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Enzymatic reduction of tartrazine by Proteus vulgaris from rats
Abstract
The reduction of the azo food colouring, tartrazine, has been studied in whole cells and cell-free preparations of Proteus vulgaris isolated from rat-intestinal contents. The ability of whole cells to reduce this colouring is dependent upon age and nutritional status. Old starved cells are most efficient. This effect is related to cell permeability. A soluble FMN-flavoprotein, which is NADPH dependent, has been isolated as a crude enzyme and found capable of reducing tartrazine. Some reaction conditions are reported. The role of intestinal flora in mammalian detoxification mechanisms and the enzymatic mechanism of bacterial reduction of azo colourings are discussed.
... According to the second mechanism of biological azo dye reduction, azo dyes are indirectly reduced by enzymatically reduced electron carriers. Early research has hypothesised that reduced flavins (FADH 2 , FMNH 2 , riboflavin) generated by flavindependent reductases can reduce azo dyes in a non-specific chemical reaction 111,282 . Flavins were indeed often found to stimulate azo dye reduction (Table 1.4) and recent research has revealed that flavin reductases are indeed 'anaerobic azoreductases' 286 . ...
... Lysis of cells would release cofactors in the extracellular environment. Hence, it has been reported several times that cell extracts or starving or lysed cells show higher azo dye reduction rates than intact or resting cells 87,213,282,286,337,348 . However, for intact cells, a membrane transport system would be a prerequisite for the reduction of azo dyes by these cofactors. ...
... In this study, the reaction rates of dye reduction are, however, discussed by comparison of the pseudo first-order reaction rate constant k c ' (in d -1 ) at a given sulphide concentration. At high sulphide levels, values between -200 and -220 mV that are known to mediate azo dye reduction in biological systems 38,58,111,161,271,282,296 . The mediating role of flavins in the chemical reduction of azo dyes by sulphide has not been reported before. ...
Azo dyes, aromatic moieties linked together by azo (-N=N-) chromophores, represent the largest class of dyes used in textile-processing and other industries. The release of these compounds into the environment is undesirable, not only because of their colour, but also because many azo dyes and their breakdown products are toxic and/or mutagenic to life. To remove azo dyes from wastewater, a biological treatment strategy based on anaerobic reduction of the azo dyes, followed by aerobic transformation of the formed aromatic amines, holds promise. However, the first stage of the process, anaerobic azo dye reduction, proceeds relatively slow. Therefore, this thesis research aimed at optimising anaerobic azo dye reduction, by studying the reaction mechanism and by consequently applying the obtained insights.In this thesis it is shown that non-adapted anaerobic granular sludge has the capacity to non-specifically reduce azo dyes. As there was no correlation between a dye's reduction rate and its molecular characteristics (i.e. its size and its number of sulphonate groups and other polar substituents), it is unlikely that the mechanism of azo dye reduction involves cell wall penetration. Moreover, the presence of bacteria is not a prerequisite: azo dyes can also be reduced by sulphide in a purely chemical reaction. As dye containing wastewater usually contains sulphate and other sulphur species that will be biologically reduced to sulphide during treatment in anaerobic bioreactors, azo dye reduction will be a combination of biotic and abiotic processes. However, it was demonstrated that under normal conditions in high-rate anaerobic bioreactors (high sludge content, moderate sulphide levels), chemical azo dye reduction by sulphide hardly contributes to the overall reaction. Anaerobic azo dye reduction is therefore mainly a biological process, either a direct enzymatically catalysed reaction involving non-specific enzymes or a reaction with enzymatically reduced electron carriers. Azo dye reduction by sludge that had not earlier been exposed to dyes was found to relate to the oxidation of endogenous substrate and, especially, to the oxidation of hydrogen when present in bulk concentrations. Enrichment was required for the utilisation of electrons from volatile fatty acids for dye reduction.Examination of the reduction of twenty chemically distinct azo dyes by anaerobic granular sludge revealed a large variation in the reaction rates. Especially reactive azo dyes with triazyl reactive groups were slowly reduced. For these common occurring reactive dyes, long contact times may be necessary to reach a satisfying extent of decolourisation. Consequently, they pose a serious problem for applying high-rate anaerobic treatment as the first stage in the biological degradation of azo dyes. However, this problem can be overcome by using redox mediators, compounds that speed up the reaction rate by shuttling electrons from the biological oxidation of primary electron donors or from bulk electron donors to the electron-accepting azo dyes.It was observed that one of the constituent aromatic amines of the azo dye Acid Orange 7 had an autocatalytic effect on the dye's reduction, probably by acting as a redox mediator. Other compounds, e.g. the artificial redox mediator anthraquinone-2,6-disulphonate (AQDS), a compound that is known to catalyse the reductive transfer of several pollutants, and the commonly occurring flavin enzyme cofactor riboflavin, were found to be extremely powerful catalysts, capable of raising the pseudo first-order reaction rate constants by orders of magnitude. Moreover, a large stimulatory effect was found for autoclaved sludge, presumably due to the release of internal electron carriers, e.g. enzyme cofactors like riboflavin, during autoclaving.AQDS was successfully applied to improve the continuous reduction of Reactive Red 2 (a reactive azo dye with a triazyl reactive group) in a lab-scale anaerobic bioreactor that was operated under moderate hydraulic loading conditions. Without AQDS, the reactor's dye removal efficiency was very low, which gave rise to severe dye toxicity towards the biological activity. Addition of catalytic concentrations of AQDS to the reactor influent caused an immediate increase of the dye removal efficiency and recovery of the methane production. Eventually, almost complete RR2 colour removal could be reached.Though effective AQDS dosage levels are low, continuous dosing has disadvantages with respect to the costs and the discharge of this biologically recalcitrant compound. Therefore, the feasibility of activated carbon (AC), which is known to contain quinone groups at its surface, to act alternatively as an insoluble/immobilised redox mediator was explored. Incorporation of AC in the sludge of lab-scale anaerobic bioreactors that treated Reactive Red 2 in synthetic wastewater containing volatile fatty acid as primary electron donor resulted in enhanced continuous dye reduction as compared to the control reactors without AC. The effect of AC was in large excess of its dye adsorption capacity. In addition, it was shown that bacteria could utilise AC as terminal electron acceptor in the oxidation of acetate. Moreover, AC catalysis of chemical azo dye reduction by sulphide was demonstrated. These results clearly suggest that AC accepts electrons from the microbial oxidation of organic acids and transfers the electrons to azo dyes, thereby accelerating their biological reduction.The research presented in this thesis makes clear that the reduction of azo dyes can be optimised by utilising redox mediators, i.e. either by continuous dosing of soluble quinones or by incorporation of AC in the sludge blanket. The potential of using redox mediators is probably not limited to enhancing azo dye reduction but may be extrapolated to other non-specific reductive (bio)transformations, e.g. reduction of halogenated or nitroaromatic compounds. The potential of using redox mediators is furthermore probably not limited to wastewater treatment but may also apply to bioremediation of soils polluted with e.g. polychlorinated solvents or nitroaromatic pesticides.
... A few studies have been conducted to determine which bacterial species may be responsible for reducing and degrading the azo compounds. However, these experiments have been conducted with facultatively anaerobic enteric bacteria, which constitute only a small portion of the total intestinal microflora (15,17,19,20). ...
... B. thetaiotaomicron (C20-14) was chosen for this study, since it is one of the predominant anaerobes in the gastrointestinal tract and is capable of reducing all the dyes listed in Table 1. Tartrazine was used as the substrate, since it is a common azo dye, and its reduction by Proteus vulgaris has been studied previously (17). ...
... The metabolism of tartrazine in animals, for example, has been studied by several investigators (11,13). The major urinary metabolite is sulfanilic acid, which is probably produced by the reduction of tartrazine by the intestinal microorganisms (17). Another possible metabolite, sulfophenyl-3-carboxy-4-aminopyrazolone, was not found. ...
