Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using different protocols of the Salmonella assay

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Abstract

Textile dyes can enter the water ecosystem through wastewater discharges potentially exposing humans through the consumption of water and food. The commercial disperse dye product CI Disperse Blue 291 containing the aminoazobenzene 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyacetanilide (CAS registry no. 56548-64-2) was tested for mutagenic activity in the Salmonella assay. We used strains with different levels of nitroreductase and O-acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) that are relevant enzymes in the activation of nitrocompounds by the intestinal microflora. The commercial product tested also was mutagenic for TA1537, TA1538, TA98 and TA100. Presence of the pKM101 plasmid and the addition of S9 enhanced the mutagenic response. Specialized strains showed that both nitroreductase and O-acetyltransferase are important in activation of the product. The highest potency obtained was 240 revertants per microgram for YG1041 in the presence of S9. Besides being able to cause frameshift mutations (hisd3052), the dye was able to cause all types of base pair substitution with a preference for TA to AT; CG to TA and CG to AT changes. With these results clearly showing that the bacterial nitroreductase and O-acetyltransferase metabolites of this compound are mutagenic, there is a need to test this dye using in vivo systems to verify possible adverse effects of this product in mammalian tissues.

Introduction

Among the several types of dyes used worldwide, disperse dyes are frequently used for dyeing polyester, nylon, cellulose acetate and acrylic fibers (Aspland, 1997). Chemical types include azo, anthraquinone, nitro, methine and quinoline compounds (Gregory, 1990). Among them, the azo dyes are the most common; and 35% of the total US production of all dyes belong to the azo class (Chung and Stevens, 1993). According to Arslan et al. (1999), 20% of the dyes used for coloring activities might be discharged as wastewaters. Therefore, humans may be exposed through the consumption of water and food making the study of the toxicological effects of these compounds very important.

The term azo dye is applied to those synthetic organic colorants that have one or more azo groups (N = N). The genotoxicity of several azo dyes, especially aminoazobenzenes (AAB), have been studied (Miller and Miller, 1961, Yahagi et al., 1975, Degawa et al., 1978, Ashby et al., 1983, Esancy et al., 1990). Several dyes from this group showed mutagenic responses in Salmonella and mammalian assay systems; and it is clear that their potencies depend on the nature and position of the substituents with respect to both the aromatic rings and the amino nitrogen atom. For example, 3-methoxy-4-aminoazobenzene (3-OMe-AAB) is a potent hepatocarcinogen in rats and a strong mutagen in bacteria, while the 2-methoxy-4-aminoazobenzene (2-OMe-AAB) is apparently a non-carcinogen and an extremely weak mutagen in bacteria (Hashimoto et al., 1977). Because minor changes in the molecule can drastically modify their mutagenic activity and carcinogenic potential, it is important that each azo dye released into the market is adequately tested. Among a special group of aminoazobenzene dyes, that have nitro and halogenated substituents and are becoming widely used, only the dinitrobromoaminoazobenzene dye Disperse Blue 79 has been extensively evaluated for genotoxicity (Federal Register, 1989).

Azo dyes entering the human body through ingestion can be metabolized to aromatic amines by the azoreductases of intestinal microorganisms. If they are nitro azo dyes, they also can be metabolized by the nitroreductases produced by the same microorganisms (Chadwick et al., 1992). Mammalian enzymes in the liver and other organs can also catalyze the reductive cleavage of the azo bond and the nitroreduction of the nitro group; however, it has been shown that the intestinal microbial azoreductase and nitroreductase play a more important role in this type of metabolism (Chung et al., 1992). In both cases, if N-hydroxylamines are formed they are capable of causing DNA damage. If they are completely reduced to aromatic amines, they can be oxidized also to N-hydroxyderivatives by P450 enzymes. The N-hydroxy radicals can be further acetylated by enzymes such as the O-acetyltransferase generating electrophilic nitrenium ions capable of reacting with DNA to form adducts (Bartsch, 1981, Arlt et al., 2002). The generation of reactive oxygen species also seems to be involved in the genotoxicity of the O-hydroxy-aromatic amines (Sweeney et al., 1994).

