Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm.Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.     

In vitro evolution of RNA aptamers recognizing carcinogenic aromatic amines

The modification of cellular DNA by environmental substances is thought to be a crucial event in chemical induced carcinogenesis. Among the environmental carcinogens, aromatic amines are known for the fact that they can induce several types of cancers through the formation of so-called DNA adducts. We took advantage of the potential of the SELEX method to select for highly specific RNA ligands that recognize specific genotoxic aromatic amines. The aromatic amine 4,4'-methylenedianiline (MDA) was used as a target. Following in vitro selection, we obtained specific MDA-binding RNA molecules based on an affinity chromatography assay. These results open the possibility of using the SELEX technique to generate RNA molecules as diagnostic tools for the detection of DNA damaging compounds and ultimately DNA adducts.     

Chemical characterization of direct-acting airborne mutagens: The functional group

Durham NC air was sampled, extracted, and bioassayed for mutagenic activity in Salmonella typhimurium TA98. Portions of the extracts were treated with sodium borohydride over copper(II) acetylacetonate to reduce any nitroaromatic substances to their corresponding amines. All of the reduced extracts were not directly mutagenic, but the 2 that were derived from cold-weather air samplings did contain substances that could be activated oxidatively. These “indirect-acting” mutagens were present in the same 2 reduced extracts that contained detectable concentrations of aromatic amines. These results suggest that a major portion of the total mutagenic activity in air-pollution particles is contributed by nitro-substituted compounds that are detectable as their corresponding amines. They also suggest that the atmospheric concentrations of these substances may be high in the colder months of Durham's year.     

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.     

Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines. Non-specific reactions with phenols and benzo[alpha]pyrene

Treatment of mutagenic primary aromatic amines with nitrous acid is known to decrease their mutagenicity. We examined some factors concerning the validity of using decreases in mutagenicity due to nitrous acid treatment as an indication of the presence of mutagenic primary aromatic amines in complex mixtures. We found that treatment of benzo[alpha]pyrene with nitrous acid for the extended periods of time previously employed leads to formation of three nitrobenzo[alpha]pyrene isomers. Some of the isomers are direct-acting mutagens for S. typhimurium with considerably greater mutagenicity than benzo[alpha]pyrene isomers. In attempts to minimize reaction of chemicals other than aromatic amines, we found that only very brief reaction periods are required for complete reaction of nitrous acid with representative aromatic amines, essentially eliminating their mutagenicity. During such brief reaction periods modification of benzo[alpha]pyrene is negligible, but phenols react readily. Chromatographic analysis indicated that reaction of nitrous acid with aromatic amines leads to the formation of families of products, thereby increasing the complexity of the mixtures in which the amines may occur. Thus, experiments examining the effects of nitrous acid on the mutagenic activity of complex mixtures must be carefully designed, and the results must be interpreted cautiously.     

Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment

Aromatic amines represent one of the most important classes of industrial and environmental chemicals: many of them have been reported to be powerful carcinogens and mutagens, and/or hemotoxicants. Their toxicity has been studied also with quantitative structure-activity relationship (QSAR) methods: these studies are potentially suitable for investigating mechanisms of action and for estimating the toxicity of compounds lacking experimental determinations. In this paper, we first summarized the QSAR models for the rodent carcinogenicity of the aromatic amines. The gradation of potency of the carcinogenic amines depended firstly on their hydrophobicity, and secondly on electronic (reactivity, propensity to be metabolically transformed) and steric properties. On the contrary, the difference between carcinogenic and non-carcinogenic aromatic amines depended mainly on electronic and steric properties. These QSARs can be used directly for estimating the carcinogenicity of aromatic amines. A two-step prediction is possible: (1) estimation of yes/no activity; (2) if the answer from step 1 is yes, then prediction of the degree of potency. The QSARs for rodent carcinogenicity were put in a wider context by comparing them with those for: (a) Salmonella mutagenicity; (b) general toxicity; (c) enzymatic reactions; (d) physical-chemical reactions. This comparative QSAR exercise generated a coherent global picture of the action mechanisms of the aromatic amines. The QSARs for carcinogenicity were similar to those for Salmonella mutagenicity, thus pointing to a similar mechanism of action. On the contrary, the general toxicity QSARs (both in vitro and in vivo systems) were mostly based on hydrophobicity, pointing to an aspecific mechanism of action much simpler than that for carcinogenicity and mutagenicity. The oxidation of the amines (first step in the main metabolic pathway leading to carcinogenic and mutagenic species) had identical QSARs in both enzymatic and physical-chemical systems, thus providing evidence for the link between simple chemical reactions and those in biological systems. The results show that it is possible to generate mechanistically and statistically sound QSAR models for rodent carcinogenicity, and indirectly that the rodent bioassay is a reliable source of good quality data.     

