Mutagenicity of azo dyes: structure-activity relationships.

Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.     

Developing azo and formazan dyes based on environmental considerations: Salmonella mutagenicity

In previous papers, the synthesis and chemical properties of iron-complexed azo and formazan dyes were reported. It was shown that in certain cases iron could be substituted for the traditionally used metals such as chromium and cobalt, without having an adverse effect on dye stability. While these results suggested that the iron analogs were potential replacements for the commercially used chromium and cobalt prototypes, characterization of potentially adverse environmental effects of the new dyes was deemed an essential step in their further development. The present paper provides results from using the Salmonella/mammalian microsome assay to determine the mutagenicity of some important commercial metal complexed dyes, their unmetallized forms, and the corresponding iron-complexed analogs. The study compared the mutagenic properties of six unmetallized azo dyes, six commercial cobalt- or chromium-complexed azo dyes, six iron-complexed azo dyes, six unmetallized formazan dyes, and six iron-complexed formazan dyes. The results of this study suggest that the mutagenicity of the unmetallized dye precursors plays a role in determining the mutagenicity of the iron-complexes. For the monoazo dye containing a nitro group, metal complex formation using iron or chromium decreased or removed mutagenicity in TA100; however, little reduction in mutagenicity was noted in TA98. For the formazan dye containing a nitro group, metal-complex formation using iron increased mutagenicity. Results varied for metal-complexes of azo and formazan dyes without nitro groups, but in general, the metal-complexed dyes based on mutagenic ligands were also mutagenic, while those dyes based on nonmutagenic ligands were nonmutagenic.     

The effect of alkoxy substituents on the mutagenicity of some phenylenediamine-based disazo dyes

16 phenylenediamine-based disazo dyes were examined in the Salmonella/mammalian microsome assay with strains TA98, TA100 and TA1538. All of the dyes contain an alkoxy group ortho to one of the azo linkages. Increasing the size of this alkoxy substituent from 1 to 4 carbons led to a decrease in mutagenic activity in certain instances while no change was noted in other cases. Comparison of the mutagenicity of the disazo dyes with their potential reductive-cleavage products suggests that (1) the reductive-cleavage products are not solely responsible for the mutagenicity of the disazo dyes, and (2) significant reductive-cleavage of the disazo dyes is not taking place in the standard Salmonella assay.     

Mutagenicity of selected sulfonated azo dyes in the Salmonella/microsome assay: use of aerobic and anaerobic activation procedures

A selection of 16 sulfonated azo dyes of both the monoazo type and diazo dyes based on benzidine, o-tolidine and o-dianisidine were assayed for mutagenicity in Salmonella typhimurium strains TA98 and TA100 employing both aerobic and anaerobic preincubation procedures. 3 food dyes, FD & C Red No. 40 and Yellows No. 5 and No. 6 were non-mutagenic in all tests. 5 dyes were mutagenic with aerobic treatment (trypan blue, Pontacyl Sky Blue 4BX, Congo Red, Eriochrome Blue Black B, dimethylaminoazobenzene) and 6 were mutagenic aerobically with riboflavin and cofactors (Deltapurpurin, trypan blue, Pontacyl Sky Blue 4BX, Congo Red, methyl orange, Ponceau 3R). Anaerobic preincubation involving enzymatic reduction of the dyes led to a different pattern of mutagenicity, with trypan blue giving much enhanced mutagenicity; Eriochrome Blue Black B, Pontacyl Sky Blue 4BX, Deltapurpurin and Congo Red exhibiting similar activity to aerobic preincubation; and methyl orange and Ponceau 3R yielding no mutagenicity. The results are interpreted with respect to an hypothesis involving partial reduction of the azo bond under differing degrees of aerobiosis via azo-anion radicals and hydrazo intermediates.     

