Formation of Nitrated and Hydroxylated Aromatic Compounds from Benzene and Peroxynitrite, A Possible Mechanism of Benzene Genotoxicity

Peroxynitrite, the reaction product of nitric oxide (NO*) and superoxide anion (O*-) produced during immune activation by a variety of inflammatory cells, may contribute to genotoxicity of benzene through its ability to carry out hydroxylation and nitration. After exposure of benzene to synthesised peroxynitrite, phenol, nitrophenols (p-nitrophenol, o-nitrophenol and m-nitrophenol) and nitrobenzene were identified in the reaction mixture by HPLC separation and single UV wavelength and diode array detection. The formation of phenol, nitrophenols and nitrobenzene showed a linear relationship with both benzene and peroxynitrite concentrations. The molar ratio for phenol/(nitrobenzene and nitrophenols) was approximately 9/5 with a total product yield of 14% hydroxylated and nitrated products as based on peroxynitrite. The physiological relevance of the chemical reaction between benzene and peroxynitrite was tested by detecting the reaction products in human neutrophils (2.5 ± 10⁷ cells/ml) incubated with 10 mM benzene for 25 min. The concentration of phenol and p-nitrophenol were found to be 1.29 ± 0.22 and 1.56 ± 0.61 μM mean ± SD) in the incubation medium of the neutrophils pretreated with phorbol myristate acetate (500 nM) for 5 min, respectively, whereas no metabolites were detected if the neutrophils were not pretreated. Nitrated aromatic compounds are known to be more carcinogenic than the parent compounds. It is reported that acute and chronic infection increases the risk of cancer at various sites; and that anti-inflammatory agents decrease benzene myelotoxicity. We suggest that the increased production of peroxynitrite during chronic inflammation combined with benzene exposure may increase the carcinogenicity of benzene by a mechanism that includes the formation of metabolites from the chemical reaction between benzene and peroxynitrite. Thus, peroxynitrite mediated hydroxylation and nitration of benzene during immune activation represent a novel in vivo mechanism for generation of proximal carcinogens of benzene.     

硝基苯类化合物微生物降解研究进展

硝基苯类化合物是一类具有稳定化学性质、高毒性和易在生物体内积累的优先污染物.微生物降解在硝基苯类化合物废水废气治理和污染环境修复方面具有明显优势.从降解菌的驯化筛选、降解途径、降解机理、共代谢、趋化性和分子遗传学角度,阐述了硝基苯类化合物微生物降解研究的最新进展,指出应进一步加强工程菌的构建及其应用开发研究.在硝基苯类化合物污染环境的微生物修复方面,共代谢和混合菌株的协同作用具有重要的应用前景.     

Fe0/厌氧微生物联合体系降解硝基苯的研究

利用Fe0/厌氧微生物联合体系降解硝基苯(NB), 结果显示, Fe0与厌氧微生物之间存在明显的协同效应, 硝基苯的降解效果随零价铁投加量的增加而提高;最佳pH值为5.0~6.0;添加少量共代谢初级基质(葡萄糖), 可以大幅度提高硝基苯的降解;较高浓度铁离子对硝基苯的降解表现出一定的抑制作用, 添加0.5 mg/L的Fe3+或Fe2+可以加快硝基苯的降解。硝基苯降解的主要产物为苯胺, 降解过程遵循一级动力学模型, 一级反应速率常数k值随硝基苯浓度的提高而降低。     

Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonas putida ZWL73

The genes encoding enzymes involved in the initial reactions during degradation of 4-chloronitrobenzene (4CNB) were characterized from the 4CNB utilizer Pseudomonas putida ZWL73, in which a partial reductive pathway was adopted. A DNA fragment containing genes coding for chloronitrobenzene nitroreductase (CnbA) and hydroxylaminobenzene mutase (CnbB) were PCR-amplified and subsequently sequenced. These two genes were actively expressed in Escherichia coli, and recombinant E. coli cells catalyzed the conversion of 4CNB to 2-amino-5-chlorophenol, which is the ring-cleavage substrate in the degradation of 4CNB. Phylogenetic analyses on sequences of chloronitrobenzene nitroreductase and hydroxylaminobenzene mutase revealed that these two enzymes are closely related to the functionally identified nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas strains JS45 and HS12. The nitroreductase from strain ZWL73 showed a higher specific activity toward 4CNB than nitrobenzene (approximately at a ratio of 1.6:1 for the recombinant or 2:1 for the wild type), which is in contrast to the case where the nitroreductase from nitrobenzene utilizers Pseudomonas pseudoalcaligenes JS45 with an apparently lower specific activity against 4CNB than nitrobenzene (0.16:1) [Kadiyala et al. Appl Environ Microbiol 69:6520–6526, 2003]. This suggests that the nitroreductase from 4-chloronitrobenzene utilizer P. putida ZWL73 may have evolved to prefer chloronitrobenzene to nitrobenzene as its substrate.Y.X. and J.-F.W. equally contributed to this work.     

