Redox-mediated oxidation and removal of aromatic amines from polluted water by partially purified bitter gourd (Momordica charantia) peroxidase

Here, the role of bitter gourd peroxidase has been investigated for the treatment of water contaminated with aromatic amines. Most of the aromatic amines were recalcitrant to the action of bitter gourd peroxidase. However, these aromatic amines were oxidized by bitter gourd peroxidase in the presence of a redox mediator, o-dianisidine HCl. The maximum oxidation of aniline was found to be in the buffer of pH 5.0 at 40 °C in the presence of 0.5 mM H₂O₂ and 0.15 mM o-dianisidine HCl. Aromatic amines oxidized and removed from wastewater were 65% aniline, 50% m-toluidine, 86% m-chloroaniline, 54% p-aminobenzoic acid, 61% diphenylamine and 95% N,N-dimethylaniline. Benzidine and p-nitroaniline were recalcitrant to the action of this enzyme even in the presence of o-dianisidine HCl. Complex mixtures of aromatic amines were treated by bitter gourd peroxidase. These mixtures were removed to varying extent, mixtures A, B and C were oxidized to 59%, 56% and 62%, respectively. Mixtures D, E and F were marginally oxidized to 30%, 14% and 16%, respectively.     

N-acetoxy-N-acetylaminoarenes and nitrosoarenes one-electron non-enzymatic and enzymatic oxidation products of various carcinogenic aromatic acethydroxamic acids

A number of carcinogenic aromatic acethydroxamic acids (e.g.N-hydroxy-N-acetyl derivatives of 2-aminofluorene, 3-aminofluorene, 4-aminostilbene, 1-aminonaphthalene, 2-aminonaphthalene, 2-aminophenanthrene, and 4-aminobiphenyl) are readily oxidized by alkaline Fe(CN)₆³⁻ or Ag₂O. The free nitroxide radicals thus formed dismutate in organic solution according to second order kinetics to yield the corresponding N-acetoxy-N-acetylaminoarenes and nitrosoarenes. The structures of the latter products were established by mass and infrared spectrum analyses. Evicence was obtained for a similar one-electron oxidation of these acethydroxamic acids with horseradish peroxidase and H₂O₂ at pH 7. One-electron oxidation of N-hydroxy-2-acetylaminofluorene was also demonstrated with lactoperoxidase and human myeloperoxidase. The possible relevance of a similar peroxidative attack in vivo to the carcinogenic activities of some aromatic amines and amides is discussed.     

Removal of phenols and aromatic amines from wastewater by a combination treatment with tyrosinase and a coagulant

Removal of phenols and aromatic amines from industrial wastewater by tyrosinase was investigated. A color change from colorless to darkbrown was observed, but no precipitate was formed. Colored products were found to be easily removed by a combination treatment with tyrosinase and a cationic polymer coagulant containing amino group, such as hexamethylenediamine-epichlorohidrin polycondensate, polyethleneimine, or chitosan. The first two coagulants, synthetic polymers, were more effective than chitosan, a polymer produced in crustacean shells. Phenols and aromatic amines are not precipitated by any kind of coagulants, but their enzymatic reaction products are easily precipitated by a cationic polymer coagulant. These results indicate that the combination of tyrosinase and a cationic polymer coagulant is effective in removing carcinogenic phenols and aromatic amines from an aqueous solution. Immobilization of tyrosinase on magnetite gave a good retention of activity (80%) and storage stability i.e., only 5% loss after 15 days of storage at ambient temperature. In the treatment of immobilized tyrosinase, colored enzymatic reaction products were removed by less coagulant compared with soluble tyrosinase. (c) 1995 John Wiley & Sons, Inc.     

Aromatic ring cleavage of β-O-4 lignin model dimers without prior demeth(ox)ylation by lignin peroxidase

Methyl oxalate of arylglycerol was formed as an aromatic ring cleavage product in degradation of arylglycerol-β-aryl ether (β-O-4) type lignin substructure model dimers by extracellular lignin peroxidase of Phanerochaete chrysosporium. The enzymatic cleavage of arylglycerol-β-(o-[²H₃]methoxyphenyl) ether indicated that the methyl group of the methyl ester was derived from the methoxy group of the β-O-4 model dimer. It is thus concluded that demeth(ox)ylation was not essential for the enzymatic aromatic ring cleavage of the methoxylated aromatic substrates, β-O-4 lignin substructure models.     