Reduction of seven azo dyes (amaranth, Ponceau SX, Allura Red, Sunset Yellow, tartrazine, Orange II, and methyl orange) was carried out by cell suspensions of predominant intestinal anaerobes. It was optimal at pH 7.4 in 0.4 M phosphate buffer and inhibited by glucose. Flavin mononucleotide caused a marked enhancement of azo reduction by Bacteroides thetaiotaomicron. Other electron carriers, e.g., methyl viologen, benzyl viologen, phenosafranin, neutral red, crystal violet, flavin adenine dinucleotide, menadione, and Janus Green B can replace flavin mononucleotide. These data suggest that an extracellular shuttle is required for azo reduction.
... Since the 20th century (1970s), the environmental importance of these quinoid compounds has received increasing attention. In 1967, Roxon et al. [1] reported that riboflavin, a quinone-like compound, can accelerate the transformation of some organic contaminants. Subsequently, an increasing number of researchers began to focus on the application of quinone compounds (e.g., anthraquinone-2,6-disulfonate, anthraquinone-2-sulfonate, humus, carbon materials, etc.) for the transformation of environmental contaminants [2]. ...
... In addition, the presence of quinoid compounds can accelerate reactions by lowering the activation energy of the total reaction and may even be a prerequisite for the reaction to take place under some situations. To date, many kinds of quinoid compounds have been proven to be capable of facilitating electron transfer in redox reactions between diverse inorganic or organic compounds, including organic matter, metals, and minerals [1,2]. Over the past twenty years, quinoid compounds have attracted more attention than before because of their significant interactions with microbes, which may further increase their environmental importance. ...
In recent years, quinoid redox mediators (QRMs) have attracted increasing attention because of their key role in wastewater biotreatment. Previous studies have shown that the anaerobic respiration of many bacteria could be coupled to the reduction and reoxidation of quinone groups. Thus, QRMs are widely involved in the microbial transformation of various organic and inorganic substances. To date, few reviews have focused on the involvement of quinoid compounds in environmental pollution biotreatment processes. In this paper, we review the different types of QRMs that interact closely with microorganisms, the characteristics of those QRMs, the involvement of QRMs during the biotransformation of recalcitrant organic pollutants, heavy metal ions and metallic oxides, and their enhancement on microbial fuel cells. Finally, the future research focus and application prospects with regard to different types of QRMs are proposed. This study can improve our understanding of QRM-mediated environmental pollution biotreatment processes and provide fundamental guidance on what kinds of QRMs are practical for engineering applications.
... As early as 1931, in a study on bacterial electricity generation, it was demonstrated that electromotively active organic compounds, such as benzoquinone, or inorganic compounds, such as ferricyanide, could increase the electric current of a bacterial half-cell (Cohen, 1931). To our knowledge, the first paper mentioning a role of quinoid enzyme cofactors in the reduction of azo dyes dates from the late 1960s ( Roxon et al., 1967). RM-catalyzed reduction of nitroaromatics has firstly been reported by Brown (1981). ...
... Tables 1-5 show that a wide range of molar mediator/dye-ratios has been applied. In several studies, two or more ratios have investigated, usually by changing the mediator concentration ( Roxon et al., 1967;Walker et al., 1971;Kudlich et al., 1997 Yemashova and Kalyuzhnyi, 2006;Hong et al., 2007). Dye reduction rates increase with increasing mediator concentrations. ...
Evidence is accumulating that inert humic substances can play important roles in the anaerobic degradation of priority pollutants by shuttling electrons. This paper reviews the roles of humus and quinone analogues as electron acceptors, redox mediators and electron donors for microbial and abiotic degradation processes. An eventual technology based on pumping quinones as terminal electron acceptors into aquifers and sediments to stimulate xenobiotic degradation offers promising potential. Also quinone redox mediators can be considered to accelerate reductive transformations (e.g. dechlorination, azo cleavage) of priority pollutants.
... As early as 1931, in a study on bacterial electricity generation, it was demonstrated that electromotively active organic compounds, such as benzoquinone, or inorganic compounds, such as ferricyanide, could increase the electric current of a bacterial half-cell (Cohen, 1931). To our knowledge, the first paper mentioning a role of quinoid enzyme cofactors in the reduction of azo dyes dates from the late 1960s (Roxon et al., 1967). RM-catalyzed reduction of nitroaromatics has firstly been reported by Brown (1981). ...
... Tables 1-5 show that a wide range of molar mediator/dye-ratios has been applied. In several studies, two or more ratios have investigated, usually by changing the mediator concentration (Roxon et al., 1967;Walker et al., 1971;Kudlich et al., 1997;Van der Zee et al., 2000;Cervantes et al., 2001b;Van der Zee et al., 2001;Rau et al., 2002;Dos Santos et al., 2003;Field and Brady, 2003;Dos Santos et al., 2004b;Albuquerque et al., 2005;Dos Santos et al., 2005b;Dos Santos et al., 2005c;Dos Santos et al., 2005d;Yemashova and Kalyuzhnyi, 2006;Hong et al., 2007). Dye reduction rates increase with increasing mediator concentrations. ...
During the last two decades, extensive research has explored the catalytic effects of different organic molecules with redox mediating properties on the anaerobic (bio)transformation of a wide variety of organic and inorganic compounds. The accumulated evidence points at a major role of electron shuttles in the redox conversion of several distinct contaminants, both by chemical and biological mechanisms. Many microorganisms are capable of reducing redox mediators linked to the anaerobic oxidation of organic and inorganic substrates. Electron shuttles can also be chemically reduced by electron donors commonly found in anaerobic environments (e.g. sulfide and ferrous iron). Reduced electron shuttles can transfer electrons to several distinct electron-withdrawing compounds, such as azo dyes, polyhalogenated compounds, nitroaromatics and oxidized metalloids, among others. Moreover, reduced molecules with redox properties can support the microbial reduction of electron acceptors, such as nitrate, arsenate and perchlorate. The aim of this review paper is to summarize the results of reductive (bio)transformation processes catalyzed by electron shuttles and to indicate which aspects should be further investigated to enhance the applicability of redox mediators on the (bio)transformation of contaminants.
... The second model is based on studies with facultative anaerobes by Roxon et al. (1967) and Walker (1971), which suggested that unspecific reduction of azo dyes was carried out by reduced flavins generated by cytosolic flavin reductases. But the inability of the reduced flavins to cross the cell membrane and the highly polar sulfonated azo dyes was found to be limiting factor of second model. ...
... Выделены чистые культуры микробов, разлагающих азокрасители. Так, показано, что Proteus vulgaris восстанавливал красители тартразин, амарант, «солнечный закат» [9], Streptococcus faecalis способен к азоредукции при добавлении НАДФ [10]. Близкая активность обнаружена у многих других видов микроорганизмов [11,12]. ...
Food additives and, in particular, food colours are becoming more widespread in all countries. The review is devoted to the least studied problem of synthetic food colours safety assessment approved for use in the Russian Federation to the analysis of their genotoxic effect (mechanisms, methods of determination and results of studies on various living objects). Presented results of the synthetic food colours genotoxicity half-century study demonstrated that among of studied colours there was none for which unambiguous research results were obtained, that allows us to conclude the possibility of their real mutagenic and/or carcinogenic danger. It is shown that the problem of the dose range selecting for genotoxicity testing, the associated problem of impurities control as well as approaches to test systems and test objects selection are the key to ensuring the genetic/carcinogenic safety of food colours. These problems are aggravated by the fact that in Russian Federation there is no unified system for food colours genetic safety assessing. So the main task of this publication is to prove the urgent necessity for elaboration of this system and outline group of main problems associated therewith.