Enhanced biotransformation of mononitrophenols by Stenotrophomonas maltophilia KB2 in the presence of aromatic compounds of plant origin

Stenotrophomonas maltophilia KB2 used in this study is known to metabolise broad range of aromatic compounds including phenol, some chloro and methylphenols, benzoic acids, catochols and others. To study the applicability of the strain for degradation of mononitrophenols in monosubstrate as well as cometabolic systems its degradation potential in the presence of mononitrophenols or different aromatic compounds of plant origin was tested. Stenotrophomonas maltophilia KB2 strain was not able to degrade any of mononitrophenols used in the single substrate experiments. Effect of additional carbon source on nitrophenols degradation revealed that presence of benzoate, 4-hydroxybenzoate or 3,4-dixydroxybenzoate stimulate transformation of 2-nitrophenol, 3-nitrophenol as well as 4-nitrophenol. Depending on growth substrate and mononitrophenol used, decrease in cometabolite concentration was from 25 to 45%. Obtained results suggest that Stenotrophomonas maltophilia KB2 strain could be potentially used for cometabolic degradation of nitrophenols in the presence of aromatic acids, for the bioremediation of contaminated sites.     

Cometabolic biotransformation of nitrobenzene by 3-nitrophenol degrading Pseudomonas putida 2NP8

A strain of Pseudomonas putida (2NP8) capable of growing on both 2-nitrophenol and 3-nitrophenol, but not on nitrobenzene (NB), was isolated from municipal activated sludge. 2-Nitrophenol was degraded by this strain with production of nitrite. Degradation of 3-nitrophenol resulted in the formation of ammonia. Cells grown on 2-nitrophenol did not degrade nitrobenzene. A specific nitrobenzene degradation activity was induced by 3-nitrophenol. Ammonia, nitrosobenzene, and hydroxylaminobenzene have been detected as metabolites of nitrobenzene degradation by cells grown in the presence of 3-nitrophenol. These results indicated a NB cometabolism mediated by 3-nitrophenol nitroreductase.     

Induction of aromatic ring: cleavage dioxygenases in <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> strain KB2 in cometabolic systems

Stenotrophomonas maltophilia KB2 is known to produce different enzymes of dioxygenase family. The aim of our studies was to determine activity of these enzymes after induction by benzoic acids in cometabolic systems with nitrophenols. We have shown that under cometabolic conditions KB2 strain degraded 0.25–0.4 mM of nitrophenols after 14 days of incubation. Simultaneously degradation of 3 mM of growth substrate during 1–3 days was observed depending on substrate as well as cometabolite used. From cometabolic systems with nitrophenols as cometabolites and 3,4-dihydroxybenzoate as a growth substrate, dioxygenases with the highest activity of protocatechuate 3,4-dioxygenase were isolated. Activity of catechol 1,2- dioxygenase and protocatechuate 4,5-dioxygenase was not observed. Catechol 2,3-dioxygenase was active only in cultures with 4-nitrophenol. Ability of KB2 strain to induce and synthesize various dioxygenases depending on substrate present in medium makes this strain useful in bioremediation of sites contaminated with different aromatic compounds.     

Characterization of the nitrobenzene-degrading strain Pseudomonas sp. a3 and use of its immobilized cells in the treatment of mixed aromatics wastewater

A bacterial strain Pseudomonas sp. a3 capable of degrading nitrobenzene, phenol, aniline, and other aromatics was isolated and characterized. When nitrobenzene was degraded, the release of NH(4) (+) was detected, but not of NO(2) (-). This result implied that nitrobenzene might have a partial reductive metabolic pathway in strain a3. However, aniline appeared as one of the metabolites during the aerobic degradation of nitrobenzene. Moreover, the appearance of 2-aminophenol during aniline degradation by strain a3 indicated that novel initial reactions existed during the degradation of nitrobenzene and aniline by strain a3. Strain a3 was immobilized in the mixed carrier of polyvinyl alcohol and sodium alginate to improve its degrading efficiency. The optimal concentrations of polyvinyl alcohol and sodium alginate in the mixed carrier were 9 and 3 %, respectively. The immobilized cells had stable degradation activity and good mechanical properties in the recycling tests. The immobilized cells also exhibited higher tolerances in acidic (pH 4-5) and highly saline (10 % NaCl) environments than those of free cells. The biodegradation of nitrobenzene mixed with aniline and phenol using immobilized cells of Pseudomonas sp. a3 was also greatly improved compared with those of free cells. The immobilized cells could completely degrade 300 mg L(-1) nitrobenzene within 10 h with 150 mg L(-1) aniline and 150 mg L(-1) phenol. This result revealed that the immobilized cells of Pseudomonas sp. a3 could be a potential candidate for treating nitrobenzene wastewater mixed with other aromatics.     

