[Show abstract][Hide abstract] ABSTRACT: Nicotine is an important environmental toxicant in tobacco waste. Shinella sp. strain HZN7 can metabolize nicotine into non-toxic compounds via variations of the pyridine and pyrrolidine pathways. However, the catabolic mechanism of this variant pathway at the gene or enzyme level is still unknown. In this study, two 6-hydroxy-nicotine degradation deficient mutants, N7-M9 and N7-W3, were generated by transposon mutagenesis. The corresponding mutant genes, designated nctB and tnp2, were cloned and analyzed. The nctB gene encodes a novel FAD-containing (S)-6-hydroxy-nicotine oxidase that converts (S)-6-hydroxy-nicotine into 6-hydroxy-N-methylmyosmine then spontaneously hydrolyzes into 6-hydroxy-pseudooxynicotine. The deletion and complementation of the nctB gene showed this enzyme is essential for nicotine or (S)-6-hydroxy-nicotine degradation. Purified NctB can also convert (S)-nicotine into N-methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB towards (S)-6-hydroxy-nicotine (Km = 0.019 mM;, kcat = 7.3 s(-1)) and nicotine (Km = 2.03 mM, kcat = 0.396 s(-1)) indicated that (S)-6-hydroxy-nicotine was the preferred substrate in vivo. NctB showed no activities towards (R)-enantiomers of nicotine or 6-hydroxy-nicotine. Strain HZN7 could degrade (R)-nicotine into (R)-6-hydroxy-nicotine without any further degradation. The tnp2 gene from mutant N7-W3 encodes a putative transposase, and its deletion did not abolish the nicotine degradation activity. This study advances the understanding of microbial diversity of nicotine biodegradation.
[Show abstract][Hide abstract] ABSTRACT: Nicotine is a significant toxic waste generated in tobacco manufacturing. Biological methods for the degradation of nicotine waste are in high demand. In this study, we report the identification and characterization of the novel nicotine-degrading strain Shinella sp. HZN7. This strain can degrade 500 mg/L nicotine completely within 3 h at 30 °C and pH values of 6.5 ∼ 8.0. The biodegradation of nicotine by Shinella sp. HZN7 involves five intermediate metabolites: 6-hydroxy-nicotine (6HN), 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine (6HPON), 6-hydroxy-3-succinoyl-pyridine (HSP), and 2,5-dihydroxypyridine, as detected by ultraviolet spectrophotometry, HPLC, and LC-MS. We generated three mutants, N7-W18, N7-X5, and N7-M17, by transposon mutagenesis, in which the nicotine-degrading pathway terminated at 6HN, 6HPON, and HSP, respectively. The production of the five intermediate metabolites and their order in the degradation pathway were confirmed in the three mutants, indicating that strain HZN7 degrades nicotine via a variant of the pyridine and pyrrolidine pathways. The mutant gene from strain N7-X5, orf2, was cloned by self-formed adaptor PCR, but the nucleotide and amino acid sequence showed no similarity to any gene or gene product with defined function. However, orf2 disruption and complementation suggested that the orf2 gene is essential for the conversion of 6HPON to HSP in strain HZN7. This is the first study to provide genetic evidence for this variant nicotine degradation pathway.
Applied Microbiology and Biotechnology 09/2013; · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,434-bp open reading frame (designated nox) was found to encode a nicotine oxidase that shows 30% amino acid sequence identity with 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans. The nox gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteria Escherichia coli DH5α (DH-nox) and Pseudomonas putida KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse-transcription PCR showed that the nox gene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in Pseudomonas strain(s) but not in E. coli. The nox-disrupted mutant of strain HZN6 lost the ability to degrade nicotine but not pseudooxynicotine. These results suggested that the nox gene is responsible for the first step of nicotine degradation. RS-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved FAD-binding GxGxxG motif and His456 are essential for nicotine degradation activity.
