Azo dyes constitute the largest group of colorants used in industry and can go through through municipal waste H2O workss about unchanged due to their opposition to aerobic intervention, which potentially exposes worlds and local biology to inauspicious effects. Unfortunately, small is known about their environmental destiny. Under anaerobiotic conditions, some azo dyes are cleaved by micro-organisms organizing potentially carcinogenic aromatic aminoalkanes. In the present survey, the azo dye Disperse Orange 1, widely used in fabric death, was tested utilizing the Comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and MicrotoxA® checks. The human hepatocarcinoma cell line ( HepG2 ) was used in the comet check and for cell viability. In the mutagenicity check, Salmonella typhimurium strains with different degrees of nitroreductase and O-acetyltransferase were used. The dye showed genotoxic effects with regard to HepG2 cells at concentrations of 1.0 and 2.0Aµg/mL. In the mutagenicity check, greater responses were obtained with the strains TA98 and YG1041, proposing that this compound chiefly induces frameshift mutants. Furthermore, the mutagenicity was greatly enhanced with the strains overproducing nitroreductase and O-acetyltransferase, demoing the importance of these enzymes in the mutagenicity of this dye. In add-on, the compound induced programmed cell death 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 harm, Ames trial, Vibrio fisheri, Daphnia similis
Man-made dyes are extensively used in fabric dyeing, paper printing and picture taking, and in the pharmaceutical, nutrient, cosmetics and crude oil merchandises industries [ 1 ] . Harmonizing to their chemical construction, 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 ] .
Presently, there are at least 3,000 azo dyes in usage [ 5 ] , stand foring about 60 % of the dyes applied throughout the universe [ 6 ] .A In the fabric industry, azo dyes are used for colourising wool, cotton, polyester and assorted other substrates [ 5 ] .
Pollution of the environment with these compounds causes, apart from ocular pollution, alterations in the biological rhythms chiefly impacting procedures of photosynthesis [ 6 ] . Furthermore, non-ionic azo dyes are considered potentially toxic [ 7 ] non merely because of the dye itself, but besides because azo compounds can bring forth carcinogenic/mutagenic merchandises, such as aromatic aminoalkanes due to the metamorphosis of enteric microflora and/or mammalian azo decrease and chemical decrease [ 1,5,6, 8-14 ] . McCann et Al. [ 10 ] reported a 90 % correlativity between carcinogenicity and mutagenicity for 61 aromatic aminoalkanes and azo dyes tested utilizing the Salmonella/microsome mutagenicity trial [ 10 ] .
It is of import to indicate out that each dye should be tested separately because the authority of these compounds is strongly dependent on the nature and place of the substituents with regard to both the aromatic rings and the amino N atom. For illustration, 3-methoxy-4-aminoazobenzene ( 3-OMe-AAB ) is a powerful hepatocarcinogen in rats and a strong mutagen in bacteriums, whereas 2-methoxy-4-aminoazobenzene ( 2-OMe-AAB ) is seemingly a non-carcinogen and an highly weak mutagen under similar conditions [ 15 ] .
The sum of azo dyes produced in the universe per twelvemonth is estimated to be over 10,000 dozenss [ 25 ] , with about 2,000 types of colour available merely for the fabric industry [ 26 ] .A About 12 % of this sum is lost to blow watercourses, and of this per centum, 20 % is released into the environment [ 27 ] , since the traditional effluent intervention engineerings have been shown to be uneffective in taking these dyes because of the chemical stableness of these pollutants [ 28 ] . In this context, it is of import to measure the azo dyes non merely from the human point of position, but besides sing the ecotoxic effects.
Sing the deficiency of surveies measuring 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 utilizing the Comet, Salmonella/microsome mutagenicity, cell viability and Daphnia similis and MicrotoxA® checks, severally. In add-on, the Salmonella/microsome mutagenicity check was besides used to analyze the function 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 construction of the dye Disperse Orange 1.
