The comet assay with 8 mouse organs: results with 39 currently used food additives

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Abstract

We determined the genotoxicity of 39 chemicals currently in use as food additives. They fell into six categories—dyes, color fixatives and preservatives, preservatives, antioxidants, fungicides, and sweeteners. We tested groups of four male ddY mice once orally with each additive at up to 0.5×LD50 or the limit dose (2000 mg/kg) and performed the comet assay on the glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow 3 and 24 h after treatment. Of all the additives, dyes were the most genotoxic. Amaranth, Allura Red, New Coccine, Tartrazine, Erythrosine, Phloxine, and Rose Bengal induced dose-related DNA damage in the glandular stomach, colon, and/or urinary bladder. All seven dyes induced DNA damage in the gastrointestinal organs at a low dose (10 or 100 mg/kg). Among them, Amaranth, Allura Red, New Coccine, and Tartrazine induced DNA damage in the colon at close to the acceptable daily intakes (ADIs). Two antioxidants (butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)), three fungicides (biphenyl, sodium o-phenylphenol, and thiabendazole), and four sweeteners (sodium cyclamate, saccharin, sodium saccharin, and sucralose) also induced DNA damage in gastrointestinal organs. Based on these results, we believe that more extensive assessment of food additives in current use is warranted.

Introduction

Food additives are used widely for various purposes, including preservation, coloring, and sweetening. Some food additives, however, have been prohibited from use because of their toxicity. AF-2, for example, was used as a food preservative in Japan from before 1965 until it was banned because of carcinogenicity in experimental animals [1].

Butter Yellow (p-dimethylaminoazobenzene), an azo compound, was listed for food use in the USA and de-listed in the same year; legislation prohibiting its use in Europe followed soon after, because it was implicated as a carcinogen in several animal species [2]. Many azo compounds, including Butter Yellow, are genotoxic in short-term tests and carcinogenic in laboratory animals [3], yet some of them are still being used. Saccharin and its salt are widely used sweeteners primarily because of their value to diabetes patients, and although sodium saccharin is carcinogenic in experimental animals, there is no clear evidence that it is in humans [4].

Although epidemiological studies of food additives are important in the assessment of toxicological risk to humans, they are difficult because exposure cannot be accurately assessed. Thus, risk assessment largely depends on laboratory toxicity studies. Currently, 337 synthesized and 488 naturally occurring food additives are permitted for use in Japan. Based on laboratory toxicity studies, some of them present a risk to our health. The usual way we evaluate chemical genotoxicity is by a battery of in vitro and in vivo genotoxicity tests [5]; the in vivo tests reflect the compound’s absorption, distribution, excretion, and metabolism. According to the data on food additive genotoxicity recently compiled by the Ministry of Health and Welfare (MHW) of Japan, some food additives currently used in Japan are positive in at least one in vitro genotoxicity study, and many have never been tested in vivo [6] in spite of the importance of in vivo genotoxicity data. The alkaline (pH >13) comet assay introduced by Singh et al. [7] is a rapid and sensitive procedure for quantitating DNA lesions in mammalian cells. It detects not only single strand breaks but also alkali-labile sites, DNA cross-linking, and incomplete excision repair sites [7], [8]. We have modified the assay by using nuclei obtained by homogenization instead cells obtained by enzyme treatment [9], and we have shown that the modified assay detects in vivo genotoxicity of various classes of chemicals [10], [11], [12]. The purpose of this study was to examine whether representative, widely used food additives induce DNA damage in mice.

Section snippets

Chemicals and animals

Table 1 lists tested food additives, their CAS numbers, and relevant data. Their chemical structures are shown in Fig. 1. Regular (GP-42) and low melting point (LGT) agarose were obtained from Nacalai Tesque (Kyoto) and diluted, respectively, to 1 and 2% in physiological saline. Male ddY mice were obtained from Japan SLC Co., Shizuoka, Japan, at 7 weeks of age and used after 1 week of acclimatization. They were fed commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) and tap

Results

An increase in DNA damage was indicated by an increase in DNA migration (Table 2). No death, morbidity, or clinical signs were observed after any treatment. Necropsy and histopathological examination of tissue sections stained by the hematoxylin–eosin and TUNEL methods revealed no treatment effect on any organ examined. Thus, any DNA damage observed was not likely to be due to general cytotoxicity (necrosis) and apoptosis.

Discussion

In vitro and in vivo genotoxicity tests detect compounds that induce genetic damage directly or indirectly by various mechanisms. Since, no single test is capable of detecting all genotoxic agents, the usual approach is to carry out a battery of tests [5]. A standard test battery includes the following: (1) a bacterial reverse mutation test; (2) a mammalian cell genotoxicity test to detect damage not detected in bacteria; and (3) an in vivo test so that additional factors (absorption,

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