Applied and Environmental Microbiology 01/2013; · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A highly effective acetochlor-degrading bacterial strain (D-12) was isolated from the soil of a pesticide factory. The strain was identified as Achromobacter sp. based on its 16S rRNA gene sequence. The strain D-12 optimally degrades acetochlor at a pH of 7.0 and a temperature of 30°C in a mineral salts medium (MSM). Approximately 95% of acetochlor was degraded by the stain treated at a concentration of 10 mg L(-1) after 5 days of incubation. A chiral high performance liquid chromatography (HPLC) system was used to study the enantioselectivity during the process. However, no obvious enantioselective biodegradation was observed. The primary biodegradation acetochlor products were identified by high-performance liquid chromatography-mass spectroscopy (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). The results indicated that the strain D-12 could be applied in the bioremediation of an acetochlor-polluted environment.
Journal of Environmental Science and Health Part B Pesticides Food Contaminants and Agricultural Wastes 01/2013; 48(11):960-6. · 1.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many of organophosphorous insecticides are chiral compounds. In this study, the enantioselective effects of organophosphate insecticide methamidophos on the coelomocytes lysosomal membrane stability of earthworm Eisenia fetida were studied: (1) The enantiomers of methamidophos were absolutely separated by high-performance liquid chromatography with a commercial chiral column; (2) The neutral red retention assay was used to judge the lysosomal membrane stability. The results showed that with the concentration increasing, lysosomal membranes have been significantly destroyed by individual stereoisomers and racemate of methamidophos. The neutral red retention times were significantly descended from 76.88 to 29.78 min. Both (+)- and (-)-methamidophos showed more prone to destroy the integrity of the lysosomal membrane than the racemate. However, the different effect between stereoisomers is slight.
Bulletin of Environmental Contamination and Toxicology 10/2012; · 1.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enantioselectivity interaction of 2,4-dichlorprop (DCPP) and catalase were studied, and it was further evaluated with the presence of humus. Both of rac-DCPP and R-DCPP can inhibit the activity of catalase with the concentrations of 0.05-80 mg L(-1), the inhibitory type of rac-DCPP was uncompetitive, and of R-DCPP was complex. The presence of humic acid has changed the inhibitory ability of DCPP on catalase, the inhibition of rac-DCPP disappeared and the inhibition type of R-DCPP mainly became uncompetitive. These results suggest that inhibition of chiral DCPP on catalase is enantioselective.
Bulletin of Environmental Contamination and Toxicology 09/2012; 89(5):945-9. · 1.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have demonstrated that pesticides could induce cytotoxicity and genotoxicity in vivo and in vitro, and that oxidative stress may be an important factor involved. However, investigations comparing the capability of different organophosphorous (OP) compounds to induce cytotoxicity, genotoxicity and oxidative stress are limited. Hence, the aim of this paper was to access the cytotoxic and genotoxic effects of five OPs or metabolites, Acephate (ACE), Methamidophos (MET), Chloramidophos (CHL), Malathion (MAT) and Malaoxon (MAO), and to clarify the role of oxidative stress, using PC12 cells. The results demonstrated that MET, MAT and MAO caused significant inhibition of cell viability and increased DNA damage in PC12 cells at 40 mg L(-1). MAO was more toxic than the other OPs. ACE, MET, MAT and MAO increased the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), and decreased the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) at 20 mg L(-1) and 40 mg L(-1) to different degrees. Pre-treatment with vitamin E(600 μM)caused a significant attenuation in the cytotoxic and genotoxic effect; pre-treatment reversed subsequent OP-induced elevation of peroxidation products and the decline of anti-oxidant enzyme activities. These results indicate that oxidative damage is likely to be an initiating event that contributes to the OP-induced cytotoxicity.