The mighty arginine, the stable quaternary amines, the powerful aromatics, and the aggressive phosphate: their role in the noncovalent minuet

In the age of proteomics, the role of certain amino acid residues and some post-translational modifications in noncovalent complex formation are gaining in importance, as the understanding of interactions between biological molecules, is at the heart of the structure function relationship puzzle. In this work, mass spectrometry is used to highlight ammonium- or guanidinium-aromatic interactions through Cation-pi bonds and ammonium- or guanidinium-phosphate interactions through salt bridge formation. Such interactions are crucial factors in certain ligand-receptor interactions and receptor-receptor interactions. In addition, the ability of phosphorylated residues and phosphorylated lipids to form noncovalent complexes with guanidinium and quaternary ammonium (mostly through Coulombic interactions) is demonstrated, and could explain the stability of certain membrane embedded protein, or a possible role for phosphorylation in protein-protein interactions. Dougherty's work demonstrates cation-pi interactions in intra-protein interactions and folding, the present work explores inter-peptide interactions, i.e., the formation of noncovalent complexes between peptides' epitopes containing adjacent aromatic residues and ones containing adjacent Arg as a model to better understand the role of cation-pi complexes in protein-protein interaction. Complexes of peptides containing aromatic residues with quaternary amines as well as the interaction of aromatic compounds, with the guanidinium group of Arg are also investigated. Considering that an inordinate number of therapeutic compounds contain aromatic rings and quaternary amines, the above-described interactions could possibly be of great importance in better understanding their mechanism of action.     

Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Identifying human cells capable of metabolizing various classes of carcinogens

Human cells that appear capable of metabolizing various classes of carcinogens have been identified using one of two methods: metabolism of tritiated benzo(a)pyrene to aqueous-acetone soluble forms or inhibition of cellular DNA synthesis. Each of the assay systems was optimized and the results on 15 human epithelial cell lines were compared. One or more cell lines were found to activate each of four classes of carcinogens examined: polycyclic hydrocarbons, aromatic amines, heterocyclic hydrocarbons, and nitrosamines. Cells that appeared capable of metabolizing polycyclic hydrocarbons or aromatic amines by these methods were also found to produce metabolites which were cytotoxic to cocultivated human xeroderma pigmentosum fibroblasts after a 48-hr exposure to the carcinogen.     

4-aminophenylalanine as a biocompatible nucleophilic catalyst for hydrazone ligations at low temperature and neutral pH

Hydrazone formation and similar reactions are highly versatile and specific, but their application to biological systems has been limited by their characteristically slow reaction kinetics at neutral pH. Catalysis of these reactions through imine formation with aromatic amines such as aniline has broadened the applicability of these reactions to biomolecular labeling. High concentrations of the catalyst are necessary, which may be incompatible with the native structure of certain proteins. In this study, we investigated the utility of 4-aminophenylalanine (4a-Phe) as a catalyst for these reactions. We find that 4a-Phe is nearly as effective as aniline in catalyzing hydrazone formation between the reactive amino acid 3-formyltyrosine (3f-Tyr) and hydrazine-containing fluorophores, both free in solution and incorporated into the protein tubulin. The catalyst 4a-Phe maintains ～70% of the catalytic efficacy of aniline and is less detrimental to the native structure of tubulin. Examination of the temperature dependence of imine formation between 3f-Tyr and 4a-Phe shows an increase in imine concentration accompanying a decrease in temperature, confirming the exothermic nature of the equilibrium reaction. Interestingly, decreasing the temperature of the 4a-Phe-catalyzed hydrazone reaction between 3f-Tyr and the fluorophore 7-hydrazinyl-4-methylcoumarin increases the overall rate of the reaction. This result indicates that the temperature dependence of the catalyst-aldehyde equilibrium is greater than the temperature dependence of the rate constant for hydrazone formation from this intermediate, and that the rate of hydrazone formation a direct function of the concentration of the intermediate imine. These results provide a platform for conducting nucleophilic catalysis under conditions that are more compatible with biomolecular targets than previously demonstrated, thereby expanding the utility of hydrazone ligations in biological systems.     

