Article pubs.acs.org/JAFC
Degradation of the Neonicotinoid Insecticide Acetamiprid via the N‑Carbamoylimine Derivate (IM-1-2) Mediated by the Nitrile Hydratase of the Nitrogen-Fixing Bacterium Ensifer meliloti CGMCC 7333 Ling-Yan Zhou,†,‡ Long-Jiang Zhang,*,‡ Shi-Lei Sun,† Feng Ge,‡ Shi-Yun Mao,† Yuan Ma,† Zhong-Hua Liu,† Yi-Jun Dai,*,† and Sheng Yuan† †
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, People’s Republic of China ‡ Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing 210042, People’s Republic of China ABSTRACT: The metabolism of the widely used neonicotinoid insecticide acetamiprid (ACE) has been extensively studied in plants, animals, soils, and microbes. However, hydration of the N-cyanoimine group in ACE to the N-carbamoylimine derivate (IM-1-2) by purified microbes, the enzyme responsible for this biotransformation, and further degradation of IM-1-2 have not been studied. The present study used liquid chromatography−mass spectrometry and nuclear magnetic resonance spectroscopy to determine that the nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 transforms ACE to IM-1-2. CGMCC 7333 cells degraded 65.1% of ACE in 96 h, with a half-life of 2.6 days. Escherichia coli Rosetta (DE3) overexpressing the nitrile hydratase (NHase) from CGMCC 7333 and purified NHase converted ACE to IM-1-2 with degradation ratios of 97.1% in 100 min and 93.9% in 120 min, respectively. Interestingly, IM-1-2 was not further degraded by CGMCC 7333, whereas it was spontaneously hydrolyzed at the N-carbamoylimine group to the derivate ACE-NH, which was further converted to the derivative ACE-NH2. Then, ACE-NH2 was cleaved to the major metabolite IM-1-4. IM-1-2 showed significantly lower insecticidal activity than ACE against the aphid Aphis craccivora Koch. The present findings will improve the understanding of the environmental fate of ACE and the corresponding enzymatic mechanisms of degradation. KEYWORDS: acetamiprid, bioefficacy, Ensifer meliloti, hydration, nitrile hydratase, IM-1-2
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INTRODUCTION
g/L) and thus has a high potential to leach into soil or runoff in surface water, resulting in soil and water pollution.12 Owing to the requirement for government registration and the correlation between the toxicity of neonicotinoid insecticides and their biotransformation,13 the biotransformation of ACE has been well-defined in plants, animals, and soils, where degradation follows three major pathways: oxidative cleavage of the (6-chloro-3-pyridyl)methyl substituent to IM-14 and subsequent oxidation to 6-chloronicotinic acid (IC-0) or acetylation to IM-1-3; N-demethylation of the methyl group at the acyclic spacer to IM-2-1 and hydration of the N-cyanoimine group to the N-carbamoylimine metabolite IM-1-2 (see Figure 1).14,15 Microbial degradation is one of the primary ways pesticides are decomposed in the soil environment and is considered to be an effective and economical method for pesticide remediation in contaminated environments.16 Therefore, microbial degradation of ACE has been extensively studied, and a few pure microbial cultures capable of degrading ACE have been isolated. The bacterium Stenotrophomonas maltophilia CGMCC 1.1788 and the white-rot fungus
Neonicotinoid insecticides, which include imidacloprid (IMI), acetamiprid (ACE), thiacloprid (THI), thiamethoxam (TMX), clothianidin, nitenpyram, and dinotefuran, are an important class of neurotoxins specifically acting as agonists of the insect nicotinic acetylcholine receptors (nAChR). They are the most widely used insecticides in the world in the past three decades.1,2 However, neonicotinoid insecticides threaten birds, honey bees, and other pollinators; therefore, the European Union (EU) banned the use of the most common nitroimine subclasses IMI, TMX, and clothianidin in several crops for a period of 2 years.3,4 ACE, (E)-N1-[(6-chloro-3pyridyl)methyl]-N2-cyano-N1-methylacetamidine, belongs to the cyanoimine subclass and has been proven to be very effective when used as a foliar spray on rice, tea, fruits, and vegetables. Therefore, ACE is very popular in rice and tea plantation areas.5,6 Although this insecticide presents lower acute toxicity to bees than IMI and is not banned in the EU area,3,7 extensive and intensive application of ACE has caused many problems, such as persistence in crops, vegetables, and tea, threat to birds, population-level effects on honey bees, and impact on the development of mammalian nervous systems.6,8−12 Moreover, ACE has a high water solubility of 4.2 g/L (the first IMI launched had a water solubility of only 0.61 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 9957
March 26, 2014 September 23, 2014 September 25, 2014 October 6, 2014 dx.doi.org/10.1021/jf503557t | J. Agric. Food Chem. 2014, 62, 9957−9964
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Figure 1. Molecular structures of ACE and its metabolites and the pathway in microbial metabolism.