Journal of Environmental Science and Health Part B Pesticides Food Contaminants and Agricultural Wastes 05/2012; 47(5):445-54. · 1.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nicotine is a natural alkaloid produced by tobacco plants, and the mechanisms of its catabolism by microorganisms are diverse. In the present study, we reported the mutation, cloning, and identification of two novel genes involved in nicotine degradation from the newly isolated Pseudomonas sp. strain HZN6. Transposon mutagenesis identified a HZN6 mutant in which the nicotine-degrading pathway was blocked at pseudooxynicotine. A 3,874-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR. Two open reading frames (designated pao and sap) were analyzed, and the deduced amino acid sequences shared 29% identity with 6-hydroxy-l-nicotine oxidase from Arthrobacter nicotinovorans and 49% identity with an aldehyde dehydrogenase from Bartonella henselae. Both pao and sap were cloned and functionally expressed in recombinant Escherichia coli BL21. The pao gene encoded a novel pseudooxynicotine amine oxidase with noncovalently bound flavin adenine dinucleotide (FAD) and exhibited substrate specificity removing the methylamine from pseudooxynicotine with the formation of 3-succinoylsemialdehyde-pyridine and hydrogen dioxide. The sap gene encoded a NADP(+)-dependent 3-succinoylsemialdehyde-pyridine dehydrogenase that catalyzed the dehydrogenation of 3-succinoylsemialdehyde-pyridine to 3-succinoyl-pyridine. Genetic analyses indicated that the pao gene played an essential role in nicotine or pseudooxynicotine mineralization in strain HZN6, whereas the sap gene did not. This study provides novel insight into the nicotine-degrading mechanism at the genetic level in Pseudomonas spp.
Applied and Environmental Microbiology 01/2012; 78(7):2154-60. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen, and energy. The strain's main intermediate metabolites were determined to be pseudooxynicotine, 3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas.
Applied Microbiology and Biotechnology 06/2011; 92(5):1023-32. · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the widespread use of synthetic pyrethroids (SPs), the various toxic effects of these compounds have attracted much interest with respect to the investigation of involved mechanisms. However, research on molecular mechanisms of enantioselective toxicity of SPs has been limited. Our previous investigations suggested that enantiomers of cis-bifenthrin (cis-BF) behaved enantioselectively in endocrine disruption and mammalian cytotoxicity. While little is known about the molecular mechanism of the enantioselective toxicity of cis-BF, recent experiments implicated oxidative stress in the cytotoxicity of many SPs. Therefore, the aim of this study was to determine whether cis-BF enantioselectively induced oxidative stress lead to enantioselective cytotoxicity. In this article, we used the rat pheochromocytoma PC12 cell line as an in vitro model to evaluate the involvement of the oxidative stress pathway in enantioselective cytotoxicity of cis-BF. Following exposure of cells to cis-BF and its enantiomers, a significant reduction in cell survival and superoxide dimutase, as well as increased production of lactate dehydrogenase, intracellular reactive oxygen species and malondialdehyde, was observed in 1S-cis-BF, while 1R-cis-BF exhibited these effects to a lesser degree. These results clearly demonstrated that enantiomer-specific cis-BF-induced oxidative damage is possibly an initiating event and contributes, at least in part, to the mechanism of cis-BF-induced enantioselective cytotoxicity. Furthermore, our study also indicated that enantioselectivity should be considered when evaluating the ecotoxicological effects and the health risks of chiral contaminants.
[Show abstract][Hide abstract] ABSTRACT: Synthetic pyrethroids (SPs) are a family of chiral insecticides with a large number of stereoisomers. Fenvalerate (FV) is one of the most potent pyrethroid insecticides, controlling a wide range of insect pests in agricultural fields, public health situations and animal houses. FV contains two chiral centers. In this study, four stereoisomers of FV were absolutely separated by high-performance liquid chromatography with a commercial chiral column, CHIRALCEL OJ-H, using n-hexane containing 1,2-dichloroethane and ethanol as mobile phase. Toxicity assays of each isomer and racemate of FV were performed using Daphnia magna (D. magna), zebrafish (Danio rerio) and zebrafish embryo-larval. In the acute toxicity of D. magna, significant differences were observed: the 24h EC(50) of alphaS-2S-FV was 51 times more toxic than the alphaR-2R-FV, and the 48 h LC(50) results showed that the alphaS-2S-FV was 99 times more toxic than alphaR-2R-FV. In the toxicity assay of D. rerio, dramatic differences were also found: the LC(50) value of alphaS-2S-FV was 17, 22, 39 and 56 times more toxic than the alphaR-2R-FV at 24, 48, 72 and 96 h, respectively. The assays of 4-day-old zebrafish embryo-larval showed that the exposure to FV enantioselectively induced crooked body, yolk sac edema and pericardial edema and that the alphaS-2S-FV was 3.8 times stronger than the other isomers in 96-h mortality. The results indicate that the enantiomeric differences should be taken into consideration in assessing the ecological effects of SPs.