Effects of industrially produced flavours with pro- and antioxidative properties on the formation of the heterocyclic amine PhIP in a model system

PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is a heterocyclic aromatic amine belonging to a class of mutagens found in food. This project studied the effects of commercially available flavours of spices on the formation of PhIP, one of the most common heterocyclic aromatic amines in heated meat and fish products. The model reactions were carried out in diethylene glycol. Highest amounts of PhIP were obtained at 200 degrees C, a heating time of 60 min and an equivalent molar ratio of phenylalanine and creatinine. With this model system, the influence of Monascus red and flavours extracted from thyme, marjoram and rosemary on the formation of PhIP was tested. The flavours were added to the model system in different amounts. The oxidative properties were determined with the rancimat method. It was shown that all tested products, independent of their pro- or antioxidative properties, increased PhIP in the model system.     

Carcinogen activation by human liver enzymes in the Ames mutagenicity test.

Liver post-mitochondrial supernatants derived from 10 individuals were used as the source of metabolic activation for carcinogens in the Ames quantitative mutagenicity test using Salmonella typhimurium TA 100. The liver samples were obtained from brain-dead donors and autopsy cases. The ability of human enzymes to activate aromatic amines ranged from the undetectable to highly active for 2-acetylaminofluorene. None of the samples exhibited any ability to activate benzidine. A generally low activity was observed in the capability of human enzymes to activate the polynuclear aromatic hydrocarbons, 3-methylcholanthrene and benzo(a)pyrene. Most samples were positive for activating 4-nitrobiphenyl. However, the highest mutagenic activity in the presence of human enzymes was consistently observed for aflatoxin B1 and sterigmatocystin. These results indicated that (a) human enzyme systems, like rodent systems, are more effective in inducing mutagenic activity from mycotoxins than aromatic amines and polynuclear aromatic hydrocarbons, and (b) samples derived from different individuals exhibited considerable variation in the ability to activate carcinogens belonging to a same class of compound.     

Nuclear amination catalyzed by fungal laccases: Comparison of laccase catalyzed amination with known chemical routes to aminoquinones

Laccases are able to initiate nuclear amination of p-hydroquinones with primary aromatic amines, resulting in the formation of the corresponding monoaminated and diaminated quinones. Two laccase catalyzed reactions are compared with established synthetic routes to aminoquinones, showing that formation of products from laccase catalyzed reaction is comparable with reaction using sodium iodate as oxidant. Advantages and disadvantages of laccase catalyzed amination are discussed. It is concluded that laccase catalysis is less suitable than sodium iodate oxidation for the amination of simple p-hydroquinones with simple amines.     

Effect of UV-light on formation of fluorescent products of lipid peroxidation

The rate of fluorescent product formation during the peroxidation of polyunsaturated linolenic acid or egg phosphatidylethanolamine and also during the oxidation of linolenic acid together with a phenylalanine and synthetic phosphatidylethanolamine 1,5--3 times more intensive after previous UV-irradiation of the unsaturated fatty acid. Schiff bases are fluorescent products in amine containing systems which are produced in the reaction of the malonaldehyde with amines. It is possible that fluorochromes produced during the only unsaturated acid oxidation are the result of the radical recombination. Accumulation of the oxidated products determined by TBA-reactive substances does not inevitably correlate with the fluorescent intensity in explored systems.     

Ionic interactions for substituted MCH1R inhibitors studied by pKa values

MCH1R inhibitors with the quinoline moiety having the aromatic amine and aliphatic amine chain were selected, and then the effect of substituents of the quinoline ring on the ionic interaction were studied by calculating pK_a values for these amines at the B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level in the gas phase and in water. For substituent with C, N, O, and S atoms next to the quinoline ring, respectively, the pK_a values of aromatic amines are estimated to be 8.98, 12.19, 4.64, and 4.33 and those of the aliphatic amines are 12.65, 10.82, 9.94, and 11.55, respectively.     