2.2 Comet check
The Comet check is used to observe genomic harm which sometimes can turn into mutant. It has been indicated as a method able to observe really little alterations in Deoxyribonucleic acid construction, such as fix activities, the mode of its wadding and its unity [ 16,17 ] . In the present work, the comet check was used with HepG2 cells derived from human hepatocarcinoma [ 18 ] . This cell line was besides used to measure the initiation of cell decease 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 % bovid foetal serum, 100 IU/mL penicillin G, 100 mg/mL streptomycin and 1 Aµg/mL amphotericin, all from Gibco ( Grand Island, USA ) at 370C, flushed with 5 % CO2 in air.
The comet check was carried out harmonizing to Tice et Al. [ 38 ] and the concentrations were chosen based on a old survey performed by Chequer et Al. [ 11 ] , in which they used micronucleous check in HepG2 cells to measure the same dye. Briefly, after 4h of exposure of the HepG2 cells ( 2 x 105 cell/well ) to the dye ( 0.05Aµg/mL, 0.1Aµg/mL, 0.2Aµg/mL, 0.4Aµg/mL, 1.0Aµg/mL, 2.0Aµg/mL and 4.0Aµg/mL dissolved in PBS ) or to PBS in different Petri-dishes ( 60 x 15 millimeters, Greiner ) , the cells were trypsinized ( 0.1 % , 4 min ) , suspended, homogenized in 1 milliliter of medium and centrifuged ( 10 min, 212 g ) . Subsequently 100 AµL of low runing agarose ( Gibco, Grand Island, USA ) was added to the cell suspension. They were so transferred to agarose-coated slides and lysed ( 2.5 M NaCl ; 100 millimeter EDTA ; 10 millimeter Tris-HCl, pH 10 ; 1 % Triton X-100 and 10 % DMSO ) for 24 hours. The slides were placed in the cataphoresis solution ( 200 millimeter EDTA plus 10 M NaOH ) for 20 min and submitted to electrophoresis ( 300 ma and 1.0 V/cm, 20 min ) . The slides were so transferred to the neutralisation solution ( 4.85 % Tris-HCl, pH 7.5 ) for 20 min, stained with ethidium bromide ( 10 Aµg/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 intervention ) . The cells were scored visually into five categories harmonizing to tail strength ( from undamaged, 0, to maximum damaged, 4 ) . The harm index ( DI ) was based on the length of migration in order to measure the overall harm caused by the compound, and to compare it with the positive ( Benzo [ a ] pyrene, 12.5 Aµg/mL, Sigma, St Louis, USA ) and negative ( PBS ) controls. The DI was calculated as follows:
DI = n1 + 2n2 + 3n3 + 4n4,
Where n1 represents the figure of cells with degree 1 harm ; n2 the figure of cells with degree 2 harm ; n3 the figure of cells with degree 3 harm ; and n4 the figure of cells with degree 4 harm.
In add-on, trypan blue was used to verify cell viability and was & gt ; 90 % in all experiments.
The statistical analysis was performed utilizing the analysis of discrepancy ( ANOVA ) , followed by Dunnett ‘s Multiple Comparison Test. The differences were considered important when P & lt ; 0.05.
2.3 Mutagenicity proving
The Salmonella/microsome mutagenicity assay or Ames trial is widely used to observe chemical mutagens and possible carcinogens [ 19-23, 39,40 ] . In the present work, this check was besides used to find the function of metabolization on the mutagenic activity of Disperse Orange 1. For this intent the traditional strains TA98, able to observe frameshift mutants ( hisD3052, rfa, I”bio, I”uvrB, pKM101 ) , TA100, able to observe base brace permutation ( hisG46, rfa, I”bio, I”uvrB, pKM101 ) , and the strains YG1041 and YG1042 ( derived from TA98 and TA100, severally ) , both of which overproduce nitroreductase and O-acetyltransferase, were employed [ 24 ] . The combination of these strains can assist clarify the part of these enzymes in the mutagenicity of nitroazo dyes.