... This H-bonding interactions make rubazoic acids derivatives as interesting compounds to probe the nature of the proton potential energy profile both in the liquid and solid states. Many of the water-soluble azo dyes are known to be degraded by intestinal microorganisms in vitro [2] and in vivo [3][4][5] and rubazoic acid formation occured as intermediate during degradation of dyes. Its formation was first of all reported by Knorr in 1887. ...
A kinetic study based on spectrophotometric and titrimetric techniques has been carried out for the formation of Rubazoic acid type derivative from 4-amino-1(4-sulphophenyl) 3-methyl-2-pyrazolin-5-one (4-amino SPMP). Physico-chemical factors like effect of Time, pH, aeration and temperature on the formation of Rubazoic acid derivative have been studied in detail. The oxidizing and diazotizing property of nitrous acid has been evaluated for 4-amino-SPMP. Moreover the diazonium compound of SPMP was isolated and its stability in solid form has also been investigated.
... It is reduced in the organism to highly sensitizing aromatic amine because it is a nitrous derivative (azo class). Its main metabolite is sulfanylic acid (Jones et al. 1964;Roxon et al. 1967;Maekawa et al. 1987;Chung et al. 1992). Some of these amines are toxic, carcinogenic, and mutagenic (Chung 2000;Zhang and Ma 2013). ...
Tartrazine is one of the most widely used food additives. The present investigation was carried out on 40 adult male albino rats. They were divided into four groups of ten animals for each. Group I was considered as a control group. Group II was treated with tartrazine daily in a dose 7.5 mg/kg body weight by oral gavage for 30 days. Group III was received 15 mg/kg body weight of tartrazine for the same period. Group IV was administered tartrazine in a dose 100 mg/kg body weight for the whole duration of the experiment. At the end of experiment, samples from the cerebellum, submandibular salivary glands, and kidneys were fixed in neutral buffered formalin 10% and prepared routinely for paraffin sectioning and staining for histopathological and immunohistochemical investigations of proliferating cell nuclear antigen “PCNA” and glial fibrillar acidic protein “GFAP”. Tartrazine-treated groups revealed histopathological degenerative changes in the obtained organs. In group II, the cerebellum showed subcortical edema, congestion of the blood vessels, cytoplasmic vacuolations, and pyknosis of the nuclei in the gray matter neurons. Concerning the submandibular glands, they expressed cytoplasmic vacuolations and pyknosis of the nuclei of the acinar cells, congestion of the interacinar blood capillaries, and degenerative changes in the striated duct. The kidneys appeared with interstitial hemorrhage and dilatation of the glomerular capillaries. The PCT and DCT showed ill-defined cell boundaries. The collecting tubules in the renal medulla appeared with flattened epithelial cells. The severity of these changes increases by increasing the dose of tartrazine in group III and reach to the highest level in group IV. The immunoexpression of the GFAP in the cerebellum of the experimental groups was intense compared to the control group. The immunoreactivity of PCNA in the nuclei of the acinar and ductal cells of the submandibular gland and the cells of the renal cortex and medulla was strong in the tartrazine-treated groups compared to the control group. The current study concluded that the tartrazine had serious effect on the cerebellum, submandibular glands, and kidneys that adversely affect the functions of these organs.
... Similarly, lignolytic enzymes secreted extracellularly by fungal strains also produce higher decolourization of azo dyes by bacterial as well as fungal culture [16,17] results in more complete degradation and avoids accumulation intermediates. Various authors [18,19] emphasized the necessity of involvement of azo reductase cytoplasmic enzyme in the decolourization of azo dyes, often assuming electron [20]. Although decolourization may result from chemical reduction with reduced coenzymes, the coenzymes depend on cytoplasmic reducing enzymes to supply electrons. ...
Natural as well as synthetic dyes are well known and used for dyeing of various materials. Most common use of dyestuffs is in textile industry. Proper decolourisation and disposal of waste water from dyeing industry is an important issue. Symmetrical nitroformazans constitute a special class of azo-hydrazone dyes which have found application in dyeing industry. These dyes are a major cause of water pollution and need to be removed or destroyed before the disposal of waste water from dyestuff industry. Several methods like physical, chemical and biological have been reported for removal of dye in waste water. The commonly used physico- chemical techniques are costly, less efficient and are liable to interference by other waste water constituents. In the present study fungus, Coriolus versicolor was used for decolourisation of 3-nitro-1,5-diarylformazans. Maximum decolourisation of dye was observed after 96 hours incubation and minimum after 24 hour incubation.
... These metabolites are absorbed and appear in the urine and only a minor proportion of any oral dose of tartrazine is absorbed intact (Jones et al., 1964;Roxon et al., 1966;Ryan et al., 1969a,b). The enzymatic azo reduction of tartrazine has been shown to be dependent on gut microorganisms (Roxon et al., 1967;Ryan et al., 1969a,b). After intraperitoneal administration of 2.4 mg/kg bw of 14C-tartrazine, between 64 and 96% of the dose was recovered unchanged in urine within 24 h in rats and rabbits and no other products were reported. ...
Tartrazine is a food colour that activates the transcriptional function of the human oestrogen receptor alpha in an in vitro cell model. Since oestrogens are cholestatic, we hypothesised tartrazine will cause periportal injury to the liver in vivo. To test this hypothesis, tartrazine was initially administered systemically to mice resulting in a periportal recruitment of inflammatory cells, increased serum alkaline phosphatase activity and mild periportal fibrosis. To determine whether an oestrogenic effect may be a key event in this response, tartrazine, sulphonated metabolites and a food additive contaminant were screened for their ability to interact with murine oestrogen receptors. In all cases, there were no interactions as agonists or antagonists and further, no oestrogenicity was observed with tartrazine in an in vivo uterine growth assay. To examine the relevance of the hepatic effects of tartrazine to its use as a food additive, tartrazine was orally administered to transgenic NF-?B-Luc mice. Pre- and concurrent oral treatment with alcohol was incorporated given its potential to promote gut permeability and hepatic inflammation. Tartrazine alone induced NF- ?B activities in the colon and liver but there was no periportal recruitment of inflammatory cells or fibrosis. Tartrazine, its sulphonated metabolites and the contaminant inhibited sulphotransferase activities in murine hepatic S9 extracts. Given the role of sulfotransferases in bile acid excretion, the initiating event giving rise to periportal inflammation and subsequent hepatic pathology through systemic tartrazine exposure is therefore potentially associated an inhibition of bile acid sulphation and excretion and not on oestrogen receptor-mediated transcriptional function. However, these effects were restricted to systemic exposures to tartrazine and did not occur to any significant effect after oral exposure.
... The study of the carcinogenetic and mutagenetic effects of Tartrazine were established by some authors which gives variable results [6][7][8][9][10][11][12][13][14][15][16] . ...
The following study is about Tartrazine (E 102) which is known as an azo dye used in food products, drugs and cosmetics. As a part of the safety assessment of Tartrazine, a 13-week subchronic oral toxicity study was performed on Wistar rats of both sexes. The animals were divided into 5 groups of 6 animals each, 3 of each sex, and fed a diet containing 5, 7.5, or 10 mg/kg b.w, of Tartrazine and 3.75 mg/kg b.w, of Sulfanilic acid. There were no treatment-related adverse effects with regard to body weight, food and water consumption. Their blood samples were analyzed for hematological measurements, Glucose, Creatinine, Blood urea nitrogen, Cholesterol total, Triglecerid, alanine aminotransferase, aspartate aminotransferase. The Stomach, Jejunum, Liver, Kidneys tissues were also processed for histological examination. The present study shows that Tartrazine and Sulfanilic acid induced a morphological change from the discoid shape to an echinocytic form in rat RBCs. Relative weights of the liver were significantly increased in group treated with 10 mg/kg b.w, of Tartrazine. Our data showed a significant increase in GLU, CREA, CHOL, TG, AST, and total Protein in serum of rats treated with Tartrazine and Sulfanilic acid compared to control rats and these significant changes were more apparent in high doses than low ones. The histopathological changes of Liver and Kidney were in accordance with the biochemical findings.
