Toxicity assessment and microbial degradation of azo dyes

Toxic effluents containing azo dyes are discharged from various industries and they adversely affect water resources, soil fertility, aquatic organisms and ecosystem integrity. They pose toxicity (lethal effect, genotoxicity, mutagenicity and carcinogenicity) to aquatic organisms (fish, algae, bacteria, etc.) as well as animals. They are not readily degradable under natural conditions and are typically not removed from waste water by conventional waste water treatment systems. Benzidine based dyes have long been recognized as a human urinary bladder carcinogen and tumorigenic in a variety of laboratory animals. Several microorganisms have been found to decolourize, transform and even to completely mineralize azo dyes. A mixed culture of two Pseudomonas strains efficiently degraded mixture of 3-chlorobenzoate (3-CBA) and phenol/cresols. Azoreductases of different microorganisms are useful for the development of biodegradation systems as they catalyze reductive cleavage of azo groups (-N=N-) under mild conditions. In this review, toxic impacts of dyeing factory effluents on plants, fishes, and environment, and plausible bioremediation strategies for removal of azo dyes have been discussed.     

The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Induction of rat liver cytosol methyl red azo-reductase by 3-methylcholanthrene assayed by a sensitive fluorometric method

A sensitive and rapid fluorometric assay has been developed to measure the reduction of methyl red by rat liver enzymes. Greater than 90% of the methyl red reductase activity is found in the cytosol rather than in microsomes. The cytosol reductase activity is induced 7 to 10-fold by pretreatment of rats with 3-methylcholanthrene while little or no increase in activity is observed after phenobarbital treatment. The cytosol reductase activity is not inhibited by oxygen. Thus, the properties of the cytosol azo-reductase are quite different from those of the microsomal azo-reductases.     

Azo reduction of methyl red by neuronal nitric oxide synthase: the important role of FMN in catalysis

Nitric oxide synthase (NOS) is composed of an oxygenase domain and a reductase domain. The reductase domain has NADPH, FAD, and FMN binding sites. Wild-type nNOS reduced the azo bond of methyl red with a turnover number of approximately 130 min(-1) in the presence of Ca(2+)/calmodulin (CaM) and NADPH under anaerobic conditions. Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding inhibitor, completely inhibited azo reduction. The omission of Ca(2+)/CaM from the reaction system decreased the activity to 5%. The rate of the azo reduction with an FMN-deficient mutant was also 5% that of the wild type. NADPH oxidation rates for the wild-type and mutant enzymes were well coupled with azo reduction. Thus, we suggest that electrons delivered from the FMN of the nNOS enzyme reduce the azo bond of methyl red and that this reductase activity is controlled by Ca(2+)/CaM.     

A continuous spectrophotometric determination of hepatic microsomal azo reductase activity and its dependence on cytochrome P-450.

1. A continuous spectrophotometric determination of rat hepatic microsomal anaerobic azo reductase activity has been developed. 2. The addition of soluble flavins (riboflavin, FMN or FAD) greatly increased this NADPH-dependent activity towards a number of azo substrates. 3. Investigations with amaranth as substrate gave an apparent Km of 34 microM and Vmax. of 4 nmol/min per mg of microsomal protein. The inclusion of a fixed concentration of FMN increased Vmax. and greatly decreased Km, the magnitude of these changes reflecting the concentration of flavin present. 4. Investigations using a fixed amaranth concentration over a range of flavin concentrations gave biphasic double-reciprocal plots with two apparent Km and Vmax. values. 5. Pretreatment of animals with cobaltous chloride, 2-allyl-2-isopropylacetamide, carbon tetrachloride, phenobarbitone and 3-methylcholanthrene altered azo reductase activity in parallel with changes in cytochrome P-450 content. 6. The significance of these results is discussed in terms of the electron-transfer components present in the hepatic microsomal fraction.     

Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers

We have detected a flavonoid 3[prime],5[prime]-hydroxylase (F3[prime],5[prime]H) in the microsomal fraction of Petunia hybrida flowers. Activity varied with the development of flowers, peaking immediately prior to and during anthesis, but was absent in mature flowers. F3[prime],5[prime]H activity in flower extracts from genetically defined floral color mutants correlated strictly with the genotypes Hf1 and Hf2. No activity was detected in flowers from mutants homozygous recessive for both alleles. F3[prime],5[prime]H activity was dependent on NADPH and molecular oxygen; there was only slight activity with NADH. The enzyme catalyzes the hydroxylation of 5,7,4[prime]-trihydroxyflavonone at the 3[prime] and 5[prime] positions, and of 5,7,3[prime],4[prime]-tetrahydroxyflavonone and dihydroquercetin at the 5[prime] position. Hydroxylase activity was inhibited by plant growth regulators (1-aminobenzotriazole and tetcyclacis) and by CO, N-ethylmaleimide, diethyldithiocarbamate, and cytochrome (Cyt) c. Activity was not affected by diethylpyrocarbonate or phenylmethylsulfonyl fluoride, but was enhanced by 2-mercaptoethanol. A polyclonal antibody that inhibits higher plant NADPH-Cyt P450 reductase inhibited the F3[prime],5[prime]H. The data are consistent with the suggestion that the P. hybrida F3[prime],5[prime]H is a monooxygenase consisting of a Cyt P450 and a NADPH-Cyt P-450 reductase. Cyts P450 were detected in microsomal membranes and in solubilized detergent extracts of these membranes. F3[prime],5[prime]H activity was sensitive to low concentrations of all detergents tested, and therefore solubilization of the active enzyme was not achieved. Reaction products other than flavanones were observed in F3[prime],5[prime]H assays and these may be formed by enzymic oxidation of flavanones. The possibility of a microsomal flavone synthase of a type that has not been described in P. hybrida is discussed.     

5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane, a structural component of the modified folate in Sulfolobus solfataricus.

The partial characterization of the modified folate present in Sulfolobus solfataricus has been carried out. Separation of ethanol-water extracts of these cells on a DEAE-Sephadex column led to the isolation of a small amount of intact oxidized cofactor, which, when subjected to reductive cleavage with Zn-HCl, produced 6-methylpterin. This indicated that the modified folate in these cells contained a nonmethylated pterin linked, via a methylene group at the C-6 position of the pterin, to an arylamine, as is found in folate. Oxidative cleavage of intact reduced cofactor produced pterin and a single arylamine. The azo dye derivative of this arylamine was prepared and purified by chromatography on a Bio-Gel P-6 column. The resulting purified compound was shown to be readily hydrolyzed in dilute acid to the azo dye derivative of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypantane, which was, in turn, readily cleaved to 5-(p-aminophenyl)-1,2,3,4- tetrahydroxypentane by Zn-HCl reduction. The stereochemistry of the resulting 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane was shown to be ribo, the same as that of the 5-(p-aminophenyl)-1,2,3,4- tetrahydroxypentane moiety found in methanopterin. The complete arylamine side chain of the modified folate thus contains 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane attached, via an acid-labile bond, to a currently unidentified substituent. The modified folate present in S. solfataricus thus contains structural features common to both folates and methanopterin.     

Studies on the mechanism of aromatase and other cytochrome P450 mediated deformylation reactions

Aromatase is a microsomal cytochrome P450 that converts androgens to estrogens by three sequential oxidations. The isolation of the 19-hydroxy and 19-oxo androgens suggests that the first two oxidations occur at the C₁₉ carbon. However, the mechanism of the third oxidation, which results in C₁₀---C₁₉ bond cleavage, has not been determined. Two proposed mechanisms which remain viable involve either initial 1β-hydrogen atom abstraction or addition of the ferric peroxy anion from aromatase to the C₁₉ aldehyde. Semiempirical molecular orbital calculations (AM1) were used to study potential reaction mechanisms initiated by initial 1β-hydrogen atom abstraction. Initially, the energetics of carbon---carbon bond cleavage of the keto and enol forms of C1-radicals were studied and were found to be energetically similar. A mechanism was proposed in which the 19-oxo intermediate is subject to initial nucleophilic attack by the protein. The geometry of the A-ring in the androgens is between that for the 1-radicals and estrogen, suggesting that some transition state stabilization for the homolytic cleavage reaction can occur.

