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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 dying, was tested using the Comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and MicrotoxÂ® 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 1.0 and 2.0Âµg/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 hours in contact with the HepG2 cells. No toxic effects were observed for either Daphnia similis or Vibrio fisheri.
Keywords: Disperse Orange 1, DNA damage, Ames test, Vibrio fisheri, Daphnia similis
Synthetic dyes are extensively used in textile dyeing, paper printing and photography, and in the pharmaceutical, food, cosmetics and petroleum products industries . According to their chemical structure, dyes may be classified into several groups such as azo, anthraquinone, benzodifuranone, quinophthalones and others . 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 3,000 azo dyes in use , representing about 60% of the dyes applied throughout the world .Â In the textile industry, azo dyes are used for coloring wool, cotton, polyester and various other substrates .
Pollution of the environment with these compounds causes, apart from visual pollution, changes in the biological cycles mainly affecting processes of photosynthesis . Moreover, non-ionic azo dyes are considered potentially toxic  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 [1,5,6, 8-14]. McCann et al.  reported a 90% correlation between carcinogenicity and mutagenicity for 61 aromatic amines and azo dyes tested using the Salmonella/microsome mutagenicity test .
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 .
The amount of azo dyes produced in the world per year is estimated to be over 10,000 tons , with about 2,000 types of color available just for the textile industry .Â About 12% of this amount is lost to waste streams, and of this percentage, 20% is released into the environment , since the traditional wastewater treatment technologies have been shown to be ineffective in removing these dyes because of the chemical stability of these pollutants . 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 MicrotoxÂ® assays, respectively. In addition, the Salmonella/microsome mutagenicity assay was also used to study the role of the enzymes nitroreductase, o-acetyltransferase, azoreductase and cytochrome P450 in the metabolic activation of this dye.
2. Material and Methods
The dye Disperse Orange 1 (4-(4-nitrofenilazo)difenilamina); CAS registry no 2581-69-3 ) purchased from Sigma (St Louis, USA) (Fig. 1), was analyzed.
Fig.1. Chemical structure of the dye Disperse Orange 1.
2.2 Comet assay
The Comet assay is used to detect genomic damage which sometimes can turn into mutation. It has been indicated as a method able to detect very small changes in DNA structure, such as repair activities, the manner of its packing and its integrity [16,17]. In the present work, the comet assay was used with HepG2 cells derived from human hepatoma . This cell line was also used to evaluate the induction of cell death by the azo dye.
The HepG2 cells were obtained from the American Type Culture Collection, N° HB 8065. The cell line was cultured for 24 hours in Dulbecco's medium (Sigma, St Louis, USA) supplemented with 10% bovine fetal serum, 100 IU/mL penicillin G, 100 mg/mL streptomycin and 1 Âµg/mL amphotericin, all from Gibco (Grand Island, USA) at 370C, flushed with 5% CO2 in air.
The comet assay was carried out according to Tice et al.  and the concentrations were chosen based on a previous study performed by Chequer et al., in which they used micronucleous assay in HepG2 cells to evaluated the same dye . Briefly, after 4h of exposure of the HepG2 cells (2 x 105 cell/well) to the dye (0.05Âµg/mL, 0.1Âµg/mL, 0.2Âµg/mL, 0.4Âµg/mL, 1.0Âµg/mL, 2.0Âµg/mL and 4.0Âµg/mL dissolved in PBS) or to PBS in different Petri-dishes (60 x 15 mm, Greiner), the cells were trypsinized (0.1%, 4 min), suspended, homogenized in 1 mL of medium and centrifuged (10 min, 212 g). Subsequently 100 ÂµL of low melting agarose (Gibco, Grand Island, USA) was added to the cell suspension. They were then transferred to agarose-coated slides and lysed (2.5 M NaCl; 100 mM EDTA; 10 mM Tris-HCl, pH 10; 1% Triton X-100 and 10 % DMSO) for 24 hours. The slides were placed in the electrophoresis solution (200 mM EDTA plus 10 M NaOH) for 20 min and submitted to electrophoresis (300 mA and 1.0 V/cm, 20 min). The slides were then transferred to the neutralization solution (4.85% Tris-HCl, pH 7.5) for 20 min, stained with ethidium bromide (10 Âµg/mL - Sigma, St Louis, USA) and examined under a fluorescence microscope (Nikon, model 027012). All experiments were carried out in triplicate and the length of DNA migration measured in 50 cells per slide (100 cells per treatment). The cells were scored visually into five classes according to tail intensity (from undamaged, 0, to maximum damaged, 4). The damage index (DI) was based on the length of migration in order to evaluate the overall damage caused by the compound, and to compare it with the positive (Benzo[a]pyrene, 12.5 Âµg/mL, Sigma, St Louis, USA) and negative (PBS) controls. The DI was calculated as follows:
DI = n1 + 2n2 + 3n3 + 4n4,
Where n1 represents the number of cells with level 1 damage; n2 the number of cells with level 2 damage; n3 the number of cells with level 3 damage; and n4 the number of cells with level 4 damage.