Roles of UDP-Glucuronosyltransferases in Chemical Carcinogenesi

Abstract

UDP-glucuronosyltransferases (UGT) play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA, and protein. They also inactivate carcinogenic, N-oxidized metabolites of aromatic amines. Furthermore, glucuronides may be stable transport forms of proximate carcinogens excreted via the CCor urinary tract, thereby liberating the ultimate carcinogen at the target of carcinogenicity. Isozymes of the UGT enzyme super family that control the glucuronidation of metabolites of aromatic hyharbons and of N-oxidized aromatic amines have been identified in rats and humans. Phenol UGT appears to be conduced with other drug-metabolizing enzymes via the Ah or dioxin receptor. This isozyme probably controls various proximate carcinogens and contributes to the persistently altered enzyme pattern, leading to the “toxin-resistance phenotype” at cancer prestages. Knowledge about UGTs in different species, their regulation, and their tissue distribution will improve the risk assessment of carcinogens.     

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.     

A new biological assay, utilizing parasitic protozoa, for screening carcinogenic substances which induce bladder cancer in man and other mammals

Injections of aromatic amines (β-naphthylamine, benzidine, O-dianisidine or N-2-fluorenyl acetamide), tryptophan metabolites (3-hydroxyanthranilic acid, xanthurenic acid or LD-kynurenine sulphate), oestrone, and nicotine, which are known bladder carcinogens in man and some other mammals induced sexual reproduction (encystation) in Opalina sudafricana when injected into its host Bufo regularis. This may be used as a new biological assay for screening substances which induce bladder cancer in man and some other mammals. It is speculated that the metabolites of the injected carcinogenic substances used in this work are excreted in the urine of the host, hydrolysed by the hydrolytic enzymes and become carcinogenic. These carcinogenic metabolites reach the parasites in the rectum of the toads and induce them to divide mitotically to form small forms which eventually encyst. It is speculated that the presence of cysts in the rectum of the injected toads is indicative that a carcinogenic effect took place in the parasites. Oestrone is the only carcinogenic substance which induced encystation in the opalinids in vitro. Urine of toads injected with β-naphthylamine, benzidine, O-dianisidine, N-2-fluorenyl acetamide, 3-hydroxyanthranilic acid, xanthurenic acid, DL-kynurenine sulphate, oestrone and nicotine induced cyst formation in the parasites in vitro.     

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.     

Bioactivation of xenobiotics by prostaglandin H synthase

Prostaglandin H synthase (PHS) catalyzes the oxidation of arachidonic acid to prostaglandin H2 in reactions which utilize two activities, a cyclooxygenase and a peroxidase. These enzymatic activities generate enzyme- and substrate-derived free radical intermediates which can oxidize xenobiotics to biologically reactive intermediates. As a consequence, in the presence of arachidonic acid or a peroxide source, PHS can bioactivate many chemical carcinogens to their ultimate mutagenic and carcinogenic forms. In general, PHS-dependent bioactivation is most important in extrahepatic tissues with low monooxygenase activity such as the urinary bladder, renal medulla, skin and lung. Mutagenicity assays are useful in the detection of compounds which are converted to genotoxic metabolites during PHS oxidation. In addition, the oxidation of xenobiotics by PHS often form metabolites or adducts to cellular macromolecules which are specific for peroxidase- or peroxyl radical-dependent reactions. These specific metabolites and/or adducts have served as biological markers of xenobiotic bioactivation by PHS in certain tissues. Evidence is presented which supports a role for PHS in the bioactivation of several polycyclic aromatic hydrocarbons and aromatic amines, two classes of carcinogens which induce extrahepatic neoplasia. It should be emphasized that the toxicities induced by PHS-dependent bioactivation of xenobiotics are not limited to carcinogenicity. Examples are given which demonstrate a role for PHS in pulmonary toxicity, teratogenicity, nephrotoxicity and myelotoxicity.     

