INTRODUCTION:

Many foods today do not display their natural colour (Ollikainen, 1982). Food producers commonly select, modify and standardize the colours that we see and come to associate with specific foods. Consumers are conditioned to expect food of certain colours and reject any deviation from their expectations (Amerine et al., 1965). Additionally, numerous sociological, psychological, technical and economic factors have also influenced the extensive use of food colours (Downham and Collins, 2000).

In India, implementation of the Prevention of Food Adulteration Act, 1954 (PFAA, 1954) was a bold step to check use of unauthorized harmful/toxic substances in food. The Prevention of Food Adulteration Act (1954) permits only eight synthetic dyes to be used as food colourants. These include Tartrazine, Sunset yellow, Carmoisine, Ponceau 4R, Erythrosine, Brilliant blue, Indigo carmine and Fast green. These colours are permitted to be used within the limit specified for the particular item. For all the dyes, the maximum limit has been fixed at 0.20 gm/kg of food. Moreover, it is mandatory to declare the addition of the artificial colour on the label of the food product (PFA, 2003). Synthetic permitted food colours are available in the market but being more costly; traders take advantage of lackadaisical approach of the law enforcing authorities and substitute it with cheap and easily available non-permitted food dyes (Giri, 1991). Non-permitted food colours have been toxicologically classified under the category C II and C III by the Joint FAO/ WHO Expert Committee on Food Additives which implies that the available toxicological data is inadequate for safety evaluation but indicates the possibility of harmful effects and for which virtually no information on long term toxicity is available. This has been the main ground for non inclusion of these dyes in the prescribed list of food colours (Singh et al., 1988). In actual practice non-permitted food dyes like auramine, methanol yellow, lead chromate, rhodamine, sudan-iii and iv, orange ii and malachite green pose serious health hazards, as they are mutagenic and potential carcinogenic and are being used as food colours in the market (Ashfag et al., 2002). In the central and sub urban areas of Kolkat few foods, manufactured by unorganized private sector and small vendors, did contain colours in higher concentration than the permitted range (Biswas et al., 1994). Studies conducted by the National Institute of Nutrition (NIN), Hyderabad have shown the use of adulterants such as Lathyrussativus and non-permitted colours in urban street foods (Bhat et al., 1994).

In spite of the stringent regulatory provisions, illegal use of unauthorized food dyes continues in India because of their low cost, ready availability and efficacy, and the likely threat it poses to human health have been a matter of continued concern (Khanna et al., 1973, 1975, 1978, 1980, 1985, 1986; Prasad and Rastogi, 1982; Wess and Archer, 1982; Singh et al., 1987; Khanna and Das, 1991; Roy and Chakraborti, 1991; Babu and Shenolikar, 1995; Dixit et al., 1995; Bhat and Mathur, 1998; Mathur, 2000 and Ashfaq and Masud, 2002). There is paucity of the literature on the toxicity caused by the use of non-permitted food colourants and no systematic studies have so far been made to study their toxicity. Thus, there is an urgent need to carry out a systematic study to evaluate the toxic behaviour of the most commonly used non-permitted food colours so that consumer awareness can be regenerate regarding the toxic effects of consuming such food dyes because consumers have an important role to play in ensuring their safety.

MATERIALS AND METHODS:

Adult male Swiss albino mice (Mus musculus) having 24±3 gm body weight and age of 4-5 weeks were selected for the present investigation. The mice were procured from Chaudhary Charan Singh (CCS) Agriculture University, Hissar. These mice were kept in clean and aseptic polypropylene cages and acclimatized to the laboratory conditions for two weeks before any experimental procedures. Animals were fed with standard mice pellet diet supplied by M/S Ashirwaad Industries, Chandigarh and water was given ad libitum.

SOURCE OF THE NON-PERMITTED FOOD: DYE: The food dye Lead chromate manufactured by Central Drug House (P) Ltd Bombay, was procured from the local market.