Ecotoxicology and Environmental Safety 08/2009; 72(7):1913-8. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chiral pesticides currently constitute about 50% of all pesticides dosage used in China, and this ratio is increasing as more complex structures are introduced. Dichlorprop methyl (DCPPM) is a chiral herbicide consisting of a pair of enantiomers. In this study, the enantiomeric separation of DCPPM was investigated by gas chromatography (GC) and high-performance liquid chromatography (HPLC) using chiral stationary phases (CSPs), and its enantiomeric degradation was characterized using a DCPPM-degrading bacterial strain isolated from an activated sludge from a textile-printing wastewater treatment plant. Baseline separation by both GC and HPLC was achieved. Incubation with DCPPM-degrading bacteria in different pH solutions showed that the R enantiomer was preferentially degraded over the S enantiomer of DCPPM. The degradation rate constant decreased with increasing pH in the order of k(pH5) approximately k(pH7) >k(pH9). In comparison, the enantioselectivity as indicated by EF followed the order of pH 7 > pH 9-pH 5.
[Show abstract][Hide abstract] ABSTRACT: Synthetic pyrethroids (SPs) are used in preference to organochlorines and organophosphates due to their high efficiency, low toxicity to mammals, and ready biodegradability. Previous studies reported that enantioselective toxicity of SPs occurs in aquatic toxicity. Several studies have indicated that SPs could lead to oxidative damage in humans or animals which was associated with their toxic effects. Little is known about the differences in the effects of chronic toxicity induced by individual stereoisomers of chiral SPs. The present study was therefore undertaken to evaluate the enantioselectivity in cytotoxicity, genotoxicity caused by bifenthrin (BF) on human amnion epithelial (FL) cell lines and pesticidal activity on target organism. The cell proliferation and cytoflow analysis indicated that 1S-cis-BF presented more toxic effects than 1R-cis-BF above the concentration of 7.5 mg L(-1) (p>0.05). FL cells incubated with 1S-cis-BF exhibited a dose-dependent accumulation of intracellular reactive oxygen species (ROS). In the comet assay, the number of cells with damaged DNA incubated with 1S-cis-BF was more than that with 1R-cis-BF (p<0.01). While the LC(50) values of enantiomer to the target pest on Pieris rapae L. show that 1R-cis-BF was 300 times more active than 1S-cis-BF. These results indicate that the enantioselective toxicity and activity of BF between non-target organism and target organism was reversal. These implications together suggest that assessment of the environmental safety and new pesticides development with chiral centers should consider enantioselectivity.
[Show abstract][Hide abstract] ABSTRACT: Chiral pollutants are receiving growing environmental concern due to differential biological activities of their enantiomers. In the present study, enantiomeric separation of the pyrethroid insecticide lambda-cyhalothrin (LCT) was investigated by high-performance liquid chromatography (HPLC) using the columns of Chiralpak AD (amylase tris[3,5-dimethyl-phenyl carbamate]), Chiralpak AS (amylase tris[(S)-1-phenyl carbamate]), Chiralcel OD (cellulose tris[3,5-dimethylphenyl carbamate]), and Chiralcel OJ (cellulose tris[4-methyl benzoate]) with different chiral stationary phases. The differential toxicities of the enantiomers in aquatic systems were evaluated using the acute zebrafish (Danio rerio) toxicity test and the zebrafish embryo test. The enantiomers of LCT were separated completely on all the columns tested and detected by circular dichroism at 236 nm. Better separations were achieved at lower temperatures (e.g., 20 degrees C) and lower levels of polar modifiers (</=5%) in mobile phase. Ethanol was found to be a good modifier of the mobile phase for all the columns, although isopropanol acted better for the Chiralcel OD column. The (-)-enantiomer was >162 times more toxic than its antipode to zebrafish in the acute test. The embryo test indicated that the exposure to LCT enantioselectively induced crooked body, yolk sac edema, and pericardial edema and that the (-)-enantiomer was 7.2 times stronger than the (+)-enantiomer in 96-h mortality. The malformations were induced by the racemate and its (-)-enantiomer at lower concentrations tested (e.g., 50 microg L(-1)), whereas the (+)-enantiomer induced malformations at relatively higher concentrations (>/=100 microg L(-1)). These results suggest that the toxicological effects of chiral pesticides must be evaluated using their individual enantiomers.