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 effect of N-nitroso carcinogens and triazenes on protein synthesis in cell-free systems

The effect of a series of carcinogenic nitrosamines, alkylnitrosoureas and alkaryltriazenes on enzymatic reactions involved in protein synthesis was studied in cell-free systems from rat liver. The addition of most compounds stimulated the formation of aminoacyl-tRNA complex in test systems from rat liver whereas analogous preparations from Escherichia coli did not show this effect. The polymerization of phenylalanine and the binding of aminoacyl-tRNA to ribosome were only slightly and apparently non-specifically inhibited in the presence of the test compounds. On the other hand, the binding of nRNA to ribosome was markedly stimulated after the addition of most carcinogens tested. It appears that the carcinogens intervene specifically with one of the early steps in peptide initiation. Since the binding of mRNA to ribosome is known to be an important rate-limiting step in protein synthesis, the N-nitroso carcinogens and triazenes may thus control the expression of genetic message at the translation level.     

Enzyme catalytic nitration of aromatic compounds

Nitroaromatic compounds are important intermediates in organic synthesis. The classic method used to synthesize them is chemical nitration, which involves the use of nitric acid diluted in water or acetic acid, both harmful to the environment. With the development of green chemistry, environmental friendly enzyme catalysis is increasingly employed in chemical processes. In this work, we adopted a non-aqueous horseradish peroxidase (HRP)/NaNO₂/H₂O₂ reaction system to study the structural characteristics of aromatic compounds potentially nitrated by enzyme catalysis, as well as the relationship between the charges on carbon atoms in benzene ring and the nitro product distribution. Investigation of various reaction parameters showed that mild reaction conditions (ambient temperature and neutral pH), plus appropriate use of H₂O₂ and NaNO₂ could prevent inactivation of HRP and polymerization of the substrates. Compared to aqueous–organic co-solvent reaction media, the aqueous–organic two-liquid phase system had great advantages in increasing the dissolved concentration of substrate and alleviating substrate inhibition. Analysis of the aromatic compounds’ structural characteristics indicated that substrates containing substituents of NH₂ or OH were readily catalyzed. Furthermore, analysis of the relationship between natural bond orbital (NBO) charges on carbon atoms in benzene ring, as calculated by the density functional method, and the nitro product distribution characteristics, demonstrated that the favored nitration sites were the ortho and para positions of substituents in benzene ring, similar to the selectivity of chemical nitration.     

A transversion mutation hypothesis for chemical carcinogenesis by N2-substitution of guanine in DNA

Several carcinogenic aromatic amines and polycyclic hydrocarbons react covalently with the exocyclic amino group (N²) of guanine in DNA. In this study, space-filling molecular models of DNA containing N²-guanyl adducts of 2-acetylaminofluorene (AAF) or benzo[a]pyrene (BP) were constructed. From these models and from available physico-chemical data, it is suggested that the N² adducts may be easily converted from the normal anti to a syn conformation (base/deoxyribose). This confuguration causes minimal distortion of the DNA model with only a 2–3 Å shift in the helical axis of symmetry. Such an alteration may account for the persistence of these adducts in DNA and for the frameshift mutations induced by these carcinogens. Additionally, the syn N²-guanyl configuration places the N-7 and O⁶ atoms of the modified syn guanine in the base pairing region such that, during replication, mispairing with N-1 and N² of an opposite guanine may occur. This would then represent a carcinogen-induced transversion mutation and may lead to neoplastic transformation.     

Quantum chemical studies of methylbenz[a]anthracenes: Metabolism and correlations with carcinogenicity

In the context of the bay region, K-region and radical cation hypotheses for polycyclic aromatic carcinogens, molecular properties were calculated for fourteen methyl derivatives of benz[a]anthracene (BA) related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2)-the stabilities of the putative carbocation ultimate carcinogens. Allvalence electron methods were used, avoiding the inherent difficulties found in the π-electron methods. The calculated substrate reactivities were found to predict major metabolites sucessfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons (PAH) towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. This latter correlation holds when both the methyl derivatives of BA and previously studied unsubstituted PAH are considered together, indicating its potential usefulness as a screening parameter for carcinogenic activity.     

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.     