... Co-factors like FADH 2 , FMNH 2 , NADH and NADPH, which provide 'H' for reduction, were located at cytoplasm (Russ et al. 2000). Lysis of cells would release co-factors at extracellular environment could be the possible reason for higher azo dye reduction rates using cell extracts or starving or lysed cells than intact or resting cells (Roxon et al. 1967;Walker 1970;Dubin and Wright 1975;Wuhrmann et al. 1980;Mechsner and Wuhrmann 1982;Russ et al. 2000). ...
Azo dyes are one of the largest groups of textile dyes
, extensively used for dyeing purposes. A significant proportion of these dyes were entering into the surrounding environment in the form of wastewater.
... According to the second mechanism of biological Azo dye reduction, Azo dyes are indirectly reduced by enzymatically reduced electron carriers. Early research has hypothesised that reduced flavins (FADH2, FMNH2, riboflavin) generated by flavindependent reductases can reduce Azo dyes in a non-specific chemical reaction (Gingell and Walker, 1971;Roxon, 1967 ). Flavins were indeed often found to stimulate Azo dye reduction and recent research has revealed that flavin reductases are indeed .anaerobic ...
One of the main problems associated with the treatment of textile wastewater is the removal of dyes. Most (60-70%) of the more than 10,000 dyes applied in textile-processing industries are Azo compounds, i.e. molecules with one or more Azo (N=N) bridges linking substituted aromatic structures. Discharge of Azo dyes is undesirable, not only for aesthetic reasons, but also because many Azo dyes and their breakdown products are toxic toward aquatic life, and mutagenic for humans. Reductive cleavage of Azo bond, leading to the formation of aromatic amines, is the initial reaction during the biological 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 nonspecific with respect to organisms as well as dyes. Various mechanisms, which include direct enzymatic and indirect (mediated) and chemical Azo dye reduction. 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 biodegradation of Azo dyes and mineralization of aromatic amines
... The study of the carcinogenetic and mutagenetic effects of Tartrazine were established by some authors which gives variable results [6][7][8][9][10][11][12][13][14][15][16] . ...
The following study is about Tartrazine (E 102) which is known as an azo dye used in food products, drugs and cosmetics. As a part of the safety assessment of Tartrazine, a 13‐week subchronic oral toxicity study was performed on Wistar rats of both sexes. The animals were divided into 5 groups of 6 animals each, 3 of each sex, and fed a diet containing 5, 7.5, or 10 mg/kg b.w, of Tartrazine and 3.75 mg/kg b.w, of Sulfanilic acid. There were no treatment‐related adverse effects with regard to body weight, food and water consumption. Their blood samples were analyzed for hematological measurements, Glucose, Creatinine, Blood urea nitrogen, Cholesterol total, Triglecerid, alanine aminotransferase, aspartate aminotransferase. The Stomach, Jejunum, Liver, Kidneys tissues were also processed for histological examination. The present study shows that Tartrazine and Sulfanilic acid induced a morphological change from the discoid shape to an echinocytic form in rat RBCs. Relative weights of the liver were significantly increased in group treated with 10 mg/kg b.w, of Tartrazine. Our data showed a significant increase in GLU, CREA, CHOL, TG, AST, and total Protein in serum of rats treated with Tartrazine and Sulfanilic acid compared to control rats and these significant changes were more apparent in high doses than low ones. The histopathological changes of Liver and Kidney were in accordance with the biochemical findings.
... acetate) coupled to an ATP-gain. Roxon et al. (1967) proposed that azo dyes act under anaerobic conditions as alternative terminal electron acceptors for a facultative anaerobic bacterium (Proteus vulgaris) fermenting glucose. ...
Azo dyes are widely used in textile finishing, and have become of concern in wastewater treatment because of their color, bio-recalcitrance, and potential toxicity to animals and humans. Thus, wastewater with azo dyes must be decolorized and furthermore mineralized in appropriate systems combining biological and chemical processes. In this study, the potential for sulfate reducing bacteria (SRB) to decolorize azo dyes was studied, employing the pure culture of Desulfovibrio desulfuricans (D. desulfuricans) with varying sulfate levels. Under sulfate-rich conditions, the sulfide produced from sulfate respiration with pyruvate (electron donor) by D. desulfuricans chemically decolorized the azo dyes C. I. Reactive Orange 96 (RO 96) and C. I. Reactive Red 120 (RR 120). Under sulfate-depleted conditions (≤0.1 mmol/L), the decolorization of RO 96 and RR 120 occurred in correlation with the fermentation of pyruvate by D. desulfuricans. It is suggested that the electrons liberated from the pyruvate oxidation were transferred via enzymes and/or coenzymes (electron carriers) to the dyes as alternative terminal electron acceptors, giving rise to decolorization, instead of to the protons (H+), resulting in the production of H2. Both
... Various authors (Roxon et al. 1967;Rafii et al. 1990;Chung and Stevens 1993) emphasized the necessity of involvement of azo reductase (cytoplasmic enzyme) in the decolorization of azo dyes, often assuming electron carriers (coenzymes) fla- Other work has focused on the final reaction consisting of a simple chemical reduction of azo dyes by electron carriers. Wuhrmann et al. (1980) proposed an intracellular chemical reduction of azo dyes by reduced flavin nucleotides (FADH 2 ). ...
To determine the role of diverse groups of bacteria in the decolorization of a monoazo dye C. I. Reactive Orange 96 (RO 96), batch experiments with an anaerobic mixed culture were made using the selective inhibitors molybdate and 2-bromoethanesulfonic acid (BES). In the presence of acetate and sulfate, inhibition of sulfate reducing bacteria (SRB) by molybdate (8 mmol/L) caused a significant decrease in the decolorization rate of RO 96. Increases in the SRB inhibitor concentration (20, 38 mmol/L) led to slower decolorization of RO 96, confirming the contribution of SRB to the decolorization. Inhibition of methane producing bacteria (MPB) with BES (10, 20 mmol/L), however, did not significantly influence the decolorization, indicating that MPB took no part in the decolorization. In the presence of lactate and sulfate, SRB also contributed to the decolorization of RO 96. The mechanism of decolorization of azo dyes based on extracellular chemical reduction with sulfide was postulated. Sulfide produced via sulfate respiration by SRB, chemically decolorizes azo dyes.
... [9][10][11][12][13] This may come about because the chemical structure of the tartrazine molecule has similar features to those of benzoates, other azo compounds, pyrazole compounds and the hydroxy-aromatic acids, which also include salicylates. 2 Furthermore, it has been established that the azo compounds can be reduced in the intestine and in the liver, 106,107 indicating that one of the several routes through which these molecules, too small to be antigenic in themselves, may act as a hapten, thus conjugating a larger molecule to form an antigenic compound. 2,108,109 A major breakthrough in the understanding of the mechanisms involved in ASA intolerance came also with discovery that aspirin, including other non-steroidal antiinflammatory drugs, inhibit the synthesis of prostaglandins, by selectively blocking the cyclo-oxinase pathway, resulting in an enhanced production of leucotrienes. ...
... In fact, the role of reduced flavins (FMN and FADH) has been shown as accelerators of the azo dye reduction by acting as electron carriers [2,6]. Also, other enzymatic cofactors, such as NADH or NADP(H) have been reported to be active electron donors for the reduction of tartrazine [14]. Artificial redox mediators, such as benzyl viologen [2,151617 or different quinones [16,18] have been reported to accelerate azo dye reduction. ...