EXPERIMENTAL PROTOCOL:

Healthy adult male Swiss albino mice, weighing 24±3 gm and age of 4-5 weeks were used in the present experiments. The animals were divided into five groups as follows:

Group I:

This group comprising of 10 mice served as control dose not received any treatment. 5 mice were sacrificed on 21^st day and the remaining 5 mice were autopsied on 42^nd day. They were offered only the pellets of standard mice feed in the manner similar to the experimental groups.

Group II and III:

The animals of this group were fed with low doses of the food dye for 21 days as short term and for 42 days as long term experiments.

Group IV and V:

The animals of this group were fed with high doses of the food dye for 21 days as short term and for 42 days as long term experiments. After 21 and 42 days of the treatment, the animals were weighed and autopsied.

Table 1: Experimental design for Lead chromate

Groups	No. of animals used	Dosage of Lead chromate (gm/kg b.wt.)
Group I(Control)	10f	Nil
Group II*	5	0.50 gm/kg b. wt. of Lead chromate
Group III**	5	0.50 gm/kg b. wt. of Lead chromate
Group IV *	5	1.0 gm/kg b. wt. of Lead chromate
Group V**	5	1.0 gm/kg b. wt. of Lead chromate

* Duration of 21 days = short term experiment;

**Duration of 42 days = long term experiment

RESULTS:

The metaphasic cells of the bone marrow of the control animals revealed 40 diploid numbers of the chromosomes. A few cells showed negligible chromosomal aberrations (Table 1 and Plate 1). A dose and time dependent increase was recorded in the number of aberrant cells which was found to be highly significant (p≤0.001) when compared with the control (Table 2 and Figure 2). The observed data (Table 2) revealed that the low dose of Lead chromate caused aberrations like centric ring, chromatid break, chromosome break, dicentrics, exchange figures and accentric fragments, with more frequency of accentric fragments and dicentrics (Fig. 2, 3). No polyploidy and pulverization were recorded in the metaphasic plates of low dose treated animals, however, aneuploidy was seen in some of the metaphasic cells of the animals which received low doses of Lead chromate for longer duration (Fig. 3).


Figure 1	Figure 2


Figure 3	Figure 4

The administration of high dose of Lead chromate caused chromosomal aberrations like dicentrics, centric ring, chromatid break, chromosome break, exchange figures and accentric fragments but their frequency was found to be more than the low dose treated animals (Fig. 4,5). Maximum chromosomal aberrations and aneuploidy were seen in the animals which received higher dose for longer duration. (Fig. 6, 7). The most frequently observed chromosomal aberrations were centric rings and accentric fragments.


Figure 5	Figure 6

Figure 7

DISCUSSION:

In the present study, the food dye lead chromate was found to exert chromotoxic effects. The chromosomal aberrations seen in the metaphasic cell plates of the bone marrow of all the experimental animals consisted mainly of centric rings, chromosome breaks, chromatid breaks, dicentrics, exchange figures and accentric fragments. A marked increase was recorded in the percentage of chromosomal aberrations in the dye treated animals. In addition, mice fed with Lead chromate for longer duration also revealed aneuploidy in some of the metaphasic plates, which finds support from the findings of Holmes et al. (2006), who reported aneuploidy in human lung cells exposed to Lead chromate. Similar chromosomal aberrations have also been reported by several other workers. Vaidya and Godbole (1978) reported that Metanil yellow has the ability to break human leucocyte chromosome in vitro, causing chromatid and isochromatid breaks. Prasad and Rastogi (1982) reported chromosomal abnormalities like breaks, gaps, centric fusion, fragments, translocation, deletion, ring etc. in albino mice after feeding of the common food colour Orange II. Wantabe et al. (1985) reported that Lead chromate in vivo gives rise to chromosomal damages in mice. Giri et al. (1986 a) studied the effects of Indigo carmine, Metanil yellow and Fast green FCF in bone marrow cells of mice and reported that all these dyes caused chromosomal aberrations. Further, Giri et al. (1986 b) also reported chromatid exchange induced by Metanil yellow and nitrite singly or in combination in the mice. Rastogi et al. (1991) have reported that even low concentration of Metanil yellow and Orange II in human lymphoblast cells were sufficient to induce mutation rates. In 1992, Giri et al. observed a significant increase in sister chromatid exchange and mitotic index in mice after in-vivo exposure of Green S. Dhatchayani et al. (2002) studied the genotoxicity of Indigo carmine in mammalian system and reported a dose dependent increase in chromosomal aberrations like ploidy, gaps and centric fusion. Alves et al. (2003) studied the mutagenicity of Annatto in mouse bone marrow cells and they reported increased frequency of micronucleated cells at highest concentrations. Srivastava et al. (2004) reported carcinogenesis, mutagenesis, chromosomal fractures and teratogenecity in various fish species and certain mammals treated with Malachite green. Turkoglu (2007) observed abnormal mitotic figures such as anaphase bridges, C-mitosis, micronuclei lagging, stickiness, breaks and unequal distribution in root tips of Allium cepa L. treated with food preservatives. The increased chromosomal aberrations in the experimental groups indicate stronger genotoxic activity of the food dye lead chromate. The most common chromosomal aberrations observed were exchange figures, centric rings and accentric fragments. The exchange figures observed in the present investigation indicates increased chromosomal recombination in the dividing bone marrow cells as a result of chemical stress caused by dye toxicity. The chromosomal aberrations like accentric fragments, centric rings and pulverization indicate direct toxic effect of this food dye on the chromosomes of the dividing cells. The chromosomal aberrations like chromosome and chromatid breaks observed in the present investigation indicates toxic action of the food dyes on DNA (Grant, 1978 and Chauhanand Sundararaman, 1990).

Similar findings were also reported by Hasegawa et al. (1984) in Chinese Hamster cells treated with Sorbic acid and its salt and Gomurgen (2005) on root tips Allium cepa L. treated with Potassium metabisulphite and Potassium nitrate. The abnormality like aneuploidy indicates disturbance in the formation of spindle fibres due to dye toxicity which resulted in unequal distribution of the chromosomes during cell division. Similar abnormalities were also observed by Turkoglu (2007) in root tips of Allium cepa L. treated with Sodium benzoate. In the present investigation aneuploidy was reported only in Lead chromate treated mice. The possible mechanism for Lead chromate induced carcinogenesis is through centrosome dysfunction, leading to the induction of aneuploidy (Holmes et al., 2006). Epel (1963); Jain and Andsorbhoy (1988); Njagi and Gopalan (1982) and Rencuzogullari et al. (2001) reported that decrease in ATP level inhibits the process of DNA synthesis. Chromosomal aberrations are believed to result from unrepaired and misrepaired DNA lesions, and inhibitors of DNA repair can induce an enhancement of the DNA damage caused by many kinds of compounds (Kihlman and Natarajan, 1984; Noviello et al., 1994). Thus, it may be possible that the chemical stress caused by the food dyes somehow inhibited the production of ATP in the mitochondria which in turn inhibited DNA synthesis, as a result of which the cells could not perform the repair processes.

CONCLUSIONS:

Food adulteration had deep roots in our society and it can be uprooted only when people are aware of toxic effects of adulteration. Therefore, in view of ensuring the safety of consumers the author suggests that in the interest of good health, one should reject artificially coloured rice, pulses, sweets, spices, green vegetables and instead use naturally coloured foods to brighten up meals. Further, there is a need to generate consumer awareness regarding the toxic effects of food dyes. Because if the consumers demand food which is not laden with undesirable colours and chemicals only then it will go a long way in solving the problem or else our future generations are certainly going to pay the price for our careless, negligent and easygoing approach to the issue.

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Received on 05.01.2017 Modified on 28.02.2017

Research J. Science and Tech. 2017; 9(2): 234-238.

DOI: 10.5958/2349-2988.2017.00042.0