A chromatographic technique for the analysis of oxidized metabolites: Application to carcinogenic N-hydroxyarylamines in urine

A new chromatographic detection method for oxidized metabolites has been developed based on the reaction of eluted compounds with an Fe⁺³-bathophenanthroline colorimetric reagent in a postcolumn reactor. The method is sensitive to N-hydroxyarylamines, aryldiamines, phenolic amines, and ascorbic acid. It has been applied to the analysis of toxic N-oxidized metabolites in rhesus monkey urine after the animals were dosed with the bladder carcinogens, 1- and 2-napthylamine. These compounds are oxidized to the corresponding N-hydroxyarylamines in the liver, conjugated as the N-glucuronide, and excreted in the urine. The N-glucuronide has been shown to undergo acidic hydrolysis in the urine to release the free N-hydroxyarylamine, an ultimate carcinogen for the induction of bladder tumors. In this study, the N-hydroxy-N-glucuronide of 2-naphthylamine was found to be excreted at a rate that was 6.8 times that of the 1-naphthylamine isomer. This is consistent with the much higher carcinogenic potency of 2-naphthylamine in a variety of species.     

Effect of jasmonic acid on the pro-/antioxidant system of wheat coleoptiles as related to hyperthermia tolerance

The effects of treatment with jasmonic acid (JA) of wheat (Triticum aestivum L, cv. Elegia) coleoptiles on the generation of superoxide anion-radical (O ₂ ^·? ), the activity of extracellular peroxidase, enzymatic and non-enzymatic components of the antioxidant system were studied. During the first hour after the start of coleoptile treatment with 1 μM JA, the generation of O ₂ ^·? was enhanced and the extracellular peroxidase was activated. During following 23 h, these effects were gradually reduced. JA-enhanced O ₂ ^·? generation was partially suppressed by coleoptile treatment with the inhibitor of peroxidase salicylhydroxamic acid, the inhibitor of NADPH-oxidase imidazol, and also the calcium chelator EGTA. Under the influence of JA treatment, antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and soluble guaiacol peroxidase) in wheat coleoptiles were activated. Treatment with JA improved coleoptile tolerance to damaging heating (10 min at 43°C); it favored the maintenance of the pools of enzymatic and non-enzymatic antioxidants. The inhibitors of NADPH-oxidase and peroxidase, and also calcium chelator reduced a positive JA influence on coleoptile thermotolerance. The role of changes in the pro-/antioxidant balance in plant tissues for the realization of stress-defensive JA effects is discussed.     

Effect of ultraviolet-B radiation on biomass production,lipid peroxidation,reactive oxygen species,and antioxidants in Withania somnifera

The present study was aimed at understanding the effects of long term supplemental UV-B (3.6 kJ m^?2 d^?1) on biomass production, accumulation of reactive oxygen species, lipid peroxidation, and enzymatic antioxidants in leaves and roots of Withania somnifera (an indigenous medicinal plant). Under the UV-B treatment, a reduction in biomass and an increased malondialdehyde content (a characteristic of lipid peroxidation) were observed in both the shoots and roots. Amongst ROS, H₂O₂ content increased under UV-B in the leaves, whereas it decreased in the roots, and superoxide radical production rate decreased in both the plant parts. The activities of all enzymatic antioxidants tested (ascorbate peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, and superoxide dismutase) increased under the UV-B treatment, the increase being greater in the roots.     

Roles of UDP-glucuronosyltransferases in chemical carcinogenesis

UDP-glucuronosyltransferases (UGT) play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA, and protein. They also inactivate carcinogenic, N-oxidized metabolites of aromatic amines. Furthermore, glucuronides may be stable transport forms of proximate carcinogens excreted via the biliary or urinary tract, thereby liberating the ultimate carcinogen at the target of carcinogenicity. Isozymes of the UGT enzyme superfamily that control the glucuronidation of metabolites of aromatic hydrocarbons and of N-oxidized aromatic amines have been identified in rats and humans. Phenol UGT appears to be coinduced with other drug-metabolizing enzymes via the Ah or dioxin receptor. This isozyme probably controls various proximate carcinogens and contributes to the persistently altered enzyme pattern, leading to the "toxin-resistance phenotype" at cancer prestages. Knowledge about UGTs in different species, their regulation, and their tissue distribution will improve the risk assessment of carcinogens.