In addition, trypan blue was used to verify cell viability and was> 90% in all experiments.
The statistical analysis was performed using the analysis of variance (ANOVA), followed by Dunnett's Multiple Comparison Test. The differences were considered significant when p < 0.05.
2.3 Mutagenicity testing
The Salmonella/microsome mutagenicity assay or Ames test is widely used to detect chemical mutagens and potential carcinogens [19-23, 39,40]. In the present work, this assay was also used to determine the role of metabolization on the mutagenic activity of Disperse Orange 1. For this purpose the traditional strains TA98, able to detect frameshift mutations (hisD3052, rfa, Î”bio, Î”uvrB, pKM101), TA100, able to detect base pair substitution (hisG46, rfa, Î”bio, Î”uvrB, pKM101), and the strains YG1041 and YG1042 (derived from TA98 and TA100, respectively), both of which overproduce nitroreductase and O-acetyltransferase, were employed . The combination of these strains can help elucidate the contribution of these enzymes in the mutagenicity of nitroazo dyes.
For the Salmonella mutagenicity assay, the standard pre incubation procedure with and without exogenous metabolic activation (S9), according to Maron and Ames  and Mortelmans and Zeiger  was employed. The dose of Disperse Orange 1 ranged from 0.05 to 3000 Âµg/plate based on preliminary assays (data not shown). Briefly, 100ÂµL overnight cultures of each strain of Salmonella typhimurium (TA98, YG1041, TA100, YG1042) (about 109 cells/mL), 500ÂµL of 0.2M sodium phosphate buffer or S9 mix and 100ÂµL of the dye concentration were incubated at 37Â°C for 30 min without shaking. After incubation, 2mL of molten top agar was added, the mixture poured onto a minimal agar plate, and the plates incubated at 37Â°C for 66 h. Colonies were counted by hand and the background was carefully evaluated.
Dimethylsulfoxide (DMSO) (Merck, Darmstadt, Germany) was used as the negative control. For TA98 and TA100, the positive controls were 4-nitroquinoline-oxide - 4NQO (5 Âµg/plate, Sigma, St Louis, USA) and 2-aminoanthracene - 2AA (25 Âµg/plate, Aldrich, Seelze, Germany). For YG1041, 4-nitro-O-phenylenediamine - 4NOP (100 Âµg/plate, Aldrich, Seelze, Germany) and 2-aminoanthracene - 2AA (0.625 Âµg/plate Aldrich, Seelze, German). For YG1042 the positive controls were 100 Âµg/plate of 2-nitrofluorene - 2NF (Aldrich, Seelze, Germany) and 2-aminoanthracene - 2AA (25 Âµg/plate, Aldrich, Seelze, Germany). The dye and the positive controls were dissolved in DMSO.
Metabolic activation was provided by Aroclor 1254 induced Sprague Dawley rat liver S9 mix (MolTox, Boone, USA), which was prepared at a concentration of 4% (v/v) according to Maron and Ames .
The samples were considered positive when a significant ANOVA and dose response was obtained from the Bernstein model . We have chosen this model because the standard method for analyzing Salmonella mutagenicity data based on fold-increases may be too insensitive for Salmonella strains with relatively high reversion frequencies, such as TA100, TA97, and TA102 . The results were expressed as the mean of number of revertants per plate Â± standard deviation.
All the experiments were performed in triplicate.
2.4 Cell viability (Cytotoxicity assay)
Cell viability was determined by the propidium iodide/annexin V assay using flow cytometry and HepG2 cells . This assay assesses the toxicity for the cell and at the same time discriminates between necrotic and apoptotic cell death. The use of annexin V in this cytotoxicity assay offered the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity, and permitted measurement of the kinetics of apoptotic death in relation to the cell cycle . Concentrations of the dye were chosen based on the higher response obtained from Salmonella mutagenicity assay
The cells (1 x 105 cells/well) were exposed to Disperse Orange 1 (2.5Âµg/mL, 5.0Âµg/mL and 10.0Âµg/mL, dissolved in PBS) for 24-72 h at 37Â°C and 5% CO2. The cells were then harvested by trypsinization, washed twice with PBS, re-suspended in the working solution of 5 Î¼g/mL propidium iodide and 0.25 Î¼g/mL annexin V (Becton Dickinson, San Diego, USA), incubated for 15 min at 37 Â°C, and analyzed with a FACSCanto flow cytometer (Becton Dickinson, San Jose, USA) using Diva software. Approximately 105 cells were analyzed after each treatment. Dulbecco's medium was used as the negative control and 100mM tert-butylhydroperoxide (tBOOH) as the positive control.