Environmental Toxicology and Chemistry 01/2008; 27(1):174-81. · 2.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Imidacloprid, the major component of many widely used insecticide formulations, is highly persistent in soils. In this study, the sorption of imidacloprid by six soils as well as its photodegradation and hydrolysis in water were studied. The soils differed significantly in organic matter content and other physical and chemical properties. Sorption increased with increasing soil organic matter content but was not significantly correlated with other soil properties. Removal of organic matter via H2O2 oxidation decreased the sorption. By normalizing the Freundlich coefficients (Kf) to organic matter contents, the variability in obtained sorption coefficient (Kom) was substantially reduced. These results indicate that soil organic matter was the primary sorptive medium for imidacloprid. The low heat of sorption calculated from Kom suggests that partition into soil organic matter was most likely the mechanism. The photodegradation and hydrolysis of imidacloprid in water followed pseudo-first-order kinetics; however, the latter process needed a six-time-higher activation energy. While both processes produced the same main intermediate, they occurred via different pathways. The hydrolysis of imidacloprid was not catalyzed by the high interlayer pH in the presence of metal-saturated clays, which appeared to result from the lack of the pesticide adsorption in the interlayers of clays.
Journal of Environmental Science and Health Part B 02/2006; 41(5):623-34. · 1.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enantioselective Degradation of 2,4-Dichlorprop methyl ester was studied by sediment bacteria. One DCPPM-degrading bacteria stain was isolated from contaminated sediments. The EF values were determined by chiral GC. The enantioselectivity of DCPPM biodegradation in aquatic phases were evaluated. Incubation with DCPPM-degrading bacteria show that the R enantiomer was preferentially degraded over the corresponding S enantiomer of DCPPM. The EF values show a much higher deviation. It indicate that the microbial degradation of DCPPM was enantioselectivety.
Huan jing ke xue= Huanjing kexue / [bian ji, Zhongguo ke xue yuan huan jing ke xue wei yuan hui "Huan jing ke xue" bian ji wei yuan hui.] 08/2005; 26(4):152-5.
[Show abstract][Hide abstract] ABSTRACT: Surfactants after entering soil may interact with other xenobiotics of sufactant and alter their behaviors. These interactions are very complex, and depend heavily on a range of parameters including surfactant concentration in soil-water, its CMC and adsorption characteristics of sufactant and pollutant etc. In this paper, batch sorption experiment was employed to study the adsorption characteristics of three types of surfactants, viz. an anionic, a cationic, and two nonionic surfactants, in 6 soils. The results showed that the adsorption isotherms of cetyltrimethylammonium bromide (CTMAB) were L-shape, with distribution coefficients (Kd) varied between 300 to 4800 L x kg(-1) in lower concentration range. The adsorption isotherms of sodium dodecyl benzene sulfonate C12 LABS, OP and Tween-20 were L- or S-shape, depending on surface-active molecular and soil properties. The affinity of anionic and nonionic surfactants to soil was weaker than that of cationic surfactant. The Kd for C12 LABS, Tween-20 and OP was within 5.3-39, 0.13-0.44 and 4.4-22.4 L x kg(-1), respectively. For sodium dodecyl benzene sulfonate, Kd was positively correlated with soil clay fraction, and the charge property of soil was also influential in its sorption. For OP and Tween-20, the empirical relations of Kd to soil properties, clay, silt, sand contents, and surface area, were established by using regression analysis. Cationic exchange capacity showed a strong positive correlation with the maximum adsorption capacity of soils, which might indicate that the ionic exchange mechanism played an important role in adsorption of CTMAB.
Ying yong sheng tai xue bao = The journal of applied ecology / Zhongguo sheng tai xue xue hui, Zhongguo ke xue yuan Shenyang ying yong sheng tai yan jiu suo zhu ban 12/2004; 15(11):2067-71.