Evaluation of new 2,2'-dimethyl-5,5'-dipropoxybenzidine- and 3,3'-dipropoxybenzidine-based direct dye analogs for mutagenic activity by use of the Salmonella/mammalian mutagenicity assay

As part of a continuing study aimed at establishing structure-activity relationships and heuristic principles useful for the design of non-genotoxic azo dyes, a series of new direct dyes based on two non-mutagenic benzidine analogs, 2,2'-dimethyl-5,5'-dipropoxybenzidine and 3,3'-dipropoxybenzidine, were evaluated for mutagenic activity in Salmonella typhimurium strains TA98 and TA100. These strains are widely used for mutagenicity screening and have been shown to detect the mutagenic activity of benzidine analogs. While some toxicity was seen with some dyes at high doses, all of the dyes examined were judged non-mutagenic with and without metabolic activation in the standard Salmonella plate-incorporation assay. The results in the standard test are consistent with the properties of the diamines themselves. However, only one of the dyes was non-mutagenic when a reductive-metabolism pre-incubation assay was used. The results of this study suggest that although benzidine analogs are potential replacements for benzidine, there is a need to understand which mutagenic products are produced when reductive metabolism is present. There is also a need to know whether or not metal complexes of these dyes are mutagenic. Such information will allow the development of new non-mutagenic azo dyes.     

假单胞菌S—42对偶氮染料的脱色和降解代谢

Pseudomonas S-42 was capable of decolorizing azo dyes such as Diamira Brilliant Orange RR(DBO-RR), Direct Brown M (DBM), Eriochrome Brown R(EBR) and so on. The cell suspension, cell-free extract and purified enzyme of Pseud. S-42 could decolorize azo dyes under similar conditions: the optimum pH and temperature laid 7.0 and 37 degrees C respectively. The efficiencies of decolorizing of DBO-RR, DBM, EBR by intact cells stood more than 90%. When the cell concentration was 15 mg(wet)/ml and the reaction time was 5 hours, the decolorizing activity for above three azo dyes by intact cells were 1.75, 2.4, 0.95 micrograms dye/mg cell, respectively. Cell-free extract and purified enzyme could well express the decolorizing activity only under the anaerobic condition and added NADH. Purified enzyme belongs to azoreductase, its molecular weight is about 34,000-2000 daltons, and its Vmax and Km for DBO-RR are 13 mumol.mg protein-1.min-1 and 54 mumol/L. The results of the detection of the biodegrading products of DBO-RR by spectrophotometric and NaNO2 reactional methods showed that the biodegradation of azo dyes was initiated by the reduction cleavage of azo bonds. It was hypothesized that biodegrading metabolism pathway of DBO-RR by Pseudomonas S-42.     

Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA 1538

A series of ten azo dyes as well as various single ring aromatic amines substituted on the benzene ring were tested for bacterial mutagenicity with Salmonella typhimurium TA 1538 using a soft-agar overlay method. Two dyes, sudan 2 and chrysoidin induced mutation but only in the presence of a rat liver preparation. Chrysoidin was the more active. Testing of its reduction products, aniline and 1,2,4-triaminobenzene showed a liver metabolite of the latter compound could be responsible for the mutagenic effect, having a comparable mutagenicity with 1,2-diamino-4-nitro-benzene, one of the mutagenic constituents of hair dyes. Structure-activity studies on a series of ring-substituted anilines indicated that mutagenic activity required at least two positions to be substituted with either amino or nitro groups, or one of each. The bacteria as well as the liver enzyme preparation may partake in the activation of these chemicals. The correlation between mutagenicity and carcinogenicity for this group of compounds is discussed.     

Mutagenicity of azo dyes in the Salmonella/microsome assay using in vitro and in vivo activation

The mutagenicity of 6 azo dyes, including direct black 38 (DB38), direct black 19 (DB19), direct brown 95 (DB95), solvent yellow 3 (SY3), trypan blue (TPB), and food black 2 (FB2), was examined in the Salmonella/microsome assay. The effect of chemical azo reduction (dithionite) and in vivo metabolism on the mutagenicity of the dyes was also studied. In vivo azo-dye metabolites were isolated from the urine of rats intubated with dyes by XAD-2 column chromatography. Urinary metabolites from all the treated animals, except animals treated with FB2, induced frame-shift mutations in strains TA1538 and TA98 in the presence of liver S9 activation. The control urine did not increase the incidence of revertants in strains TA1538 and TA98. Thus, XAD-2 chromatography can be used to isolate genotoxic metabolites from the urine of animals intubated with azo dyes.     

Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay

We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.     

Decolorization and degradation of Disperse Blue 79 and Acid Orange 10, by Bacillus fusiformis KMK5 isolated from the textile dye contaminated soil

The release of azo dyes into the environment is a concern due to coloration of natural waters and due to the toxicity, mutagenicity and carcinogenicity of the dyes and their biotransformation products. The dye degrading bacterial strain KMK 5 was isolated from the textile dyes contaminated soil of Ichalkaranji, Maharashtra, India. It was identified as Bacillus fusiformis based on the biochemical and morphological characterization as well as 16S rDNA sequencing. KMK 5 could tolerate and degrade azo dyes, Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) under anoxic conditions. Complete mineralization of DB79 and AO10 at the concentration of 1.5g/l was observed within 48h. This degradation potential increased the applicability of this microorganism for the dye removal.     

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes

We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis.     

Evaluation of azo food dyes for mutagenicity and inhibition of mutagenicity by methods using Salmonella typhimurium

The mutagenicity of 4 azo dyes (FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Red No. 40 and amaranth) that are widely used to color food has been evaluated. 4 different methods were used: (1) the standard Ames plate-incorporation assay performed directly on the dyes in the absence of S9 and in the presence of rat- or hamster-liver S9; (2) application of the standard plate assay to ether extracts of aqueous solutions of the dyes; (3) a variant of the standard assay, using hamster liver S9, preincubation, flavin mononucleotide (FMN) and other modifications designed to facilitate azo reduction; and (4) reduction of the dyes with sodium dithionite, followed by ether extraction and the standard plate assay. Assays that include chemical reduction (methods 3 and 4) were included because azo compounds ingested orally are reduced in the intestine with the release of free aromatic amines. No mutagenic activity was seen for any of the azo dyes tested by using the standard Ames plate assay (method 1). Ether extracts of some samples of FD&C Yellow No. 6, FD&C Red No. 40 and amaranth were active (method 2), but only at high doses, generally 250 mg-equivalents or more per plate. These results indicate the presence of low levels of ether-extractable mutagenic impurities. The FMN preincubation assay (method 3) gave negative results for all dye samples tested. Most batches of FD&C Red No. 40 tested had mutagenic activity that was detectable when the ether extract of less than 1 mg of dithionite-reduced dye was plated in the presence of S9 (method 4). This finding implies that an impurity in these samples of FD&C Red No. 40 can be reduced to yield an ether-extractable mutagen. Dithionite-reduced samples of FD&C Yellow No. 6 and amaranth showed ether-extractable mutagenic activity only at much higher doses than those at which activity was seen with most dithionite-reduced samples of FD&C Red No. 40 (method 4). FD&C Yellow No. 5 showed no mutagenic activity with this method. Mutagenic activity was not detected when FD&C Red No. 40 was tested by using the azo reduction preincubation assay with FMN (method 3).(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of alkoxy substituents on the mutagenicity of some aminoazobenzene dyes and their reductive-cleavage products

15 aminoazobenzene dyes and 7 of their reductive-cleavage products were examined in the Salmonella/microsome assay with strains TA98, TA100, TA1535, TA1537 and TA1538. Dyes tested included 5 derivatives of 4-aminoazobenzene with different alkoxy substituents (-OCH3, -OCH2CH3, -OCH2CH2 CH3, -OCH2CH2CH2CH3 or -OCH2CH2OH) in the 8-position as well as the corresponding derivatives of 4-[(4-aminophenyl)azo]-N,N-diethylaniline and 4-[(4-aminophenyl)azo]-N,N-bis(2-hydroxyethyl)aniline. In general, as the size of the substituent ortho to the primary amino group of the dyes was increased, the mutagenicity decreased. A similar trend was observed for the reductive-cleavage products. The results from the latter aspect of this study suggest that the mutagenicity of aminoazobenzene dyes can not be accounted for solely from the properties of their reductive-cleavage products.     