Biomineralization of 3-nitrotoluene by Diaphorobacter species

Three bacterial strains utilizing 3-nitrotoluene (3-NT) as a sole source of carbon, nitrogen and energy were isolated from an industrial wastewater treatment plant. Biochemical tests and 16S rDNA sequence analysis revealed that the isolated strains belonged to Diaphorobacter sp. Detailed studies were carried out with Diaphorobacter sp. strain DS2. Degradation of 3-NT by Diaphorobacter sp. strain DS2 was accompanied by the release of nitrite in the culture broth with increase in biomass. Total organic carbon analysis confirmed the extensive mineralization of 3-NT. The strain could degrade 3-methylcatechol, 4-methylcatechol and catechol easily suggesting that the degradation pathway could involve these as possible intermediates. Successful PCR amplification of the oxygenase large subunit and the presence of high activity for catechol 2,3-dioxygenase in the crude cell lysate further confirmed that the degradation of 3-NT occurred through (methyl)catechol intermediates in strain DS2. The strain DS2 was found to degrade other isomers of mononitrotoluene (2-NT and 4-NT) and nitrobenzene as well.     

Draft genome sequence of the nitrophenol-degrading actinomycete Rhodococcus imtechensis RKJ300

We report the 8.231-Mb genome sequence of Rhodococcus imtechensis RKJ300, isolated from pesticide-contaminated soil in Punjab, India. The genome sequence of the strain RKJ300 will be helpful in exploring the molecular pathways involved in the degradation of nitrophenols.     

Process for degradation of nitrobenzene: combining electron beam irradiation with biotransformation

Electron beam irradiations of aqueous solutions containing 15-30 mg/L of nitrobenzene at 60 kGy dose removed 78% of the contaminant. Three mononitrophenols were detected as by-products of electron beam treatment of nitrobenzene. A mixed culture enriched on a mixture of 2-, 3-, and 4-nitrophenol degraded both the residual nitrobenzene and the nitrophenol products. Percentage removal of nitrobenzene increased with increasing electron beam dose. This observation led to the conceptual design of a two-stage electron beam microbial process for degradation of nitrobenzene. Three groups of pure isolates were characterized from the mixed culture based on their abilities to grow on cor- responding nitrophenol substrates: Group A, 2NP(-)3NP(-)4NP(+); Group B, 2NP(+)3NP(+)4NP(-); and Group C, 2NP(-)3NP(+)4NP(-). Bacteria that grew on 3-NP transformed nitrobenzene into ammonia in the electron beam-treated nitrobenzene samples.     

Reduction of nitrobenzene with H2 using a microbial consortium

Proof of concept was obtained that nitrobenzene can be reduced to aniline by a mixed reductive microbial culture using H2 as the sole electron donor source. In a continuous-flow anaerobic bioreactor, both pH and temperature affected nitrobenzene reduction with optima of pH 6.5-6.8 and 30 degrees C. The efficiency of nitrobenzene degradation increased with H2 up to 10% (v/v). An increase in sulfate concentration decreased the removal rate of nitrobenzene.     

Biodegradation of nitrobenzene by a sequential anaerobic-aerobic process

Nitrobenzene was completely degraded by mixed cultures using a sequential anaerobic-aerobic treatment process. Under anaerobic conditions in a fixed-bed column aniline was formed from nitrobenzene through gratuitous reduction by cells of sewage sludge. This reaction was accelerated by the addition of glucose. Complete mineralization of aniline was accomplished by subsequent aerobic treatment using activated sludge as inoculum. The maximum degradation rate of nitrobenzene (4.5 mM) in the two-stage system was 552 mg l^–1d^–1, referring to 154 mg of nitrobenzene per gram of glucose. In a second experimental phase glucose as cosubstrate and H-donor was replaced by synthetic waste containing ethanol, methanol, isopropanol and acetone. Again, nitrobenzene (1.9 mM) was completely degraded (maximum degradation rate of 237 mg l^–d^–1, referring to 251 mg per gram of solvents). The major advantage of the described two-stage process is that the reduction of nitrobenzene by anaerobic pretreatment drastically reduces emission by stripping during aerobic treatment.Abbreviations HRT hydraulic retention time - OD₅₄₆ optical density at 546 nm     

Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and a Rhodococcus sp

A single microorganism able to mineralize chloronitrobenzenes (CNBs) has not been reported, and degradation of CNBs by coculture of two microbial strains was attempted. Pseudomonas putida HS12 was first isolated by analogue enrichment culture using nitrobenzene (NB) as the substrate, and this strain was observed to possess a partial reductive pathway for the degradation of NB. From high-performance liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance analyses, NB-grown cells of P. putida HS12 were found to convert 3- and 4-CNBs to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For the degradation of CNBs, Rhodococcus sp. strain HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with P. putida HS12 to give a coculture. This coculture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. A degradation pathway for 3- and 4-CNBs by the two isolated strains was also proposed.     