Effect of Hormone Treatment on Growth Bud Formation and Free Amine and Hydroxycinnamoyl Putrescine Levels in Leaf Explant of Nicotiana tabacum Cultivated in Vitro

Foliar explants of Nicotiana tabacum cv Xanthi n.c. were cultured on four different media: a basal medium, basal medium plus benzyladenine, basal medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and the basal medium containing both hormones. No differentiation or cell division occurred in leaf explants cultured on the basal medium. Addition of benzyladenine caused the formation of buds on the explants, while 2,4-D caused callus formation and proliferation. Likewise, only callus was formed when explants were cultured on medium containing both hormones, but growth was significantly greater than that of callus grown on a medium containing 2,4-D alone. The levels of amines and hydroxycinnamoyl putrescines were determined in the four types of explants. In nongrowing explants, amines (except an aromatic amine, tyramine) and hydroxycinnamoyl putrescines were always at a low level and only small changes in their concentrations were observed. In callus cultures, amine (except an aromatic amine, phenethylamine) and hydroxycinnamoyl putrescine levels were higher than those found in bud cultures. In all the media, transitory accumulation of aromatic amines occurred after a few days of culture. Higher levels of hydroxycinnamoyl putrescines were attained in callus cultures with a slow growth rate (2,4-D alone) than in callus cultures with a fast growth rate (benzyladenine + 2,4-D). The formation of buds was accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels. Increasing levels were found during the first 14 days in culture when cell multiplication was rapid, followed by a sharp decline after 20 days in culture as the rate of cell division decreased and differentiation took place. The relationship among amines, hydroxycinnamoyl putrescines, and cell division and bud formation is discussed.     

Application of a novel bacterial consortium for mineralization of sulphonated aromatic amines

A novel bacterial consortium (TJ-2) for mineralization of aromatic amines resulting from decolorization of azo dyes was developed. Three bacterial strains were identified as Pseudomonas pseudoalcaligenes (TJ-21,EU072476), Pseudomonas citronellolis (TJ-22,EU072477) and Pseudomonas testosterone (TJ-23,EU072477) by 16S rRNA gene sequence analysis. Aromatic amine mineralization under aerobic conditions was observed to be significantly higher with the consortium as compared to pure strains indicating complementary interactions among these strains. It was observed that more than 90% mineralization of aromatic amines was achieved within 18 h for different initial aromatic amines concentrations. It was also observed that aromatic amine mineralization depends upon the structure of aromatic amine. Para- and meta-hydroxy substituted aromatic amine were easily mineralized as compared to ortho-substituted which undergoes autoxidation when exposed to oxygen. The consortium was capable of mineralizing other aromatic amines, thus, conferring the possibility of application of TJ-2 for the treatment of industrial wastewaters containing aromatic amines.     

The physiological activity of volatile substances of plants in air and water media

Physiological effects of volatile substances released by the overground as well as by the underground organs of higher plants were studied. The activity of the volatile substances was tested both when these substances were allowed to act directly in the air and when they were dissolved in water in the form of solutions. Plants which do not contain essential oils or which are not rich in them as well as those abounding in essential oils and other volatiles were used in the experiments. The physiological activity of the volatile substances was tested on rye seedlings. The overground as well as underground mature organs of the tested plants were found to release volatile substances causing, when acting directly, in the majority of cases an inhibition of the growth in length and of the formation of dry matter in rye seedlings. A pronounced inhibition of the growth of rye seedlings was brought about especially by the volatile substances of “aromatic” plants such as common dill, wild thyme, yarrow milfoil, garden thyme, marjoram, etc. The volatile substances released by the organs of “non-aromatic” plants like sugar-beet, common sunflower, quackgrass, etc., were found to bring about a significant inhibition of the growth of rye seedlings, too. The volatile substances released by the plant organs were found to be altogether absorbable in water and physiologically active also in the form of water solutions. With the exception of volatile substances from hemp and quackgrass leaves, which brought about a mild stimulation of the dry matter formation in rye seedlings, inhibitory effects of these solutions were found to prevail in all cases. Most effective were the solutions of the volatiles from some of the “aromatic plants”. An assay for olefines in the atmosphere of the experimental vessels demonstrated that in almost all cases ethylene is being released by the plant organs.     

Enzyme polymorphisms influencing the metabolism of heterocyclic aromatic amines

Heterocyclic aromatic amines are dietary carcinogens possibly involved in human carcinogenesis, DNA-adduct formation being an obligatory step in this multistage process. Heterocyclic amine binding to DNA largely depends on the balance between metabolic activation and detoxification pathways and DNA repair efficiency. Several genes coding for metabolic enzymes are polymorphic, which affects gene expression and/or enzyme activity. This paper briefly reviews the effect of polymorphisms of activating/detoxifying enzymes on the metabolism of heterocyclic amines. Despite some epidemiological evidence of an association between genetic polymorphisms and susceptibility to cancer possibly resulting from dietary exposure to heterocyclic aromatic amines (HA), the genetic polymorphisms had only slight effects on biomarker levels, suggesting the existence of further unknown factors.