The azo dye Disperse Orange 1 induces DNA damage and cytotoxic effects but does not cause ecotoxic effects in Daphnia similis and Vibrio fischeri

Abstract

Azo dyes constitute the largest group of colorants used in industry and can pass through municipal waste water plants nearly unchanged due to their resistance to aerobic treatment, which potentially exposes humans and local biota to adverse effects. Unfortunately, little is known about their environmental fate. Under anaerobic conditions, some azo dyes are cleaved by microorganisms forming potentially carcinogenic aromatic amines. In the present study, the azo dye Disperse Orange 1, widely used in textile dyeing, was tested using the comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and Microtox^® assays. The human hepatoma cell line (HepG2) was used in the comet assay and for cell viability. In the mutagenicity assay, Salmonella typhimurium strains with different levels of nitroreductase and o-acetyltransferase were used. The dye showed genotoxic effects with respect to HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0 μg/mL. In the mutagenicity assay, greater responses were obtained with the strains TA98 and YG1041, suggesting that this compound mainly induces frameshift mutations. Moreover, the mutagenicity was greatly enhanced with the strains overproducing nitroreductase and o-acetyltransferase, showing the importance of these enzymes in the mutagenicity of this dye. In addition, the compound induced apoptosis after 72 h in contact with the HepG2 cells. No toxic effects were observed for either D. similis or Vibrio fischeri.

Introduction

Synthetic dyes are extensively used in textile dyeing, paper printing and photography, and in the pharmaceutical, food, cosmetics and petroleum products industries [1]. According to their chemical structure, dyes may be classified into several groups such as azo, anthraquinone, benzodifuranone, quinophthalones and others [2]. Of these, the azo compounds, characterized by the presence of one or more azo groups, are the most used [3], [4].

Currently, there are at least 3000 azo dyes in use [5], representing about 60% of the dyes applied throughout the world [6]. In the textile industry, azo dyes are used for coloring wool, cotton, polyester and various other substrates [5].

Pollution of the environment with these compounds causes, apart from visual pollution, changes in the biological cycles mainly affecting processes of photosynthesis [7]. Moreover, non-ionic azo dyes are considered potentially toxic [8] not only because of the dye itself, but also because azo compounds can generate carcinogenic/mutagenic products, such as aromatic amines due to the metabolism of intestinal microflora and/or mammalian azo reduction and chemical reduction [1], [5], [9], [10], [11], [12], [13], [14], [15]. McCann et al. [11] reported a 90% correlation between carcinogenicity and mutagenicity for 61 aromatic amines and azo dyes tested using the Salmonella/microsome mutagenicity test [11].

It is important to point out that each dye should be tested individually because the potency of these compounds is strongly dependent 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, whereas 2-methoxy-4-aminoazobenzene (2-OMe-AAB) is apparently a non-carcinogen and an extremely weak mutagen under similar conditions [16].

The amount of azo dyes produced in the world per year is estimated to be over 10,000 tons [17], with about 2000 types of color available just for the textile industry [18]. About 12% of this amount is lost to waste streams, and of this percentage, 20% is released into the environment [19], since the traditional wastewater treatment technologies have been shown to be ineffective in removing these dyes because of the chemical stability of these pollutants [20]. In this context, it is important to evaluate the azo dyes not only from the human point of view, but also considering the ecotoxic effects.

Considering the lack of studies evaluating the toxic effects of Disperse Orange 1 (Fig. 1), in the present work the genotoxic, mutagenic, cytotoxic and ecotoxic effects of this chemical were evaluated using the comet, Salmonella/microsome mutagenicity, cell viability and Daphnia similis and Microtox^® assays, respectively. In addition, the Salmonella/microsome mutagenicity assay was also used to study the role of the enzymes nitroreductase, o-acetyltransferase, and cytochrome P450 in the metabolic activation of this dye.