Degradation of the azo dye Acid Orange 7 (AO7) in batch anaerobic unstirred assays is described. Experiments were carried out: (i) under abiotic conditions, (ii) with active biomass using only the dye as organic substrate, and (iii) with the dye and different cosubstrates. Non-adapted biomass was used. The results obtained indicate that AO7 was only removed in the presence of active biomass, the removal rates being higher in the presence of a cosubstrate. The highest removal rates were obtained with a high concentration of glucose, 2 g l−1, which indicates that apart from the positive influence of the presence of an excess of reducing equivalents, the improvement of mass transfer conditions in the medium as a consequence of the high biogas production is also a key topic. AO7 yields sulphanilic acid (SA), which was not further degraded and was accumulated in the medium in stoichometric amounts. The other compound resulting from AO7 breakdown, 1-amino-2-naphthol (1A2N), was not detected most likely because of its low stability. However, the detection of 1,2-naphthoquinone (12NQ), a compound generated after the oxidation of 1A2N gives evidence of this mechanism.
... specific processes make use of low molecular weight mediators (e.g. flavins or quinones) which are enzymatically reduced in the cells. In earlier studies with facultative anaerobic bacteria, it was repeatedly suggested that reduced flavins generated by cytosolic flavin-dependent reductases were responsible for the unspecific reduction of azo dyes (Roxon et al,. 1967). Russ et al (2000) later reinforced these earlier findings by demonstrating the ability of cytosolic flavin reductases to act as in-vitro azoreductases using a recombinant flavin reductase in different genetic backgrounds. It was also shown that the addition of external redox mediators e.g. benzene viologen, or quinones to strictly ana ...
The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
... s well are recorded. Anaerobic decolourization of azo dyes by bacteria, the reduction equivalents generated by the oxidation of auxiliary substrates, i.e. organic carbon complexes act as electron donor through NAD(P) + reduce the azo bond via primarily assuming electron carriers (coenzymes) flavins nucleotides (FMN, FAD) or riboflavin as cofactors (Roxon et. al. 1967;Zimmermann et al. 1982, Rafii et at 1990, Chung and Stevens 1993. So incite that 46 reactions may be multi steps and complex to fulfill intracellular donor to xenobiotics (dyes) in the system The study of enzymes and enzyme-catalyzed reactions has contributed greatly to our understanding of microbial metabolism (Philips, 1999). It is alw ...
The method “Intact cell assay” was adopted to assess the influencing factors on the process of decolourization ability of the enterobacteriaceaea specie isolates under predisposed environment. Taken into account several ingredient are added individually as well in combinations in the assay reaction. Values of dye decolourized in μg ml-1 with Multi vitanmins solution (MVS) with glucose 42.53; MVS 36.00; Riboflavin (RF) 39.00; Yeast extract 36.00 and B- complex 23.00 from the initial dye of 46.00μg ml-1 were observed , where as glucose, Ascorbic acid, Cysteine, Cetyl-trimethyl-ammonium bromide (CTAB), Sodium molybdate, Biotin, KNO3, NaNO2, folic acid and 1-amino-2- naphtho-4-sulfonic acid (ANSA) does not shows influence on to the process, but, some of them showed inhibitory effect toward the decolourization. It was observed that the riboflavin addition at 19.95 n moles ml-1 in the reaction mixture, rate of decolourization was suddenly change from 0.019 ΔA/minutes to 0.20 ΔA/minutes, which is extremely high by 10 fold fast and subsequently remains faster i.e 0.2 ΔA/minutes without further additional RF in the same assay mixture. Rate of decolourization with different concentrations of riboflavin i.e. 13.0 n moles ml-1 to 59.9 n moles ml-1 showed second order kinetic. This indicates that the minimum amount of RF is essential to trigger the process of decolourization by the intact cells under the assay condition. While five different azo dyes were subjected, showed diverse behavior on to the rate of decolourization. Results of entire study incite on role of riboflavin could to a certain extent act as a redox mediator in the reaction(s) process and electron mediator between intracellular pool to the dye available at periplasmic redox sink
The gut microbiota in human gene pool and contributes to xenobiotic metabolism. Azo dye compounds represent a large group of colouring chemicals which are widely used in many industries. Although the commonly used azo dyes are not mutagenic in the standard Ames Plate Assay they are reduced by azo reductases from intestinal bacteria and, to a lesser extent, by enzymes of the cytosolic and microsomal fractions of the liver.
The first catabolic step in the reduction of azo dyes, which is accompained by a decrease in the visible light absorbance of the dye and then decolorization of the dye, is the reduction of the azo bridge to produce aromatic amines.
In this study, we developed a plate assay capable of detecting the specific bacteria from human intestinal flora that participate in the reduction of azodyes. The methods we used to conclude the result by the optical density value by using the galvanometer.
Key words: Human gut microbes, azoreductase activity, ames plate assay method, decolorizarion of dye, optical density value (OD Value), galvanometer.
Предмет дослідження. Одним із найважливіших і складних завдань виокремлюється забезпечення населення земної кулі продуктами харчування. Будучи одним з найважливіших чинників навколишнього середовища, харчування від моменту народження до кінця життя людини впливає на його організм. Мета. Проаналізувати дані літературних джерел із результатами наукових досліджень щодо використання синтетичного харчового барвника тартразину (Е102) у харчовій промисловості. Інгредієнти харчових речовин, потрапляючи в організм людини з їжею й перетворюючись в процесі метаболізму в результаті складних біохімічних реакцій на структурні елементи клітин, забезпечують людський організм пластичним матеріалом і енергією, створюють необхідну фізіологічну і розумову працездатність, визначають здоров'я, активність і тривалість життя людини, його здібність до відтворення. Тому стан харчування визнано одним з найважливіших чинників, що визначають здоров'я нації. Продукти харчування повинні не тільки задовольняти потреби людини в основних харчових речовинах і енергії, а й виконувати профілактичні та лікувальні функції. Серед речовин, що визначають зовнішній вигляд харчових продуктів, найважливіше місце належить харчовим барвникам. Згідно з Директивою Європейського парламенту і Ради ЄС 94/36 харчові барвники – це хімічні синтетичні речовини або природні сполуки, які надають чи посилюють колір харчового продукту або біологічних об'єктів; не вживаються зазвичай як харчовий продукт або складова частина їжі. Методи. Під час роботи використовували аналітичні Методи. Результати. Проаналізовано та узагальнено літературні дані щодо властивостей, добування та використання синтетичного харчового барвника тартразину (Е102) у технологіях виробництва продуктів харчування. Зроблено висновок про доцільність та безпечність застосування тартразину в харчовій промисловості. Сфера застосування результатів. Можливість додавання його у ряд харчових продуктів у безпечних кількостях, які не спричиняли б токсичної дії на живий організм, синтетичного харчового барвника тартразину (Е102).
Food color additives are used to make food more appetizing. The United States Food and Drug Administration (FDA) permitted nine artificial colorings in foods, drugs, and cosmetics, whereas the European Union (EU) approved five artificial colors (E-104, 122, 124, 131, and 142) for food. However, these synthetic coloring materials raise various health hazards. The present review aimed to summarize the toxic effects of these coloring food additives on the brain, liver, kidney, lungs, urinary bladder, and thyroid gland. In this respect, we aimed to highlight the scientific evidence and the crucial need to assess potential health hazards of all colors used in food on human and nonhuman biota for better scrutiny. Blue 1 causes kidney tumor in mice, and there is evidence of death due to ingestion through a feeding tube. Blue 2 and Citrus Red 2 cause brain and urinary bladder tumors, respectively, whereas other coloring additives may cause different types of cancers and numerous adverse health effects. In light of this, this review focuses on the different possible adverse health effects caused by these food coloring additives, and possible ways to mitigate or avoid the damage they may cause. We hope that the data collected from in vitro or in vivo studies and from clinical investigations related to the possible health hazards of food color additives will be helpful to both researchers and the food industry in the future.