Phanerochaete sordida YK-624 metabolize ACE via Ndemethylation to IM-2-1.17,18 The bacteria Ochrobactrum sp. D-12, Pigmentiphaga sp. AAP-1, D-2, Pseudoxanthomonas sp. AAP-7, Rhodococcus sp. BCH2, and Stenotrophomonas sp. THZXP and the yeast Rhodotorula mucilaginosa IM-2 metabolize ACE via the proposed N-deacetylation, C-demethylation, or oxidative cleavage of the N-cyanoimine group to IM-1-4, which is further metabolized by Rhodococcus sp. BCH2 to IC-0 or by R. mucilaginosa IM-2 to IM-1-3.10,19−23 Notably, the formation pathway of the N-carbamoylimine metabolite IM-1-2 in plants, animals, and soils was not detected in the microbial ACE degradation pathways listed above. Nevertheless, although the metabolism of ACE in plants, animals, and soil has been clearly described, as have a few cases of microbial degradation, the corresponding enzymatic mechanism of ACE degradation by potentially useful environmental microorganisms has not yet been explored. The seven major commercial neonicotinoid insecticides contain three structural components: an N-heterocyclyl-methyl moiety (A), a heterocyclic or acyclic spacer (B), and an Nnitroimine, nitromethylene, or N-cyanoimine pharmacophore (C).14 It is interesting that the same structural components in related chemical compounds are metabolized by the same microbes; for example, S. maltophilia CGMCC 1.1788 hydroxylates the same site of component B, that is, the imidazolidine ring in IMI and imidaclothiz and also the thiazolidine ring of THI.24 Pseudomonas sp. 1G transforms IMI and TMX at the component C N-nitroimine group via a nitro reduction pathway.25 THI has the same N-cyanoimine group as ACE, and we isolated four THI-degrading bacteria, Variovorax boronicumulans CGMCC 4969, Ensifer meliloti CGMCC 7333, Ensifer adhaerens 6315, and Ensifer sp. SCL3-19, with the ability to hydrate THI by converting an N-cyanoimine group to an amide metabolite.26,27 Therefore, we examined these four
strains for their ability to degrade ACE and identified the metabolic pathway of ACE transformation used by these ACEdegrading bacteria. Metabolites were then tested for bioefficacy against the horse bean aphid, and the corresponding enzymatic conversion of ACE was further studied. The present study will help to elucidate the environmental fate of ACE, the toxicity of its metabolites, and the mechanism of enzymatic ACE degradation.