For the Salmonella mutagenicity check, the standard pre incubation process with and without exogenic metabolic activation ( S9 ) , harmonizing to Maron and Ames [ 39 ] and Mortelmans and Zeiger [ 40 ] was employed. The dosage of Disperse Orange 1 ranged from 0.05 to 3000 Aµg/plate based on preliminary checks ( informations non shown ) . Briefly, 100AµL nightlong civilizations of each strain of Salmonella typhimurium ( TA98, YG1041, TA100, YG1042 ) ( about 109 cells/mL ) , 500AµL of 0.2M Na phosphate buffer or S9 mix and 100AµL of the dye concentration were incubated at 37A°C for 30 min without agitating. After incubation, 2mL of molten top agar was added, the mixture poured onto a minimum agar home base, and the home bases incubated at 37A°C for 66 h. Colonies were counted by manus 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 Aµg/plate, Sigma, St Louis, USA ) and 2-aminoanthracene – 2AA ( 25 Aµg/plate, Aldrich, Seelze, Germany ) . For YG1041, 4-nitro-O-phenylenediamine – 4NOP ( 100 Aµg/plate, Aldrich, Seelze, Germany ) and 2-aminoanthracene – 2AA ( 0.625 Aµg/plate Aldrich, Seelze, German ) . For YG1042 the positive controls were 100 Aµg/plate of 2-nitrofluorene – 2NF ( Aldrich, Seelze, Germany ) and 2-aminoanthracene – 2AA ( 25 Aµg/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 ) harmonizing to Maron and Ames [ 39 ] .
The samples were considered positive when a important ANOVA and dose response was obtained from the Bernstein theoretical account [ 41 ] . We have chosen this theoretical account because the standard method for analysing Salmonella mutagenicity informations based on fold-increases may be excessively insensitive for Salmonella strains with comparatively high reversion frequences, such as TA100, TA97, and TA102 [ 422 ] . The consequences were expressed as the mean of figure of revertants per home base A± criterion divergence.
All the experiments were performed in triplicate.
2.4 Cell viability ( Cytotoxicity assay )
Cell viability was determined by the propidium iodide/annexin V assay utilizing flow cytometry and HepG2 cells [ 42 ] . This check assesses the toxicity for the cell and at the same clip discriminates between necrotic and apoptotic cell decease. The usage of annexin V in this cytotoxicity assay offered the possibility of observing early stages of programmed cell death before the loss of cell membrane unity, and permitted measuring of the dynamicss of apoptotic decease in relation to the cell rhythm [ 42 ] . Concentrations of the dye were chosen based on the higher response obtained from Salmonella mutagenicity check
The cells ( 1 x 105 cells/well ) were exposed to Disperse Orange 1 ( 2.5Aµg/mL, 5.0Aµg/mL and 10.0Aµg/mL, dissolved in PBS ) for 24-72 H at 37A°C and 5 % CO2. The cells were so harvested by trypsinization, washed twice with PBS, re-suspended in the on the job solution of 5 I?g/mL propidium iodide and 0.25 I?g/mL annexin V ( Becton Dickinson, San Diego, USA ) , incubated for 15 min at 37 A°C, and analyzed with a FACSCanto flow cytometer ( Becton Dickinson, San Jose, USA ) utilizing Diva package. Approximately 105 cells were analyzed after each intervention. Dulbecco ‘s medium was used as the negative control and 100mM tert-butylhydroperoxide ( tBOOH ) as the positive control.
The statistical analysis was carried out utilizing one-way ANOVA, presuming equality of discrepancy with the Dunnet station hoc trial for pair-wise comparing. Consequences with P & lt ; 0.05 were considered statistically important.
2.5. Aquatic Acute Toxicity assay
2.5.1 Aquatic being civilizations
Daphnia similis, obtained from the CETESB Ecotoxicology Laboratory, were maintained at 20 A± 2°C, with light strength of 1000 lx, under a 16:8 H ( light/dark ) photoperiod. The Daphnias were fed daily with the green algae Selenastrum capricornutum and a mixture of trout nutrient and barm [ 32 ] .
2.5.2 Daphnia similis toxicity assay
The genus Daphnia is comprised of microcrustaceans extensively used in trials to measure the ague and chronic toxicity of chemical agents and wastewaters. Several writers have evaluated Daphnia similis as a trial species for usage in ecotoxicological checks [ 29-31 ] , demoing that this coinage can be used in ecotoxicological checks every bit good as Daphnia magna [ 32 ] .