The statistical analysis was carried out using one-way ANOVA, assuming equality of variance with the Dunnet post hoc test for pair-wise comparison. Results with P < 0.05 were considered statistically significant.
2.5. Aquatic Acute Toxicity assay
2.5.1 Aquatic organism cultures
Daphnia similis, obtained from the CETESB Ecotoxicology Laboratory, were maintained at 20 Â± 2°C, with light intensity of 1000 lux, under a 16:8 h (light/dark) photoperiod. The Daphnias were fed daily with the green algae Selenastrum capricornutum and a mixture of trout food and yeast .
2.5.2 Daphnia similis toxicity assay
The genus Daphnia is comprised of microcrustaceans extensively used in tests to evaluate the acute and chronic toxicity of chemical agents and effluents. Several authors have evaluated Daphnia similis as a test species for use in ecotoxicological assays [29-31], showing that this specie can be used in ecotoxicological assays as well as Daphnia magna .
The experiment was performed with 6 different doses (0.001 to 10 mg/L) based on cell viability assay so that the higher concentration in both assays was 10Âµg/mL (equal 10mg/L) with 4 replicates. For each replicate five young organisms (6 - 24 h old) were exposed to the dye for 48 h in a 10 mL glass tube. After the 48-h exposure period, the immobilized Daphnia were counted. The test was considered valid if the immobilization rate was less than 10% in the negative control group . The results were statistically analyzed using the Spearman trimmed method, which has good statistical properties, is easy to use, and is recommended for accurate and precise calculation of LC50 values and their 95% confidence interval end points .
2.5.3 Microtox bioassay
MicrotoxÂ® is an acute toxicity test based on inhibition of the bioluminescence of the marine bacterium Vibrio fischeri NRRL B 11 177 . Since the bioluminescence of Vibrio fischeri is directly linked to its respiratory activity, it provides a good indicator of metabolic activity and has been found to be well correlated with several in vivo toxicity tests [34-37].
The acute toxicity tests were performed using the Microtox procedure with the Microbics Model 500 analyzer (AZUR Environmental, Carlsbab, CA) and the procedures outlined in the Microtox Users Manual using the "81.9% Basic Test" protocol .
Freeze-dried luminescent bacteria, Vibrio fischeri, were reconstituted and exposed to a series of nine diluted dye solutions, osmotically adjusted with 22% sodium chloride (NaCl). The resulting decrease in bioluminescence was measured after 5 and 15 min at a constant temperature of 15Â°C. The data for fifteen minutes are here reported, and the toxicity was measured as percent inhibition of light emission of the dye treated aliquot according to Lin and Chao , corrected for loss of light in the control as follows:
% inhibition = 100 x (I0 x It) / I0,
where I0 = initial bacterial luminescence and It = luminescence after introduction of the dye into the bacterial suspension. All the Microtox data were recorded and analyzed by MicrotoxOmni on-line software, which is able to calculate Percent when only one usable gamma is calculated from data [Azur Environmental, 1998], and the results expressed as percent inhibition of light emission . A 2% NaCl solution was used as the negative control and zinc sulfate heptahydrate (ZnSO4.7H2O) 100mg/L as the positive control.
3.1 Comet assay
The results of the dose response experiment are shown in Fig. 2. At the lower concentrations of Disperse Orange 1 (0.05 and 0.1 Âµg/mL), no genotoxic effect was observed for the HepG2 cells. However, a significant increase in the migration of DNA fragments was detected for all the other concentrations tested (0.2, 0.4, 1.0, 2.0 and 4.0 Âµg/mL).
The cell viability in all the dye concentrations was higher than 90% (data not shown).
3.2 Mutagenicity assay
Disperse Orange 1 showed mutagenic activity for strains TA98, YG1041, TA100 and YG1042. The mutagenicity was greatly increased using strain YG1041 and S9 decreased the effect observed in all cases. Under the conditions tested, the dye tested negative for TA100 with S9 (Table 1).
3.3 Cell viability (Cytotoxicity assay)
Fig. 3 shows cell death by apoptosis caused by Disperse Orange 1 after 72 hours in contact with the HepG2 cells. In the period from 24 to 48 hours of incubation, the dye did not induce cell death (data not shown).
3.4 Aquatic Acute Toxicity assay
The dye Disperse Orange 1 did not cause a significant toxic effect on Dapnhia similis. Table 2 shows the results obtained for this experiment, based on the percent immobilization of these microorganisms as induced by the dye.