Tartrazine (TZ) is an azobenzene artificial yellow dye for foods, drugs, and cosmetics. Quercetin is member of the flavonoid family. The current study investigated the protective effect of quercetin nanoparticles (QNPs) against TZ. Male albino rats were divided into group1: control, group 2: TZ (50 mg/kg), group 3: QNPs (5 mg/kg), and group 4: QNPs+TZ. The results of the present study revealed that, the oral administration of TZ dye caused a significantly increased liver biomarkers. Also, a marked effect on lipid profile and blood parameters were shown. In addition TZ induced an elevation in the examined oxidative stress biomarkers and decrease in glutathione peroxidases and acetylcholine esterase. TZ diminished exploration and rearing in open field test as well as elevation in GABA content and apoptosis as well as changes in tissues by histopathological examination. In fact, the results showed good influence of QNPs in improving injuries associated with TZ administration. Keywords: Tartrazine Dye, Quercetin Nanoparticles, Liver-Brain injuries.
An investigation was carried out into the genetic activity of DAB, CDA (Sample A), BZD and DAT in the strain JD1 of Saccharomyces cerevisiae.
Using a protocol developed to enhance the endogenous cytochrome P450 levels present in growing cells, it was found that all four compounds gave a positive response both in the presence and absence of a S9 preparation.
Synthetic dyes
usually consist of aromatic rings, methyl, methoxy, sulfo or nitro group. They are not readily degradable and can not be removed from water by conventional wastewater treatment systems (Anliker in Biodegradation of heterocyclic dyes by Phanerochaete chrysosporium 1990. Utah State University, Logan, 1979).
Studies on the fate of azo and nitro compounds in the body have had a profound influence on the fields of chemotherapy, toxicology, and drug metabolism. For example, recognition that Prontosil was reductively cleaved to sulfanilamide in the body (Tréfouël et al., 1935) led to the development of a large series of sulfa drugs, as well as to the realization that substances which possess little biological activity in vitro may be transformed in the body to substances which exert potent therapeutic effects. Drugs may also undergo biotransformation to toxic derivatives. For example, it is now accepted that methemoglobinemia caused by certain aromatic nitro compounds and aromatic amines arises from the corresponding phenylhydroxy and nitroso compounds formed by the reduction of the nitro compounds or the N-hydroxylation of the amines. It is also now believed that the hepatomas caused by various azo dyes, including butter yellow, are mediated by N-hydroxylated metabolites.
A bacterial species was isolated from textile industrial effluent treatment plant, Jetpur, Dist. Rajkot (Gujarat India), characterized and exploited for its dye degradation potential on textile dyes Golden-HR and Magenta-HB under shaking, static, aerobic and anaerobic conditions. Results obtained indicated the remarkable decolorization potential of the isolate under static, anaerobic and facultative anaerobic condition at 37°C (near natural conditions). The degradation study was carried out using UV-Visible spectroscopy, thin layer chromatography and ring cleavage reaction.
Efforts were made to isolates indigenous microbes from the textile dye contaminated site. A total of 35 strains of organisms were isolated based on colonies and morphological charactreristics and were primary screened for their ability to decolourized the textile dye Remazol magenta HB under static culture conditions in presence of three different nutrients in minimal salt medium. Primary screened out 10 isolates were further screened for decolourisation ability against sixty different commercial textile dyes and the most potent six isolates have been further studied for their specific requirements to obtain maximum declourization capability in cultured condition. Results of dyes acted by the isolates were observed that the Iso-32 strain has completely decolourized maximum number of dyes i.e. thirty one dyes followed by Iso-26 showed twenty nine days and other four stains acted on more than twenty days. Most potential of these six strains Bio-1 to Bio-6, were subjected for optimization of influences of various nutrient ingredients supplements such as carbon sources, nitrogen sources and nutrient and/or growth factors. Results were found that in presence of the yeast extract, beef extract, multivitamin solution and riboflavin individual in minimal salt media showed maximum near about to 100% decolourized within 24 hours of incubation at 30°C temperature under static condition. In case of inorganic nitrogen sources conclusive results were seen with ammonium sulfate, di-ammonium hydrogen phosphate and ammonium chloride, whereas, nitrate and nitrite salts of ammonia, the process of decolourization was very less (10% to 46%), suggesting that NO3- or NO2- might have been adverse effects on decolourisation. Among the various sugars tested at varies concentrations for their effect on dye decolourization by the six isolates glucose, sucrose, lactose, and mannitol were showed maximum at about 0.5% concentration except Bio-6, which required 1.0% to get maximum decolourisation.
In the context of food toxicology the gastrointestinal tract is of major importance in that it is the primary site of exposure to potential toxicants and the concentrations involved are likely to be higher than those in other tissues. This can lead to localised lesions due to irritation or to more specific toxic effects. Furthermore, dietary components may undergo metabolism in the gut by endogenous mammalian enzymes or by the gut microflora and this may significantly influence the toxicological sequelae. Pre-existing enteropathies or enzyme deficiencies can alter the course of both these events.
Tartrazine is a yellow food colorant, widely used in food products, drugs and cosmetics. The acceptable daily intake (ADI) oh human is 0-7.5 mg/kg body weight. The objective of our study was to examine the toxicity of Tartrazine and its main metabolite Sulphanilic acid to the nematode Caenorhabditis elegans, Brine Shrimp larvae (Artemia Salina) and KGN granulosa cell line; in the aim to develop our knowledge about their toxicity effects. In this research, toxicity of Tartrazine and Sulphanilic acid were examined to the nematode Caenorhabditis elegans with Escherichia coli as a food source. Our results showed that from a 3 mM concentration of Tartrazine, and 1mM of Sulphanilic supplementation can disrupt the cell cycle nematode C. elegans even if it does not cause death. Different concentrations of Tartrazine and Sulphanilic acid (1, 2.5, 5, 7.5, 10, 25 50, 75, 100 µg/ml) were tested for their toxicity in a short term bioassay using Brine Shrimp (Artemia salina). The Brine Shrimp were hatched in artificial sea water and exposed to the Tartrazine and Sulphanilic acid after 48 hours. LC50 values were calculated after probit transformation of the resulting data. Tartrazine did not show any significant toxicity against Brine Shrimp but Sulphanilic acid was mildly toxic (LC50 value (μg/ml) of ~82.3 μg/ml). The Brine Shrimp assay proved to be a convenient and rapid system for toxicity assessment. The human KGN ovarian granulosa-like tumor cell culture line has been used as an in vitro system for determination of the effects of Tartrazine and sulphanilic acid, the result showed that Tartrazine and Sulphanilic acid were unaffected after 24 h of treatment exposure.
A number of poly(ether-ester) azo polymers consisting of various concentrations of 4-{2-[2-(2-hydroxyethoxy) ethoxy]ethoxy} benzoic acid (HEEEBA), 4-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}phenylazobenzoic acid (HEEEPABA) and either 12-hydroxydodecanoic or 16-hydroxyhexadecanoic acid were synthesized. The polymers were assessed in order to determine whether they are biodegradable by the azoreductase present in the rat caecum. The polymers containing HEEEBA showed satisfactory degradation (up to 48%), while those without showed poor degradation, due to poor wettability. When 1% polysorbate 80 (a wetting agent) was added to the polymers in the degradation medium, all polymers showed excellent degradation. Azo polymers with HEEEBA have the potential to be used as colon-specific drug release materials.
The microbial reduction of relatively simple azo dyes was investigated. The reduction in the initial step of the microbial degradation of the dyes was microorganism‐specific or dye‐specific, because the reduction of the dyes was directly dependent not only on the presence of azoreductase in the microorganisms, but on the permeation of the dye molecules into the cells. The cell permeability barrier of the dye molecules was observed for both sulfonated azo dyes and unsulfonated azo dyes. It was also observed that microbial azoreductase exhibit a narrow specificity.