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MATERIALS AND METHODS
Chemicals. ACE was provided by Professor Jueping Ni of the Jiangsu Pesticide Research Institute, Nanjing, China (98% purity). HPLC grade acetonitrile (part no. AB1124-134) and methanol (part no. MS1922-001) were purchased from TEDIA (Fairfield, OH, USA). All other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Strains and Media. The wild bacteria E. meliloti CGMCC 7333,27 Ensifer sp. SCL3-19,27 E. adhaerens CGMCC 6315,28 and V. boronicumulans CGMCC 496926 were isolated from soil and identified by 16S rRNA gene analysis. Ensifer sp. SCL3-19 was stored in our laboratory, and the other strains were deposited in the China General Microbiological Culture Collection Center (CGMCC) (Beijing, China). Escherichia coli Rosetta (DE3), the host for nitrile hydratase (NHase) gene expression, was maintained in our laboratory. Luria− Bertani (LB) medium (pH 7.2) for cell cultivation contained 10.0 g of peptone, 5.0 g of yeast extract, and 10.0 g of NaCl in 1.0 L of deionized water. Biodegradation of ACE by the Four THI-Degrading Bacteria, Resting E. coli Rosetta (DE3) Cells Overexpressing the NHase Gene from CGMCC 7333, and Purified NHase. To evaluate the ability of resting bacterial cells to biotransform ACE, a single bacterial colony grown on an LB agar plate was inoculated into a 100 mL flask containing 10 mL of LB broth. The broth was incubated in a rotary shaker at 220 rpm and 30 °C for 24 h. Then, 1 mL of this culture was inoculated into a 500 mL flask containing 100 mL of LB broth and 0.1 mmol/L CoCl2 and incubated using the same culture conditions for 12 h. Subsequently, 5 mL of cultivated broth was sampled, and cells were 9958
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ln(I/I0) against time using the equation ln(I/I0) = −kt, where I0 and I represent the initial and residual concentrations. The half-life was calculated as
harvested with centrifugation at 6000g for 10 min. The cell pellet was washed with 0.2 mol/L sodium phosphate buffer (pH 7.5) and resuspended in a 50 mL plastic centrifuge tube containing 5 mL of the same buffer with 500 mg/L ACE. These cell suspensions were used for resting-cell ACE transformation. Meanwhile, phosphate buffer inoculated with either bacteria or dissolved ACE alone was used as control. The NHase gene cluster from CGMCC 7333 was 1671 bp in length and contained α- and β-subunits of 642 and 660 bp in length, respectively, and an accessory protein gene of 387 bp in length.27 The GenBank accession numbers for the NHase α-subunit, β-subunit, and accessory protein-coding genes from E. meliloti CGMCC 7333 are KF601242, KF601243, and KF601244. Overexpression of recombinant NHase in E. coli Rosetta (DE3) and purification of the NHase protein with His-tag affinity chromatography were performed according to the manufacturer’s (Takara Bio Co., Dalian, China) protocols. The ACE biotransformation protocol was identical for the THI-degrading bacteria. To test the transformation of ACE by NHase, 10 μL of NHase was sampled and added to 1 mL of 0.2 mol/L phosphate buffer (pH 7.5) with 500 mg/L of ACE added in a 1.5 mL plastic centrifuge tube and covered. Transformations were conducted under the above cultivation conditions for the indicated times. The samples were centrifuged at 10000g for 10 min to remove residual cells. The supernatant was collected, subjected to filtration with a 0.22 μm pore-size membrane, and diluted 10-fold for HPLC analysis of substrates and metabolites. Preparation of Metabolites. ACE biotransformation by resting E. coli Rostta (DE3) cells overexpressing the NHase from E. meliloti CGMCC 7333 was scaled to a 1 L flask containing 300 mL of transformation broth and cultivated in a rotary shaker at 220 rpm and 30 °C until the ACE substrate was completely converted as determined by HPLC analysis. Cell residue was removed by centrifuging the mixture at 10000g for 20 min. The supernatant was extracted three times with equal volumes of ethyl acetate. The organic fraction was dehydrated with anhydrous sodium sulfate, after which the organic phase was filtered using a 0.22 μm pore-size organic membrane. Samples were then concentrated in a rotary vacuum evaporator at a temperature of 40 °C and crystallized in a vacuum dryer. The metabolite crystals were checked for purity using HPLC. HPLC, Liquid Chromatography−Mass Spectrometry (LCMS), and Nuclear Magnetic Resonance (NMR) Analyses. An Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a