3.4.2 Microtox bioassay
Similar to Daphnia, the dye did not cause significant toxicity with respect to Vibrio fischeri. Table 2 shows the inhibition of light emission by Vibrio fischeri in the presence of the dye, studied for 15 minutes. A tendency for an increase in % inhibition of light emission was observed, but the effect was not sufficient to calculate the EC50 values. EC50 for positive control was 3.045mg/L (95% confidence range: 1.952 to 4.750).
The comet assay is used routinely for the detection of DNA damage . Disperse Orange 1 showed genotoxic effects on the HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0Âµg/mL, but it appears that at the highest concentration tested (4.0Âµg/mL) the damage response had saturated despite cell viability being around 90%, which might be due to cytotoxic effects. During evaluation of the slides, an increase in completely fragmented cells was observedÂ at the higher doses, which might suggest that the cytotoxic effects were starting to occur and the Trypan Blue assay was not sensitivity enough to detect it. Chequer et al.,  observed a similar phenomenon for the micronucleus assay of azo dyes, using the same cell line.
This cytotoxic effect was confirmed by the results obtained in the cell death assay. The results showed that Disperse Orange 1 induced apoptosis after 72 hours of exposure at doses above 2.5 Âµg/mL. It is important to point out that in the comet assay the cell exposure was 4 hours, whilst in this assay, the exposure period was 72 hours.
The dye Disperse Orange 1 was mutagenic for the strains TA98, YG1041, TA100 and YG1042 (Table 1). This suggests that the dye is able to induce both base pair substitutions and frameshift mutations. However, comparing the mutagenic potencies detected with the different strains tested, the hypothesis was raised that this dye induces mainly frameshift mutations.
Moreover, the mutagenic potency was about 545 times higher for the strain YG1041 than for TA98 (Table 1), clearly demonstrating the importance of nitroreduction and acetylation in the mutagenicity of this dye, since YG1041 is able to produce high levels of nitroreductase and O-acetyltransferase. The mechanism of mutagenicity of these products could be nitroreduction by nitroreductases forming N-hydroxylamines that could induce the DNA damage . The role of nitroreductase in the metabolism and bioactivation of 1-nitropyrene (1-NP) by intestinal flora has been well established. Nitroreduction can occur as a two-electron transfer (nitro - nitroso - hydroxylamine - amino) or through one-electron transfers, forming an anion free radical intermediate . Considering that the response detected for YG1041 was much higher than for TA98, the product of the nitroreductase (probably a hydroxylamine) may have been acetylated by the O-acetyltransferase, generating very reactive species. According to a study carried out by Watanabe et al.  using the Salmonella strain YG1024, O-acetyltransferase is not specific for oxygen and can act on the amine group leading to the production of reactive species.
With all the strains, the addition of exogenous metabolic activation (S9 mixture) decreased the mutagenicity of the dye. The action of the P450 isoforms probably generated more stable products, less likely to interact with DNA. According to Chung and Cerniglia , sulfonation, carboxylation, deamination or substitution of an ethyl alcohol or acetyl group for the hydrogen in the amino group will lead to a decrease or diminishing of mutagenic activity, which could explain the decrease in mutagenic activity after treatment with S9.
The present results show that Disperse Orange 1 is a mutagenic dye. Other authors have shown that azo dyes can cause harmful effects, mainly for the genetic material. The genotoxicity of 24 azo compounds selected from the IARC (International Agency for Research on Cancer), groups 2A, 2B, and 3 was determined by the comet assay in eight mouse organs. For 17 azo compounds, the assay was positive for at least one organ . The azo dye Sudan I showed a genotoxic effect with HepG2 cells using the comet and micronucleous assays .
Disperse Orange 1 did not cause acute toxicity to both Daphnia similis or Vibrio fischeri, what can be attributed to its chemical structure, since some dyes have shown to be toxic to Daphnia magna. In a study performed by Bae and Freeman , they found that copper-complexed dyes and Direct Blue 218 were very toxic to daphnids. The authors suggest that heavy metals like copper molecules inside dye structure play an important role for the evaluation of aquatic toxicity of dye solutions. In other study, Wang et al.  evaluated reactive dyestuffs and found that the most toxic azo dye was Ambifix yellow VRNL, and the least toxic one was Procion blue HERD.
The dye Disperse Orange 1 induces damage in DNA, mainly frameshift mutations. The enzymes nitroreductase, N-acetyltransferase and O-cetyltransferase were shown to be very important in the mutagenicity of this dye. In addition, this compound causes cell death by apoptosis and DNA breakage in the human cell HepG2. No toxic effects were detected for Daphnia similis and Vibrio fisheri, but it is important to add that other ecotoxic tests should be done in order to evaluate the impact of this dye on the biota. Thus the results of this study emphasize the need for the development of non mutagenic dyes and for investment in new methods for the treatment of effluents, in order to prevent the possibly deleterious effects of these compounds for both humans and aquatic organisms.