Evolution of catabolic pathways for synthetic compounds: bacterial pathways for degradation of 2,4-dinitrotoluene and nitrobenzene

The pathways for 2,4-dinitrotoluene (2,4-DNT) and nitrobenzene offer fine illustrations of how the ability to assimilate new carbon sources evolves in bacteria. Studies of the degradation pathways provide insight about two principal strategies for overcoming the metabolic block imposed by nitro- substituents on aromatic compounds. The 2,4-DNT pathway uses novel oxygenases for oxidative denitration and subsequent ring-fission. The nitrobenzene pathway links facile reduction of the nitro- substituent, a novel mutase enzyme, and a conserved operon encoding aminophenol degradation for mineralization of nitrobenzene. Molecular genetic analysis with comparative biochemistry reveals how the pathways were assembled in response to the recent appearance of the two synthetic chemicals in the biosphere.     

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.     

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.     

假单胞菌XN-1硝基苯降解性质粒的提取及研究

假单胞菌XN 1对有机污染物硝基苯具有降解性 ,并且对氨苄青霉素具有抗性。检测和提取了假单胞菌XN 1细胞内的质粒 ,得到了一个约 2 2kb大小的质粒pXN 1。质粒消除实验证实这个质粒与硝基苯降解性有关 ,而与抗生素抗性无关。XN 1和其自发突变株XN 1 2和XN 1 3特性的差异和质粒检测的差异之间存在对应关系 ,并且得到了一个比 pXN 1小几个kb的衍生质粒 pXN 1 3。     

Enhanced Photodegradation of PNP on Soil Surface under UV Irradiation with TiO2

Photodegradation of p-nitrophenol (PNP) on soil surface was investigated to explore the photochemical remediation of soil polluted by nitrophenols. Soil samples spiked with PNP were irradiated by UV light with and without the addition of TiO ₂ . The addition of 0.5–2 wt% TiO ₂ enhanced PNP photodegradation with approximately 1.36 times increase in apparent rate of PNP disappearance. Soil moisture, humic acid and soil pH were important factors influencing the rate of PNP photodegradation. Increase in soil moisture improved the degradation significantly, whereas humic acid reduced the degradation rate. Changes in soil pH resulted in different degradation rates, and higher degradation efficiencies were observed under alkaline condition.     

Novel partial reductive pathway for 4-chloronitrobenzene and nitrobenzene degradation in Comamonas sp. strain CNB-1

Comamonas sp. strain CNB-1 grows on 4-chloronitrobenzene (4-CNB) and nitrobenzene as sole carbon and nitrogen sources. In this study, two genetic segments, cnbB-orf2-cnbA and cnbR-orf1-cnbCaCbDEFGHI, located on a newly isolated plasmid, pCNB1 (ca. 89 kb), and involved in 4-CNB/nitrobenzene degradation, were characterized. Seven genes (cnbA, cnbB, cnbCa, cnbCb, cnbD, cnbG, and cnbH) were cloned and functionally expressed in recombinant Escherichia coli, and they were identified as encoding 4-CNB nitroreductase (CnbA), 1-hydroxylaminobenzene mutase (CnbB), 2-aminophenol 1,6-dioxygenase (CnbCab), 2-amino-5-chloromuconic semialdehyde dehydrogenase (CnbD), 2-hydroxy-5-chloromuconic acid (2H5CM) tautomerase, and 2-amino-5-chloromuconic acid (2A5CM) deaminase (CnbH). In particular, the 2A5CM deaminase showed significant identities (31 to 38%) to subunit A of Asp-tRNAAsn/Glu-tRNAGln amidotransferase and not to the previously identified deaminases for nitroaromatic compound degradation. Genetic cloning and expression of cnbH in Escherichia coli revealed that CnbH catalyzed the conversion of 2A5CM into 2H5CM and ammonium. Four other genes (cnbR, cnbE, cnbF, and cnbI) were tentatively identified according to their high sequence identities to other functionally identified genes. It was proposed that CnbH might represent a novel type of deaminase and be involved in a novel partial reductive pathway for chloronitrobenzene or nitrobenzene degradation.