reverse-phase HC-C18 guard column (4.6 × 12.5 mm, 5 μm particle size, part no. 520518-904, Agilent Technologies, Wilmington, DE, USA) and an HC-C18 column (4.6 × 250 mm, 5 μm particle size, part no. 588905-902, Agilent Technologies, Wilmington, DE, USA) was used to analyze the transformation of ACE and its metabolites. Elution was conducted at a flow rate of 1 mL/min in a mobile phase that contained water, acetonitrile, and 0.01% acetic acid (water/acetonitrile, 65:35). The signal was monitored at a wavelength of 235 nm using an Agilent G1314A UV detector. LC-MS was conducted using an Agilent 1290 infinity LC with a G1315B diode array detector and an Agilent 6460 triple-quadrupole LC-MS system equipped with an electrospray ion (ESI) source (Agilent Technologies, Wilmington, DE, USA) that was operated in the positive ion mode. The column, mobile phase, and its proportion were similar to those mentioned for HPLC. The flow rate of elution was set to 0.6 mL/min, and the monitored wavelength of the metabolites formed in the biotransformation of ACE and IM-1-2 was set to 270 and 250 nm, respectively. 13C and 1H NMR spectra for the metabolite were obtained in DMSO-d6 using a Bruker AV-400 NMR spectrometer (Bruker, Faellanden, Switzerland) operating at 100 and 400 MHz, respectively. Chemical shifts were referenced against internal tetramethylsilane. Several NMR techniques were used to assign proton and carbon atom chemical shifts (δ), including distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple bond correlation (HMBC), and heteronuclear singlequantum correlation spectroscopy (HSQC). Half-Life of ACE Degradation. Half-life values for the degradation of ACE and its metabolites were determined by plotting
t1/2 = (ln 2)/k where t1/2 is the half-life and k is the apparent elimination constant. In all cases, the first-order equation provided a satisfactory fit for the data (r > 0.9) and thus provided the basis for the half-life calculation.21 Bioefficacy of ACE Metabolites. Bioefficacy assays were conducted at the National Pesticide Research and Development South Centre, Nanjing, China, during October 2013. Horse bean plants and the aphid Aphis craccivora Koch were used for bioefficacy tests. Oral ingestion and contact bioassays were performed using methods described by Buchholz and Nauen.29
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RESULTS Biodegradation of ACE by Resting V. boronicumulans CGMCC 4969, E. meliloti CGMCC 7333, E. adhaerens
Figure 2. HPLC spectra of ACE biodegradation by resting E. meliloti CGMCC 7333 cells: (A) phosphate buffer containing 500 mg/L ACE and CGMCC 7333; (B) phosphate buffer containing 500 mg/L ACE; (C) phosphate buffer containing CGMCC 7333. Elution rate of the mobile phase was 1 mL/min, and the detection wavelength was 235 nm; the OD600 value of the resting cell suspensions was 5; and the sampled time was 96 h. 9959
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Figure 3. LC-MS data for the biotransformation of ACE by E. meliloti CGMCC 7333: (A) HPLC spectrum of ACE degradation for 4 days; (B, C, D) mass data of the metabolite peaks P1 and P2 and the substrate ACE at retention times of 4.30, 5.33, and 12.89 min, respectively. Elution rate of the mobile phase was 0.6 mL/min, and the detection wavelength was 250 nm.
Table 1. 13C NMR and 1H NMR Chemical Shift Assignments for Metabolites in DMSO-d6 position C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 N11
13
1
C NMR 149.4 133.7 139.5 124.5 149.5 49.2 36.1 160.8 16.4 165.4
bacterium alone (Figure 2C). After transformation for 48 h, another new small peak (P2) with a retention time of 2.96 min was observed in the transformation broth. Peaks P1 and P2 with retention times of 4.30 and 5.33 min, respectively, were observed in LC-MS analysis of the biotransformation of ACE in 96 h (Figure 3A). As shown in Figure 3B, metabolite P2 displayed a protonated parent ion (M + H) at m/z 157 and a major fragment ion (M − CH4N) at m/z 126. Thus, P2 could be assigned to IM-1-4 (Figure 1), a major metabolite that appears in soil, plant, and microbial metabolism of ACE.10,20,23,30 Metabolite P1 (Figure 3C) displayed a protonated parent ion (M + H) at m/z 241, a sodiated adduct (M + Na) at m/z 263, a fragment ion (M − O) at m/z 224, an unknown fragment ion at m/z 198, and a fragment ion (M − C4H8N3O) at m/z 126. The unknown fragment ion at m/z 198 also appeared in the spectrum of the gas chromatography−mass spectrometry (GC-MS) analysis of IM-1-2 