Literature regrading azo dye carcinogenicity was examined to establish, if possible, guidelines to predict the human health risks of new azo dyes. Three different mechanisms for azo dye carcinogenicity were identified, all involving metabolic activation to reactive electrophilic intermediates that covalently bind DNA. In the order of decreasing number of published references, these mechanisms are 1. Azo dyes that are toxic only after reduction and cleavage of the azo linkage to give aromatic amines, mostly via intestinal anaerobic bacteria. The aromatic amines are met‐abolically oxidized to reactive electrophilic species that covalently bind DNA.2. Azo dyes with structures containing free aromatic amine groups that can be meta‐bolically oxidized without azo reduction.3. Azo dyes that may be activated via direct oxidation of the azo linkage to highly reactive electrophilic diazonium salts.Each mechanism may be compound specific, thus azo toxicity is probably caused by more than one mechanism. Although it is not possible to predict azo dye carcinogenicity with absolute certainty, it is possible to establish certain guidelines. Because some species of intestinal anaerobic bacteria (and in some cases, hepatic azo reductases) may reduce any azo compound to aromatic amines, those containing aromatic amine subgroups known to be carcinogenic, such as benzidines, must be suspect. Information about human carcinogenicity of other specific aromatic amines is scant, and various short‐term mutagenicity tests may provide some guidance. Other in vitro tests can directly assay new azo dyes. Although it is unlikely that azo dyes can be developed that can be guaranteed not to generate constituent aromatic amines, it may be possible to select aromatic amines that are not toxic.
The surface reduction behavior of nine monoazo dyes at the static mercury drop electrode has been studied by adsorptive square wave voltammetry. Three types of surface reduction behavior are seen among the nine azo dyes. Two of them are modeled as a two-step mechanism in which the first step is a quasi-reversible reduction followed by a totally irreversible reduction. For those dyes having strong electron-donating groups such as amino or dimethylamino, the second reduction occurs at a potential almost the same as that of the first, and only one peak is observed. The second step gradually separates from the first step as the pH-value of the solution increases. For those azo dyes having methoxy or hydroxy substituents, two peaks are observed; one is quasi-reversible, the other is totally irreversible. The second, irreversible step gradually overlaps the first step as the pH-value of the solution decreases. The third type voltammogram of these monoazo dyes displays a single irreversible peak that is modeled as a four-electron one-step irreversible reduction. These surface reduction behaviors are quantitatively explained by the proposed models. Kinetic parameters for these surface reactions are obtained by non-linear least squares (COOL) analysis of the voltammograms. These techniques are useful both for establishing mechanisms and chemical analysis.
Investigation into the biodegradative capability of several actinomycetes led to the discovery of many strains possessing an ability to decolorize a variety of azo and triphenylmethane dyes. Of particular interest is an Amycolatopsis sp. isolate which displayed the ability to decolorize the azo dye amido black. Through the screening of a DNA library a 4.5 kbp fragment coding for the potential decolorization gene was identified. The sequencing of this gene fragment led to the prediction of seven open reading frames encoding a polyprenyl synthetase, cupin-2 conserved barrel domain, transcriptional regulator, membrane protein, DNA-damage inducible gene and two hypothetical proteins. A gene coding for a protein consisting of 312 amino acids with 77 % similarity to a conserved hypothetical protein in Amycolatopsis mediterranei was tentatively identified as the enzyme of interest. This is the first report of an amido black biodegrading gene identified in this species.
The degradation of several azo dyes by cell-free extract from Aeromonas hydrophila var. 24B has been studied. The compounds 4-dimethylaminoazobenzene, 4-diethylaminoazobenzene, 2′-carboxy-4-dimethylaminoazo-benzene, 2′-carboxy-4-diethylaminoazobenzene, 4′-carboxy-4-dimethylaminoazobenzene, and 2′-methoxy-carbonyl-4-dimethylaminoazobenzene were appreciably degraded by the cell-free extract. Azobenzene and 4-carboxyazobenzene were not degraded. Aniline was detected by gas chromatography-mass spectrometry as a degradation product from 4-aminoazobenzene and 4-dimethylaminoazobenzene. 4-DimethyIaminoaniline was detected by gas chromatography-mass spectrometry and spectrophotofluorimetry as a degradation product from 2′-carboxy-4-dimethylaminoazobenzene. The cell-free extract had high substrate specificity for 2′-carboxy-4-dimethylaminoazobenzene.
Abstract The degradation of Fast Red E, Amaranth, Ponceau 4R and Ponceau 6R in anaerobic incubates of rat caecal extract was studied by spectrophotometric scanning. The compounds underwent reduction of the azo linkage and thus loss of colour. The reaction rates seem to depend on the number rather than position of the sulphonate groups in the molecule.
Permeabilization of cells of B. cereus and other bacterial strains by toluene treatment significantly increased the passage of sulfonated and carboxylated azo dyes from the external medium into the cells with a concomittant increase of the reduction rate of the dyes. Dyes which are not reduced at all by intact cells were readily decolorized. The reduction rate of sulfonated compounds was consistently larger than of their carboxylated analogues, once the dyes had entered the cells.
Investigations on the effects of pH, temperature, type and concentration of respiration substrates and oxygen tension on the reduction rate of derivatives of 1-phenylazo-2-naphthol and of a variety of textile dyes served as a basis for establishing a bioassay for strictly reproducible measurements of the microbial reduction rate of azo dyes. Standard organism was a strain of Bacillus cereus isolated from soil. Dye reduction occurred with the standard organism and other facultatively or obligatory aerobic bacteria in exclusively anoxic conditions. In principle, first order kinetics of decolorization were found. Reduction products may however inhibit the reaction. All dyes not measurably reduced by living cells of B. cereus were decolorized by cell extracts of the same species. Dyes adsorbed by the cell walls were in most cases reduced at slow rates and did not influence the simultaneous reduction of non-adsorbable dyes in the medium. The observations confirm the hypothesis advanced by Gingell and Walker (1971) of an intracellular, non-enzymatic reduction of azo compounds by reduced flavin nucleotides. The rate of permeation of the dyes through the cell membrane is the primordial ratelimiting step in the microbial decolorization of azo dyes. Sulfonic acid substitution seems to be an effective inhibitor of permeation.
p-Aminoazobenzene (PAAB) was degradated byBacillus subtilis. Both aniline and p-phenylenediamine as degradative compounds from PAAB were identified by thin layer chromatographic-, and high performance liquid chromatographic-methods. This fact suggests that the first degradative reaction of PAAB byB. subtilis is reductive fission of azo bond in PAAB.
Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All
the strains could decolorize azo dye up to 900mgl−1, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten
kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed
azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.
This work was undertaken to study some factors affecting the bacterial reduction (cleavage) of azo compounds, knowledge of which will be of use in the development of azo cross-linked polymers for colon-specific drug delivery. A common colonic bacterium, Bacteroides fragilis was used as test organism and the reduction of azo dyes amaranth, Orange II and tartrazine were studied; also a model azo compound, 4,4′-dihydroxyazobenzene. It was found that the azo compounds were reduced at different rates and the rate of reduction could be correlated with the half-wave (redox) potential of the azo compounds. 4,4′-Dihydroxyazobenzene (E1/2 −470 mV) was reduced at the fastest rate of 0.75 mol l−1 h−1, amaranth (E1/2 −568 mV) at 0.30 mol l−1 h−1, Orange II (E1/2 −648 mV) at 0.2 mol l−1 h−1 and tartrazine (E1/2 −700 mV) at 0.08 mol l−1 h−1. Similar observations were made with another colonic bacterium Eubacterium limosum. Reduction of 4,4′-dihydroxyazobenzene did not occur under conditions of aeration, but was enhanced by the low molecular weight electron carrier benzyl viologen, with time for 50% azo reduction being decreased from 120 min to 30 min. These studies with a common, numerically important, colonic bacterium indicate that the reduction of an azopolymer may be influenced by the chemical nature of the azo compound used as cross-linker.