The possible role of azoreduction in the bacterial mutagenicity of 4-dimethylaminoazobenzene (DAB) and 2 of its analogues (6BT and 5I)

The extent to which azoreductive cleavage contributes to the bacterial mutagenicity of 3 azo compounds has been investigated. The compounds studied were the rodent-liver carcinogens 4-dimethylaminoazobenzene (DAB) and 6-dimethylaminophenylazobenzthiazole (6BT), and the reported non-carcinogenic isostere 5-dimethylaminophenylazoindoline (5I). Although each of these compounds is mutagenic to Salmonella when evaluated using a pre-incubation protocol and in the presence of an induced rat-liver S9 mix, the constituent amines (cleavage products) were essentially inactive. It is therefore concluded that the mutagenic response reported for DAB, 6BT and 5I is related to metabolic activation of the intact molecules. In addition, the non-mutagenicity of 4'-phenyl-4-dimethylaminoazobenzene (4PhDAB) suggests that azoreductase activity is low in the Salmonella preincubation assay, at least as conducted in this laboratory. In the case of 4PhDAB, less than 1.4% azoreduction would yield sufficient quantities of the derived amine, 4-aminobiphenyl, for a positive mutagenic response to have been observed.     

Chemical characterization of a dye processing plant effluent--identification of the mutagenic components

This work shows the chemical characterization of a dye processing plant effluent that was contributing to the mutagenicity previously detected in the Cristais river, S?o Paulo, Brazil, that had an impact on the quality of the related drinking water. The mutagenic dyes Disperse Blue 373, Disperse Orange 37 and Disperse Violet 93, components of a Black Dye Commercial Product (BDCP) frequently used by the facility, were detected by thin layer chromatography (TLC). The blue and orange dyes were quantified by high performance liquid chromatography (HPLC/DAD) in a raw and treated effluent samples and their contribution to the mutagenicity was calculated based on the potency of each dye for the Salmonella YG1041. In the presence of S9 the Disperse Blue 373 accounted for 2.3% of the mutagenic activity of the raw and 71.5% of the treated effluent. In the absence of S9 the Disperse Blue 373 accounted for 1.3% of the mutagenic activity of the raw and 1.5% of the treated effluent. For the Disperse Orange 37, in the presence of S9, it contributed for 0.5% of the mutagenicity of the raw and 6% of the treated effluent. In the absence of S9; 11.5% and 4.4% of the raw and treated effluent mutagenicity, respectively. The contribution of the Disperse Violet 93 was not evaluated because this compound could not be quantified by HPLC/DAD. Mutagenic and/or carcinogenic aromatic amines were also preliminary detected using gas chromatograph/mass spectrometry in both raw and treated and are probably accounting for part of the observed mutagenicity. The effluent treatment applied by the industry does not seem to remove completely the mutagenic compounds. The Salmonella/microsome assay coupled with TLC analysis seems to be an important tool to monitor the efficiency of azo dye processing plant effluent treatments.     

Azoreductase and dye detoxification activities of Bacillus velezensis strain AB

Azo dyes are known to be a very important and widely used class of toxic and carcinogenic compounds. Although lot of research has been carried out for their removal from industrial effluents, very little attention is given to changes in their toxicity and mutagenicity during the treatment processes. Present investigation describes isolation of a Bacillus velezensis culture capable of degrading azo dye Direct Red 28 (DR28). Azoreductase enzyme was isolated from it, and its molecular weight was found to be 60 kDa. The enzyme required NADH as cofactor and was oxygen-insensitive. Toxicity and mutagenicity of the dye during biodegradation was monitored by using a battery of carefully selected in vitro tests. The culture was found to degrade DR28 to benzidine and 4-aminobiphenyl, both of which are potent mutagens. However, on longer incubation, both the compounds were degraded further, resulting in reduction in toxicity and mutagenicity of the dye. Thus, the culture seems to be a suitable candidate for further study for both decolourization and detoxification of azo dyes, resulting in their safe disposal. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