reported by Yeter and Aydin.31 The mass of metabolite P1 was enhanced by the addition of a water molecule to the substrate ACE (M + H) at m/z 223 (Figure 3D) and was identical to the mass data reported for IM-1-2 by Yeter and Aydin31 and Kamel et al.32 Resting E. coli Rosetta cells overexpressing CGMCC 7333 NHase also hydrolyzed ACE to a metabolite with the same HPLC retention time as metabolite P1, and this metabolite showed the same mass as P1 in LC-MS analysis (data not shown). This E. coli strain transformed ACE completely in 2 h and was thus used for preparation of metabolite P1. The purified P1 was then used for NMR analysis. As is shown in Table 1, there are 10 carbon atoms in the 13C NMR spectrum, which was identical to the parent ACE. Therefore, the previously reported metabolites IM-2-1 (nine carbon atoms), IM-1-4 (seven carbon atoms), and IM-1-3 (nine carbon atoms) could be excluded.17,20,23 There were 13 protons in the 1H NMR spectrum of the ACE metabolite P1, which was an
H NMR
8.33 (d), 1H, J = 1.6 Hz 7.76 (dd), 1H, J = 8.0 Hz, J = 1.6 Hz 7.48 (d), 1H, J = 8.0 Hz 4.61 (s), 2H 2.91 (s), 3H 2.12 (s), 3H 6.28, 6.13 (b), 2H
CGMCC 6315, and Ensifer sp. SCL3-19 Cells. The four THI-hydrating bacterial strains V. boronicumulans CGMCC 4969,26 E. adhaerens CGMCC 6315,28 E. meliloti CGMCC 7333, and Ensifer sp. SCL3-1927 were tested for ACE degradation. After transformation for 60 h, V. boronicumulans CGMCC 4969, E. meliloti CGMCC 7333, and E. adhaerens 6315 degraded 23 ± 2, 45 ± 5, and 8 ± 1% of ACE, respectively, whereas Ensifer sp. SCL3-19 did not degrade ACE. E. meliloti CGMCC 7333, which degraded THI at the highest level,27 also degraded ACE the most efficiently of the tested bacteria. Therefore, this bacterium was used for further studies. Identification of Metabolites Formed during ACE Biotransformation. ACE biotransformation by resting E. meliloti CGMCC 7333 cells resulted in the formation of an apparent new peak (P1) with an HPLC retention time of 3.78 min (Figure 2A). This peak could be detected in 12, 24, 36, 48, and 60 h, and no comparable peak was observed upon analysis of controls containing ACE (Figure 2B) or inoculated 9960
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amide moiety. On the basis of MS and NMR data, ACE metabolite P1 was the N-carbamoylimine derivate previously called IM-1-2.15 Kinetics of ACE Transformation by Resting E. meliloti CGMCC 7333 Cells, the E. coli Rosetta (DE3) Strain Overexpressing the NHase Gene from E. meliloti CGMCC 7333, and Purified NHase. Time course analyses of ACE hydration by resting E. meliloti CGMCC 7333 cells (Figure 4A) indicated that the quantity of ACE declined from 2.12 to 0.74 mmol/L in 96 h with an ACE degradation ratio of 65.1%. ACE degradation fitted first-order dissipation kinetics (R = 0.99) with a half-life of 63.0 h. For ACE biodegradation, the molar conversion rates (the amount of IM-1-2 formed divided by the amount of ACE lost) at 12, 24, 36, 48, 72, and 96 h were 96.0, 94.6, 90.5, 73.7, 57.2, and 43.8%, respectively. In the initial 36 h, the molar conversion rate was >90%, which indicated that ACE hydration was the primary pathway of ACE degradation. As is shown in Figure 4B, the quantity of ACE in the NHase overexpressing E. coli Rosetta (DE3) reaction declined from 2.04 to 0.06 mmol/L in 100 min with a degradation ratio of 97.1%. The half-life of ACE degradation was calculated to be 19.7 min (R = 0.99). The molar conversion rates of ACE degradation by NHase overexpressing E. coli Rosetta cells at 20, 40, 60, 80, and 100 min were 87.7, 92.1, 86.8, 91.3, and 93.3%, respectively. The quantity of ACE in the pure NHase reaction (Figure 4C) declined from 1.98 to 0.12 mmol/L in 120 min with a degradation ratio of 93.9%. The half-life of ACE degradation was calculated to be 30.1 min (R = 0.99). The molar conversion rates of ACE degradation by NHase at 10, 20, 40, 60, 80, 100, and 120 min were 98.1, 96.8, 87.2, 88.3, 91.0, 94.9, and 98.2%, respectively. These results indicated that the NHase from E. meliloti CGMCC 7333 was responsible for the conversion of ACE to IM-1-2. Degradation of IM-1-2. We previously reported that E. meliloti CGMCC 7333 hydrolyzed THI to THI amide with a molar conversion rate of >90% at every sampled time.27 Interestingly, the molar conversion rate of ACE degradation declined significantly after transformation for 36 h. This result indicated that the IM-1-2 formed by hydration of ACE by E. meliloti CGMCC 7333 may have been further