The Salmonella strains used in the Ames test belong to the group of Enterobacteria that are able to produce nitroreductase and azoreductase. However, for Salmonella to produce the levels of azoreductase needed to produce sufficient amounts of mutagenic products, the test must be performed under reductive conditions, with the addition of flavin mononucleotide (FMN) and other cofactors (Prival and Mitchell, 1982, Prival et al., 1984). Some azo dyes only exhibit mutagenic activity when the azo bond undergoes reductive cleavage. Others, depending on their chemical structure, continue to lack mutagenic activity when tested under reductive conditions (Ashby et al., 1983). When the nitrogen–nitrogen double bond undergoes reductive cleavage, genotoxic aromatic amines may be produced, which can be more or less mutagenic than the parental dye. If the azo dye has one or more nitro substituents, the endogenous nitroreductase will reduce the nitro groups to hydroxylamines or aromatic amines. Conventional Salmonella strains like TA98, although able to reduce nitro compounds, have lower nitroreductase activity than their parental strain LT2. This reduced activity is due to the deletion of one major nitroreductase gene (Porwollik et al., 2001). Therefore, other strains of Salmonella were developed, lacking completely (e.g., TA98 NR and TA100 NR) or overproducing (e.g., YG1021, YG1026) this enzyme. Results of tests employing these strains are useful for understanding the role of the nitro group reduction in the mutagenicity of certain aromatic compounds. With the same objective, strains of Salmonella lacking the O-acetyltransferase activity (e.g. TA98 DNP6) or overproducing this enzyme (e.g. YG1024 and YG1029) are available. Additionally, strains overproducing both nitroreductase and O-acetyltransferase (e.g., YG1041 and YG1042) were developed. The combination of these strains can help elucidate the role of these enzymes in the mutagenicity of nitroazodyes. Since these bacterial enzymes play a very important role in human intestinal metabolism (Chadwick et al., 1992, Chung et al., 1992), the Salmonella assay seems to be a relevant tool for studying the mutagenicity of nitro-azo compounds when one wants to predict their possible effects on human health following oral ingestion.

Because azo disperse dyes themselves are hydrophobic compounds, they have a significant potential to sorb onto sediments in the aquatic environment, leaving little dye in the aqueous phase. However, this is not true for the commercial forms of disperse dyes, which contain dispersing agents that enhance their solubility in aquatic systems (Garrison and Hill, 1972, Maguire and Tkacz, 1991). Thus, it is important to study the toxicological effects of the dyes themselves and the corresponding commercial products. The CI Disperse Blue 291 is an azo dye used all around the world and no information about its metabolism or mutagenic activity was found in the literature.

Therefore the objective of this work was to study the genotoxic activity and the mutation spectra of the commercial product CI Disperse Blue 291 (a dinitrobromo aminoazobenzene dye frequently used worldwide) using different Salmonella strains under oxidative and reductive conditions. The aim is to understand the influence of the nitroreductase, azoreductase, cytochrome P450 enzymes, and O-acetyltransferase in the metabolic activation of this disperse dye.

Section snippets

Materials and methods

We tested the commercial product CI Disperse Blue 291 obtained from Clariant. This dye contains the dye compound 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyacetanilide (CAS registry no. 56548-64-2) (Fig. 1).

Results

We studied the mutagenicity of the commercial product CI Disperse Blue 291 (30–50% of the dye product) to evaluate its mutagenic potential and to better understand the compound’s mode of action and its activation pathways. We used several strains of Salmonella that are able to detect base pair substitution mutations (TA1535, TA100) and frameshift mutations (TA1537, TA1538, TA98) in the presence and absence of rat liver S9 (Fig. 2, Fig. 3). All the strains showed positive responses with the CI

Discussion

The commercial product studied in this work showed direct-acting mutagenic activity with all strains of Salmonella typhimurium tested, except for TA1535. According to the classification of Claxton et al. (1991), the potency of this product can be considered moderate (10–100 revertants/μg). The mutagenic potency of the commercial dye CI Disperse Blue (13.7 revertants per μg) is in the same order of magnitude of some purified aminoazobenzene dyes such as 3-methoxy-4-aminoazobenzene (M4AA),

Acknowledgments

The authors thank Julian Preston, Dennis Pagano, and Kathleen M. Schenck for helpful comments on this manuscript. This article does not necessarily reflect the views of CETESB nor the US EPA and no official endorsement should be inferred. The information in this document has been funded in part by the US Environmental Protection Agency and by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). It has been subjected to review by the National Health and Environmental Effects Research

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