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 checks was 10Aµg/mL ( equal 10mg/L ) with 4 replicates. For each replicate five immature beings ( 6 – 24 H old ) were exposed to the dye for 48 H in a 10 milliliter glass tubing. After the 48-h exposure period, the immobilized Daphnia were counted. The trial was considered valid if the immobilisation rate was less than 10 % in the negative control group [ 43 ] . The consequences were statistically analyzed utilizing the Spearman trimmed method, which has good statistical belongingss, is easy to utilize, and is recommended for accurate and precise computation of LC50 values and their 95 % assurance interval terminal points [ 44 ] .
2.5.3 Microtox bio-assay
MicrotoxA® is an acute toxicity trial based on suppression of the bioluminescence of the marine bacteria Vibrio fischeri NRRL B 11 177 [ 33 ] . Since the bioluminescence of Vibrio fischeri is straight linked to its respiratory activity, it provides a good index of metabolic activity and has been found to be good correlated with several in vivo toxicity trials [ 34-37 ] .
The acute toxicity trials were performed utilizing the Microtox process with the Microbics Model 500 analyser ( AZUR Environmental, Carlsbab, CA ) and the processs outlined in the Microtox Users Manual utilizing the “ 81.9 % Basic Test ” protocol [ 45 ] .
Lyophilized luminescent bacteriums, Vibrio fischeri, were reconstituted and exposed to a series of nine diluted dye solutions, osmotically adjusted with 22 % Na chloride ( NaCl ) . The ensuing lessening in bioluminescence was measured after 5 and 15 min at a changeless temperature of 15A°C. The information for 15 proceedingss are here reported, and the toxicity was measured as percent suppression of light emanation of the dye treated aliquot harmonizing to Lin and Chao [ 37 ] , corrected for loss of visible radiation in the control as follows:
% suppression = 100 x ( I0 x It ) / I0,
where I0 = initial bacterial luminescence and It = luminescence after debut of the dye into the bacterial suspension. All the Microtox informations were recorded and analyzed by MicrotoxOmni online package, which is able to cipher Percentage when merely one useable gamma is calculated from informations [ Azur Environmental, 1998 ] , and the consequences expressed as per centum suppression of light emanation [ 46 ] . A 2 % NaCl solution was used as the negative control and Zn sulphate heptahydrate ( ZnSO4.7H2O ) 100mg/L as the positive control.
3.1 Comet check
The consequences of the dose response experiment are shown in Fig. 2. At the lower concentrations of Disperse Orange 1 ( 0.05 and 0.1 Aµg/mL ) , no genotoxic consequence was observed for the HepG2 cells. However, a important addition in the migration of Deoxyribonucleic acid fragments was detected for all the other concentrations tested ( 0.2, 0.4, 1.0, 2.0 and 4.0 Aµg/mL ) .
The cell viability in all the dye concentrations was higher than 90 % ( informations non shown ) .
3.2 Mutagenicity assay
Disperse Orange 1 showed mutagenic activity for strains TA98, YG1041, TA100 and YG1042. The mutagenicity was greatly increased utilizing strain YG1041 and S9 decreased the consequence observed in all instances. Under the conditions tested, the dye tested negative for TA100 with S9 ( Table 1 ) .
3.3 Cell viability ( Cytotoxicity assay )
Fig. 3 shows cell decease by programmed cell death 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 non bring on cell decease ( informations non shown ) .
3.4 Aquatic Acute Toxicity assay
The dye Disperse Orange 1 did non do a important toxic consequence on Dapnhia similis. Table 2 shows the consequences obtained for this experiment, based on the per centum immobilisation of these micro-organisms as induced by the dye.
3.4.2 Microtox bio-assay
Similar to Daphnia, the dye did non do important toxicity with regard to Vibrio fischeri. Table 2 shows the suppression of light emanation by Vibrio fischeri in the presence of the dye, studied for 15 proceedingss. A inclination for an addition in % suppression of light emanation was observed, but the consequence was non sufficient to cipher the EC50 values. EC50 for positive control was 3.045mg/L ( 95 % assurance scope: 1.952 to 4.750 ) .
The comet check is used routinely for the sensing of DNA harm [ 38 ] . Disperse Orange 1 showed genotoxic effects on the HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0Aµg/mL, but it appears that at the highest concentration tested ( 4.0Aµg/mL ) the harm response had saturated despite cell viability being about 90 % , which might be due to cytotoxic effects. During rating of the slides, an addition in wholly disconnected cells was observedA at the higher doses, which might propose that the cytotoxic effects were get downing to happen and the Trypan Blue check was non sensitivity plenty to observe it. Chequer et al. , [ 11 ] observed a similar phenomenon for the micronucleus check of azo dyes, utilizing the same cell line.
