-5 cm, bulbs were transferred to small containers with the test solutions. The concentrations of the dyes used were 200, 500 and 1000 ppm, since 200 p p m is the m a x i m u m level of dye permitted to be added to foods in India. Roots were treated for 6, 12 and 24 h for each dose. For each experiment, 10 bulbs were taken. In one set of experiments, root tips, after exposure to the respective test solutions, were pretreated in 0.05% aqueous solution of colchicine (Merck) for 1.5 h at 20 ° C, fixed and squashed according to the conventional aceto-orcein schedule. In another set, treated roots were allowed to recover in K n o p ' s solution for 24 h and then slides were prepared as mentioned earlier. One negative control with distilled water and a positive control with 0.25% ethyl methanesulfonate (EMS) for 6 h were set up. Mitotic aberrations were evaluated according to Chauhan et al. (1986) with minor modifications. 600 metaphase cells were scored for chromosomal aberrations from 6 root tips and 1000 cells for micronucleus formation from 10 root tips from different bulbs for each set of experiments. Data were analysed by the A N O V A test and then compared by means of Duncan's new multiple range test at the 95% confidence limit (Harter, 1960; Kotz and Johnson, 1982). Results Table 2 shows the incidence of chromosomal aberrations and polyploidy after exposure to different food dyes on root meristema~ic cells of Allium cepa. A significant increase in polyploid cells was observed with almost all the dyes. N o n e

of the concentrations of the 4 permitted dyes induced chromosome breakage at the shorter (6 and 12 h) treatment periods. However, fast green F C F and metanil yellow induced a few breaks only at 1000-ppm treatment during this period. 24 h of exposure to the dyes resulted in breaks in all cases, depending upon the concentrations used. In the case of micronucleus formation similar results were obtained, i.e., the highest frequency was noted with the 24-h treatment period. There was an increase in polyploidy with dose and time of treatment (Table 3). The frequency of breaks in cells allowed to recover was lower than that in cells scored immediately. However, all 3 concentrations of the 5 dyes used induced higher numbers of micronuclei in the recovered cells. EMS, the positive control, induced polyploidy, breaks and micronuclei in root-tip cells under the conditions used. Discussion In general, the cytogenetic effects of the dye samples observed were both time- and dosedependent. Although reports on the mutagenic and genotoxic effects of these dyes are contradictory (see Table 1), the present investigation and earlier observations (Girl et al., 1986a,b) indicate that these dyes exert effects on chromosomes and cell division. An increase in the incidence of polyploid cells was detected which may be due to an effect of the dyes on the SS- and SH-containing proteins of the spindle apparatus. No chromatid breaks or fragments were observed except in the positive control. Chromosomal breaks were also absent at the lower concentrations of dyes and the shorter treatment periods. These observations suggest that a comparatively longer treatment period, in which one cell cycle is completed (i.e., 24 h), m a y lead to considerable damage to chromosomes and chromatin material. These data fully support earlier work on bone marrow cells of rats and mice (Girl et al., 1986a,b; Girl and Das, 1987; Giri et al., 1987) where a significant increase in chromosomal aberrations and sister-chromatid exchanges was obtained following acute and chronic treatment with these dyes. The frequency of micronucleus formation also showed an increasing trend like that of the chromosomal breaks. Micro-

318

nuclei are the result of acentric chromosome fragments as well as failure of normal spindle function (Degrassi and Rizzoni, 1982; Schmid, 1982). Since acentric fragments were very rare in the present experiment, the micronucleus formation may be due to the disturbance of normal spindle function only. With regard to the relative genotoxic effects of these dyes on plant genetic systems, metanil yellow stands first followed by fast green FCF, orange G, tartrazine and indigo carmine (see Tables 2 and 3). Since these dyes are extensively used throughout the world, further studies are essential to evaluate their mutagenic, carcinogenic and genotoxic effects.

Acknowledgements The authors are grateful to Prof. A.K. Sharma, INSA Golden Jubilee Professor and Programme Coordinator, Centre of Advanced Studies in Cell and Chromosome Research for encouragement and facilities provided. Thanks are due to Prof. (Mrs.) Archana Sharma and Dr. Mandira Sarma for their encouragement and help. Financial assistance from the Department of Environment, Government of India is thankfully acknowledged.

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