More recently, studies on liver microsomal cytochrome P450 mediated deformylation of xenobiotic aldehydes supports mechanisms involving an alkyl peroxy intermediate formed by addition of the ferric peroxy anion from aromatase to the C₁₉ aldehyde. Although this intermediate could proceed through several different concerted or non-concerted pathways, one non-concerted pathway involves the heterolytic cleavage of the dioxygen bond resulting in an active oxygenating species (iron-oxene) and a diol. The diol could then undergo hydrogen atom abstraction followed by homolytic carbon---carbon bond cleavage as in the mechanisms modeled previously. When this cleavage was modeled for seven aldehydes, a good correlation with reported experimental aldehyde turnover numbers was obtained. However, when dialkoxy derivatives of the aldehydes are subject to microsomal metabolism, the rates of carbon---carbon cleavage products do not approach the rates of deformylation of the aldehyde analog.     

Simple bi- and tricyclic inhibitors of human steroid 5alpha-reductase

A number of tricyclic thiolactams, bicyclic lactams, and bicyclic thiolactams have been prepared and evaluated in vitro as inhibitors of types 1 and 2 steroid 5alpha-reductase. The tricycles with an 8-chloro substituent in the C-ring are nM (IC50) inhibitors of type 1 steroid 5alpha-reductase (SR). In all the cases studied, lactams are more potent than the corresponding thiolactams. Activity against type 2 SR is greatly enhanced by a styryl (or azo) substituent on the aryl ring of the tri- and bicycles and also a related tricyclic aryl acid.     

Multiple catalytic properties of the purified and reconstituted cytochrome P-450 (P-450sccII) system of pig testis microsomes

The catalytic properties of the testis microsomal P-450, termed P-450sccII, have been studied in a refined assay system which consists of P-450sccII (13 nmol of P-450 heme/mg of protein) and its reductase has been purified extensively from pig testis. The results indicated that P-450sccII was highly active in catalyzing hydroxylation of 11 beta-hydroxyprogesterone at the 17 alpha-position to give 21-deoxycortisol and cleavage of 17 alpha-hydroxyprogesterone at the 17-20 bond to give androstenedione with turnover numbers of 25 and 30 mol/min X mol of P-450, respectively. In contrast, many physiologically important corticosteroids we tested were found to be poor substrates for both the hydroxylase and lyase reactions. The possible reason for the importance of these substrate specificity of P-450sccII in production of both corticosteroids and androgens in the endocrine tissues is discussed. P-450sccII also catalyzed conversion of testosterone to androstenedione, but 18O experiments failed to show incorporation of atmospheric oxygen into the androstenedione formed. However, this does not preclude the possibility that the P-450-bound intermediate gem-diol stereoselectively dehydrates to give the nonlabeled ketosteroid. In addition to these steroid-oxidizing activities, P-450sccII revealed considerable specificities toward various xenobiotics, suggesting that P-450sccII and liver microsomal P-450 are basically similar as regards enzymatic functions and activities.     

The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase

The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.     

Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.     

Bacterial decolorization and degradation of azo dyes

Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries such as textile, food, cosmetics and paper printing. They are generally recalcitrant to biodegradation due to their xenobiotic nature. However microorganisms, being highly versatile, have developed enzyme systems for the decolorization and mineralization of azo dyes under certain environmental conditions. Several genera of Basidomycetes have been shown to mineralize azo dyes. Reductive cleavage of azo bond, leading to the formation of aromatic amines, is the initial reaction during the bacterial metabolism of azo dyes. Anaerobic/anoxic azo dye decolorization by several mixed and pure bacterial cultures have been reported. Under these conditions, this reaction is non-specific with respect to organisms as well as dyes. Various mechanisms, which include enzymatic as well as low molecular weight redox mediators, have been proposed for this non-specific reductive cleavage. Only few aerobic bacterial strains that can utilize azo dyes as growth substrates have been isolated. These organisms generally have a narrow substrate range. Degradation of aromatic amines depends on their chemical structure and the conditions. It is now known that simple aromatic amines can be mineralized under methanogenic conditions. Sulfonated aromatic amines, on the other hand, are resistant and require specialized aerobic microbial consortia for their mineralization. This review is focused on the bacterial decolorization of azo dyes and mineralization of aromatic amines, as well as the application of these processes for the treatment of azo-dye-containing wastewaters.     