Improved delivery systems are needed for drugs currently in use to treat localized diseases of the colon. One promising approach is to deliver the drugs specifically to the colon, an approach that has gained importance recently in the treatment of these diseases. The advantages of targeting drugs specifically to the diseased colon include fewer systemic side effects, a need for lower doses of drugs, and maintenance of the drug in its intact form close to the target site. The potential for colon-specific delivery of therapeutic proteins and peptides is also of interest. To achieve colon-specific drug delivery following oral administration, the drug needs to be protected from absorption by the upper gastrointestinal tract and from degradation by the upper gastrointestinal tract environment, allowing the drug to be abruptly released into the proximal colon. One strategy for targeting orally administered drugs to the colon exploits carriers that are degraded specifically by colonic bacteria and utilizes microbially degradable polymers/drugs, especially azo-cross-linked polymers/drugs. Prodrugs utilizing azo linkages are sulfasalazine, ipsalazine, balsalazine, and olsalazine. These were developed for delivery of 5-amino salicylic acid to the colon for localized chemotherapy of inflammatory bowl disease. The azo-conjugation approach utilizes the ability of the colonic environment to cleave these conjugates and protects the drug from absorption or degradation in the upper gastrointestinal tract. It is believed that flavin mediators present in the colon and azo-reductase enzymes released from colonic bacteria are responsible for the degradation of azo-aromatic compounds for site-specific delivery of the drug to the colon.
Azo dyes are a widespread class of poorly biodegradable industrial pollutants. In anaerobic environments, azo bonds are reductively cleaved yielding carcinogenic aromatic amines, many of which are assumed to resist further metabolism by anaerobes. Here we report for the first time that an azo dye compound is completely biodegradable in the absence of oxygen. A pharmaceutical azo dye, azo-disalicylate, constructed from two 5-aminosalicylic acid (5ASA) molecules was mineralized in an adapted methanogenic consortium to CH4 and NH3 with transient accumulation of 5ASA as a degradation intermediate in both batch assays and continuous bioreactors. The anaerobic metabolism of 5ASA was shown to provide the electrons required for the initial reductive cleavage of the azo group. Our results suggest that it is possible to design azo dyes that are fully mineralized in the environment; thereby, avoiding accumulation of notorious toxic intermediates.
Publisher Summary This chapter discusses the determination of triphosphopyridine and diphosphopyridine nucleotide oxidases; and describes the assay methods for old yellow enzyme and new yellow enzyme. In the case of old yellow enzyme, the assay method is based on the manometric determination of the oxygen uptake in the following system: glucose-6-phosphate-Zwischenferment-TPN-yellow enzyme-molecular oxygen. Under the specified conditions the concentration of TPN is high, its reduction proceeds rapidly, and the concentration of the yellow enzyme is ratedetermining in the consumption of oxygen. Catalase, present as an impurity in the Zwischenferment preparation, is inhibited by cyanide to prevent the decomposition of H 2 O 2 . In the case of new yellow enzyme, G-6-P is oxidized by molecular oxygen, and the oxygen consumption, which is a function of the concentration of the new yellow enzyme, is measured manometrically. Both the leuco forms of the old and the new yellow enzyme are rapidly oxidized by methylene blue, but the new yellow enzyme is reduced four times as fast by TPNH, and it is oxidized seven times as slowly by molecular oxygen as the old yellow enzyme. This difference in activity permits a quantitative distinction of the two enzymes under the test conditions outlined in the chapter.
A bacterium isolated from rats and identified as a Proteus species has been found to reduce tartrazine. The reaction has been followed by disappearance of the colouring with formation of sulphanilic acid. The other product of reduction is 1-p-sulphophenyl-3-carboxy-4-amino-5-pyrazolone. The conditions of the reaction have been studied. It is inhibited by oxygen and is glucose dependent. Cell suspensions which have previously been incubated with air or oxygen reduce tartrazine faster than other preparations examined. The particulate fraction of the pre-incubated preparation requires cofactors present in the soluble fraction to effect the reduction.
A study has been made of the metabolic behavior of twelve compounds which have been used as food colors. The metabolites have been identified by paper chromatography and by isolation, with subsequent characterization by melting point and by ultraviolet and infrared spectroscopy. When azo dyes were administered only small amounts (3%) were excreted in the urine, some 60–80% of the color being excreted as the component amines and derivatives thereof. These colors with a nonsubstituted benzene nucleus were hydroxylated and excreted as o- and p-aminophenol. The color erythrosin was largely excreted, in rats, in the feces (approximately 60%). A small amount of dye was excreted in the bile, but there was no evidence for the urinary excretion of the color. The significance of these observations in relation to the toxicologic properties of the colors and their metabolites is discussed.
The excretion and metabolism of tartrazine has been studied in the rat, rabbit and man. The nature of the excretory products depends upon the route of administration. Intraperitoneal injection leads to the excretion of unchanged colouring. Oral dosage results in the excretion of equivalent amounts of sulphanilic acid which is partly conjugated. A quantitative excretion study has been carried out. The reasons for the resistance of tartrazine to the action of mammalian azo reductase are discussed.
Untersuchungen an Ratten uher Keimzahlversch~ebungen ~m Kot und im Dickdarminhalt nach dem Tode. Zenbtl. Bakt. ParasttKde (Abt. I :Ongmale)
- H Haenal
- C Kunde
Haenal, H. & Kunde, C. (1956). Untersuchungen an Ratten uher Keimzahlversch~ebungen ~m Kot und im Dickdarminhalt nach dem Tode. Zenbtl. Bakt. ParasttKde (Abt. I :Ongmale). 166, 48.
Is azo reductase NADPH-cyt0chrome-c reductase? Pharmacologist 7, 147 Electron transport in cell fractmns of Proteus vulgarts
- P H Hernandez
- P Mazel
- J R Gdlette
Hernandez, P. H., Mazel, P. & Gdlette, J. R. (1965). Is azo reductase NADPH-cyt0chrome-c reductase? Pharmacologist 7, 147. Jones, C. W. & King, H. K. (1964). Electron transport in cell fractmns of Proteus vulgarts. Biochem. J. 91, I 0P.
Removal of nucleoprotein by manganese chloride from protein mixtures Protein measurement with the Folin Phenol reagent The survival of starved bacteria
- S Korkes
- A Campfllo
- I C Gunsalus
- S Ochoa
- O H Lowry
- N J Rosenbrough
- A L Farr
- J R Randall
Korkes, S., del Campfllo, A., Gunsalus, I. C. & Ochoa, S. (1951). Removal of nucleoprotein by manganese chloride from protein mixtures. J. biol. Chem. 193, 721. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. & Randall, J. R. (1951). Protein measurement with the Folin Phenol reagent. J. biol. Chem. 193, 265. Postgate, J. R. & Hunter, J. R. (1962). The survival of starved bacteria. J. gen Mtcrobiol. 29, 233.
The biuret reactlon in the determination of serum protein Microorganisms Indigenous to Man
- H W Robinson
- C G Hogden
- T Rosehury
Robinson, H W. & Hogden, C. G. (1940). The biuret reactlon in the determination of serum protein. J. biol. Chem. 135, 707. Rosehury, T. (1962) Microorganisms Indigenous to Man. McGraw-Hill, New York.
Untersuchungen an Ratten über Keimzahlverschiebungen im Kot und im Dickdarminhalt nach dem Tode
- Haenal
Indentification of Enterobacteriaceae
- Edwards
Removal of nucleoprotein by manganese chloride from protein mixtures
- Korkes
Composition, alteration and effects of the intestinal flora
- Dubos
Electron transport in cell fractions of Proteus vulgaris
- Jones
The survival of starved bacteria
- Postgate