微生物对偶氮染料的脱色及其基因工程研究进展

偶氮染料广泛应用在纺织印染、造纸印刷等行业中。染料废水的排放将会导致严重的环境污染,使用微生物处理染料废水是解决此问题的有效方法。该文概述了微生物对偶氮染料的脱色的研究,包括细菌对偶氮染料的脱色,真菌对偶氮染料的脱色,脱色产生的芳香胺并进一步被降解,以及基因工程技术在微生物对偶氮染料脱色的研究进展。     

Synthesis of beta-cyclodextrin and starch based polymers for sorption of azo dyes from aqueous solutions

Three beta-cyclodextrin (polymers 1-3) and a starch-based (polymer 4) polymers were synthesized using hexamethylene diisocyanate (HMDI) as a cross-linking agent in dry dimethylformamide and used as a sorbent for the removal of some selected azo dyes from aqueous solutions. The cross-linked polymers were characterized by Fourier transform infrared spectroscopy, thermogravimetric and differential scanning calorimetric analysis. Results of sorption showed that cyclodextrin and starch based polymers can be effectively used as a sorbent for the removal of anionic azo dyes. The Influence of the amide groups and the chemical structure of azo dyes are also studied. Results of sorption experiments showed that these adsorbent exhibited high sorption capacities toward Direct Violent 51 (80% for polymer 1, 69% for polymer 2, 70% for polymer 3 and 78% for polymer 4). The sorption capacity of dyes on the polymers was dependent on the presence of sulfonate groups of the anionic dyes. In order to explain the results an adsorption mechanism mainly physical adsorption and interactions such as hydrogen bonding, ion-exchange due to the nature of the polymer network and the formation of an inclusion complex due to the beta-CD molecules through host-guest interaction is proposed.     

Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters

The azoreductase PpAzoR from Pseudomonas putida shows a broader specificity for decolourization of azo dyes than CotA-laccase from Bacillus subtilis. However, the final products of PpAzoR activity exhibited in most cases a 2 to 3-fold higher toxicity than intact dyes themselves. We show that addition of CotA-laccase to PpAzoR reaction mixtures lead to a significant drop in the final toxicity. A sequential enzymatic process was validated through the use of 18 representative azo dyes and three model wastewaters that mimic real dye-containing effluents. A heterologous Escherichia coli strain was successfully constructed co-expressing the genes coding for both PpAzoR and CotA. Whole-cell assays of recombinant strain for the treatment of model dye wastewater resulted in decolourization levels above 80% and detoxification levels up to 50%. The high attributes of this strain, make it a promising candidate for the biological treatment of industrial dye containing effluents.     

Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus.

Five 14C-radiolabeled azo dyes and sulfanilic acid were synthesized and used to examine the relationship between dye substitution patterns and biodegradability (mineralization to CO2) by a white-rot fungus and an actinomycete. 4-Amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid were used as representative compounds having sulfo groups or both sulfo and azo groups. Such compounds are not known to be present in the biosphere as natural products. The introduction of lignin-like fragments into the molecules of 4-amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid by coupling reactions with guaiacol (2-methoxyphenol) resulted in the formation of the dyes 4-(3-methoxy-4-hydroxyphenylazo)-[U-14C]benzenesulfonic acid and 4-(2-sulfo-3'-methoxy-4'-hydroxy-azobenzene-4-azo)-[U-14C]benzenesulf oni c acid, respectively. The synthesis of acid azo dyes 4-(2-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid and 4-(4-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid also allowed the abilities of these microorganisms to mineralize these commercially important compounds to be evaluated. Phanerochaete chrysosporium mineralized all of the sulfonated azo dyes, and the substitution pattern did not significantly influence the susceptibility of the dyes to degradation. In contrast, Streptomyces chromofuscus was unable to mineralize aromatics with sulfo groups and both sulfo and azo groups. However, it mediated the mineralization of modified dyes containing lignin-like substitution patterns. This work showed that lignocellulolytic fungi and bacteria can be used for the biodegradation of anionic azo dyes, which thus far have been considered among the xenobiotic compounds most resistant to biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)