degraded. Therefore, IM-1-2 was used as a substrate for transformation by E. meliloti CGMCC 7333 to explore whether further IM-1-2 degradation was mediated by the bacterium. As shown in Figure 5, both the experimental group with bacterial inoculation and the control without CGMCC 7333 inoculation showed apparent IM-1-2 degradation. After incubation for 8 days, the amount of IM-1-2 was reduced by 1.40 and 1.32 mmol/L with bacterial inoculation and with IM-1-2 alone, respectively, and the corresponding degradation rates were 67.5 and 62.9%, respectively. The half-lives for IM-1-2 degradation for the experimental and control groups were fitted into firstorder dissipation kinetics (R 2 = 0.9986 and 0.9936, respectively) and were calculated to be 4.9 and 5.5 days, respectively. There was no significant difference (P > 0.05) in IM-1-2 degradation between the experimental and control groups at any sampled time. These results indicate that IM-1-2 degradation did not result from bacterial transformation and instead resulted from spontaneous chemical degradation. As shown in Figure 6A, three new peaks with retention times of 4.26, 4.53, and 6.84 min, respectively, were observed in the LC-MS analysis of the control group sample without bacterial inoculation after incubation for 8 days. It can be observed that
Figure 4. Time course of ACE degradation by E. meliloti CGMCC 7333 (A), E. coli Rosetta overexpressing NHase (B), and pure NHase (C). Preparation of resting E. meliloti CGMCC 7333 and E. coli Rosetta (DE3) cells is described under Materials and Methods. The OD600 value of resting cell suspensions was 5. The NHase concentration was 67.9 μg/mL in 0.2 mol/L phosphate buffer (pH 7.5) with the addition of 0.1 mmol/L CoCl2, and the reaction conditions were the same as for the resting-cell transformation of ACE. Average values and standard deviations were calculated from three parallel cultures tested three times (n = 9).
Figure 5. Degradation of IM-1-2 by E. meliloti CGMCC 7333. IM-1-2 was used as a substrate for transformation. The transformation conditions were the same as for ACE. The control was not inoculated with bacteria, and therefore any IM-1-2 degradation was spontaneous.
increase of 2 protons relative to the ACE substrate. The 2 new protons had chemical shifts of δ 6.13 and 6.28, respectively. These two protons had no correlation with the carbon atom in HSQC analysis. Therefore, they were assigned to the protons of the amide moiety. Meanwhile, a chemical shift was not observed for the C10 carbon atom of ACE in the 13C NMR spectrum, and a new chemical shift (δ 165.4) appeared in metabolite P1 that could be assigned to the carbon atom of the 9961
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Figure 6. LC-MS data for the degradation of IM-1-2 in phosphate buffer without CGMCC 7333 inoculation: (A) HPLC spectrum of IM-1-2 degradation for 8 days without bacterial inoculation (substrate IM-1-2 peaked at a retention time of 5.29 min); (B, C, D) mass data of the metabolite peaks M1, M2, and M3 at retention times of 4.26, 4.53, and 6.84 min, respectively. The elution rate of the mobile phase was 0.6 mL/min, and the detection wavelength was 270 nm.
Laboratory Bioassays of IM-1-2 against the Horse Bean Aphid A. craccivora. The insecticidal activities of metabolites produced by microbial transformation contribute to the bioefficacy persistence of neonicotinoid insecticides.8 ACE is recommended as a plant foliar spray, and IM-1-2 is one of its plant metabolites.33 Therefore, we examined the bioefficacy of IM-1-2 against the horse bean aphid A. craccivora. As is shown in Table 2, ACE showed perfect and acute insecticidal activity in both the contact and oral ingestion bioassays. At applied concentrations of 0.2500, 0.0625, and 0.0156 ppm, aphid mortalities were 100, 100, and 87.14%, respectively. At the same tested concentrations, the mortality rates of IM-1-2 were only 12.00, 7.69, and 8.22%, respectively. Even when the concentration of IM-1-2 was increased to 1 ppm, the mortality level was only 13.56%. Laboratory bioassays indicated that IM1-2 had dramatically reduced activity against the aphid A. craccivora. Similar results were observed for the bioassay of the THI amide, which reduced insecticidal activity by at least 1 order of magnitude when compared with the THI parent.19 The above data indicated that the replacement of the crucial pharmacophore cyano group in ACE and THI with an amide group results in a significant reduction in insecticidal activity.