This cytotoxic consequence was confirmed by the consequences obtained in the cell decease check. The consequences showed that Disperse Orange 1 induced programmed cell death after 72 hours of exposure at doses above 2.5 Aµg/mL. It is of import to indicate out that in the comet assay the cell exposure was 4 hours, whilst in this check, 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 bring on both basal brace permutations and frameshift mutants. However, comparing the mutagenic authorities detected with the different strains tested, the hypothesis was raised that this dye induces chiefly frameshift mutants.
Furthermore, the mutagenic authority was approximately 545 times higher for the strain YG1041 than for TA98 ( Table 1 ) , clearly showing the importance of nitroreduction and acetylation in the mutagenicity of this dye, since YG1041 is able to bring forth high degrees of nitroreductase and O-acetyltransferase. The mechanism of mutagenicity of these merchandises could be nitroreduction by nitroreductases organizing N-hydroxylamines that could bring on the DNA harm [ 47 ] . The function of nitroreductase in the metamorphosis and bioactivation of 1-nitropyrene ( 1-NP ) by enteric vegetation has been good established. Nitroreduction can happen as a two-electron transportation ( nitro – nitroso – hydroxylamine – amino ) or through one-electron transportations, organizing an anion free extremist intermediate [ 48 ] . Sing that the response detected for YG1041 was much higher than for TA98, the merchandise of the nitroreductase ( likely a hydroxylamine ) may hold been acetylated by the O-acetyltransferase, bring forthing really reactive species. Harmonizing to a survey carried out by Watanabe et Al. [ 49 ] utilizing the Salmonella strain YG1024, O-acetyltransferase is non specific for O and can move on the aminoalkane group taking to the production of reactive species.
With all the strains, the add-on of exogenic metabolic activation ( S9 mixture ) decreased the mutagenicity of the dye. The action of the P450 isoforms likely generated more stable merchandises, less likely to interact with DNA. Harmonizing to Chung and Cerniglia [ 5 ] , sulfonation, carboxylation, deaminization or permutation of an ethyl intoxicant or ethanoyl group group for the H in the amino group will take to a lessening or diminishing of mutagenic activity, which could explicate the lessening in mutagenic activity after intervention with S9.
The present consequences show that Disperse Orange 1 is a mutagenic dye. Other writers have shown that azo dyes can do harmful effects, chiefly for the familial stuff. 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 check in eight mouse variety meats. For 17 azo compounds, the check was positive for at least one organ [ 50 ] . The azo dye Sudan I showed a genotoxic consequence with HepG2 cells utilizing the comet and micronucleous checks [ 51 ] .
Disperse Orange 1 did non do acute toxicity to both Daphnia similis or Vibrio fischeri, what can be attributed to its chemical construction, since some dyes have shown to be toxic to Daphnia magna. In a survey performed by Bae and Freeman [ 52 ] , they found that copper-complexed dyes and Direct Blue 218 were really toxic to daphnids. The writers suggest that heavy metals like Cu molecules inside dye construction play an of import function for the rating of aquatic toxicity of dye solutions. In other survey, Wang et Al. [ 53 ] evaluated reactive dyestuffs and found that the most toxic azo dye was Ambifix xanthous VRNL, and the least toxic one was Procion bluish HERD.
The dye Disperse Orange 1 induces harm in DNA, chiefly frameshift mutants. The enzymes nitroreductase, N-acetyltransferase and O-cetyltransferase were shown to be really of import in the mutagenicity of this dye. In add-on, this compound causes cell decease by programmed cell death and DNA breakage in the human cell HepG2. No toxic effects were detected for Daphnia similis and Vibrio fisheri, but it is of import to add that other ecotoxic trials should be done in order to measure the impact of this dye on the biology. Thus the consequences of this survey stress the demand for the development of non mutagenic dyes and for investing in new methods for the intervention of wastewaters, in order to forestall the perchance hurtful effects of these compounds for both worlds and aquatic beings.