Enzymatic reduction and oxidation of fibre-bound azo-dyes

A new customer and environmental friendly method of hair bound dye decolouration was developed. Biotransformation of the azo-dyes Flame Orange and Ruby Red was studied using different oxidoreductases. The pathways of azo dye conversion by these enzymes were investigated and the intermediates and metabolites were identified and characterised using UV–vis spectroscopy, high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Laccase from Pycnoporus cinnabarinus, manganese peroxidase (MnP) from Nematoloma frowardii and the novel Agrocybe aegerita peroxidase (AaP) were found to use a similar mechanism to convert azo dyes. They N-demethylated the dyes and concomitantly polymerized them to some extent. On the other hand the mechanism for cleavage of the azo bond by azo-reductases of Bacillus cereus and B. subtilis was based on reduction of the azo bond at the expense of NAD(P)H.     

ortho-Substituted C-aryl glucosides as highly potent and selective renal sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors

A series of 2-substituted C-aryl glucosides have been synthesized and evaluated for inhibition of hSGLT1 and hSGLT2. Introduction of an appropriate ortho substituent at the proximal phenyl ring adjacent to the glycosidic bond was found to improve SGLT2 inhibitory activity and dramatically increase selectivity for hSGLT2 over hSGLT1. Selected compounds were investigated for in vivo efficacy.     

A new method for assaying rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and its application in a study of the effect of dietary cholesterol on this effect of dietary cholesterol on this enzyme.

A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.     

Monoazo and diazo dye decolourisation studies in a methanogenic UASB reactor

Mixed anaerobic bacterial consortia have been show to reduce azo dyes and batch decolourisation tests have also demonstrated that predominantly methanogenic cultures also perform azo bond cleavage. The anaerobic treatment of wool dyeing effluents, which contain acetic acid, could thus be improved with a better knowledge of methanogenic dye degradation. Therefore, the decolourisation of two azo textile dyes, a monoazo dye (Acid Orange 7, AO7) and a diazo dye (Direct Red 254, DR254), was investigated in a methanogenic laboratory-scale Upflow Anaerobic Sludge Blanket (UASB), fed with acetate as primary carbon source. As dye concentration was increased a decrease in total COD removal was observed, but the acetate load removal (90%) remained almost constant. A colour removal level higher than 88% was achieved for both dyes at a HRT of 24h. The identification by HPLC analysis of sulfanilic acid, a dye reduction metabolite, in the treated effluent, confirmed that the decolourisation process was due mainly to azo bond reduction. Although, HPLC chromatograms showed that 1-amino-2-naphthol, the other AO7 cleavage metabolite, was removed, aeration batch assays demonstrated that this could be due to auto-oxidation and not biological mineralization. At a HRT of 8h, a more extensive reductive biotransformation was observed for DR254 (82%) than for AO7 (56%). In order to explain this behaviour, the influence of the dye aggregation process and chemical structure of the dye molecules are discussed in the present work.     

Ring cleavage of sulfur heterocycles: how does it happen?

Sulfur heterocycles are common constituents of petroleum and liquids derived from coal, and they are found in some secondary metabolites of microorganisms and plants. They exist primarily as saturated rings and thiophenes. There are two major objectives driving investigations of the microbial metabolism of organosulfur compounds. One is the quest to develop a process for biodesulfurization of fossil fuels, and the other is to understand the fates of organosulfur compounds in petroleum- or creosote-contaminated environments which is important in assessing bioremediation processes. For these processes to be successful, cleavage of different types of sulfur heterocyclic rings is paramount. This paper reviews the evidence for microbial ring cleavage of a variety of organosulfur compounds and discusses the few well-studied cases which have shown that the C-S bond is most susceptible to breakage leading to disruption of the ring. In most cases, the introduction of one or more oxygen atom(s) onto the adjacent C atom and/or onto the S atom weakens the C-S bond, facilitating its cleavage. Although much is known about the thiophene ring cleavage in dibenzothiophene, there is still a great deal to be learned about the cleavage of other sulfur heterocycles.     

Oxygen-Insensitive Nitroreductases NfsA and NfsB of Escherichia coli Function under Anaerobic Conditions as Lawsone-Dependent Azo Reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.