Table 2. Bioefficacy of IM-1-2 against the Horse Bean Aphid A. craccivora mortalitya (%)
compound
content (mg/L)
total insects tested
IM-1-2
1.0000 0.2500 0.0625 0.0156
59 50 65 73
ACE
0.2500 0.0625 0.0156
51 67 70
100.00 ± 0.00 100.00 ± 0.00 87.14 ± 4.57
control
0
71
5.63 ± 0.94
13.56 12.00 7.69 8.22
± ± ± ±
5.85 1.24 0.85 4.22
Mortality is expressed using the mean ± standard error from three parallel tests. Mortality was examined at 72 h after application of ACE or IM-1-2.
a
M1 (Figure 6B) has a protonated parent ion (M + H) at m/z 157 and a major fragment ion (M − CH4N) at m/z 126. These mass data were identical to product P2 (Figure 3B), which was thus assigned to IM-1-4. M2 has a protonated parent ion (M + H) at m/z 198 and could be attributed to the metabolite resulting from cleavage of the N-carbamoylimine group in IM1-2. This metabolite has been detected in mice and plant metabolism of ACE and was named ACE-NH.33 M3 had a parent ion (M + H) at m/z 200 and could be attributed to the metabolite resulting from the reduction of the CNH group in ACE-NH to form a CNH2 group. M3 was not reported previously33 and was named ACE-NH2. Metabolites M1, M2, and M3 will be purified and subjected to NMR analyses in future studies.
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DISCUSSION ACE is stable in buffered solutions at pH 5.0−8.0 and in sunlight. It is degraded slowly at pH 9 and 45 °C.34 However, ACE was degraded rapidly by aerobic soil metabolism, with a half-life ranging from 1 to 8.2 days in studies of various U.S., European, and Chinese soils and varied from 16 to 17 days under field conditions in Indian soils.17,32 In our present study, ACE was readily degraded by E. meliloti CGMCC 7333, the 9962
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reverse the selective toxicity of neonicotinoid insecticides to insects over vertebrates.38 As shown in Figure 7, IM-1-2 can be spontaneously converted to the imine group containing metabolite ACE-NH and, therefore, ACE degradation via IM1-2 pathway to form ACE-NH may increase the risk of ACE toxicity to vertebrates. Our present results may help to explore the mechanism of the adverse effect of ACE and IMI on human health.11 The molecular structures of ACE and THI have 6-chloro-3pyridinylmethyl and N-cyanoamidine groups in common, but differ in their cyclic thiazolidine and acyclic methyl moieties (Figure 1). E. meliloti CGMCC 7333 hydrolyzes ACE and THI to the corresponding N-carbamoylimine derivatives despite the molecular differences between these two molecules. Ensifer sp. SCL3-19 hydrolyzes only the cyclic THI, not the acyclic ACE. We speculate that this difference in substrate selectivity between E. meliloti CGMCC 7333 and Ensifer sp. SCL3-19 may be attributed to differences in NHase structure between the two Ensifer species. The full NHase gene from Ensifer sp. SCL3-19 is being cloned and expressed, and site-specific mutation of various NHase genes will be conducted in the future to explore NHase function and the specific hydrolysis of the neonicotinoid insecticides ACE and THI. Among ACE-degrading bacteria, Rhodococcus sp. BCH2 has been reported to degrade ACE, and three metabolites were observed in HPLC spectra. Two of these metabolites were determined to be IM-1-4 and IC-0 by GC-MS analysis. The authors assumed that ACE was degraded via IM-1-2 because they found an N-carbamoylimine group in the extraction of the mixed metabolites by Fourier transform infrared spectrum analysis, although they did not identify IM-1-2 itself by LC-MS or NMR analysis.10 The present findings suggest that IM-1-2 cannot be directly detected as a product of ACE metabolism by Rhodococcus sp. BCH2, perhaps because IM-1-2 is hydrolyzed in this preparation process. In addition, it is known that the NHase of Rhodococcus catalyzes the conversion of the nitrile compound acrylonitrile to acrylamide in industrial processes,39 and therefore it has been suggested that ACE degradation by Rhodococcus sp. BCH2 requires further study. In conclusion, our present study first found that the nitrogenfixing bacterium E. meliloti CGMCC 7333 and its NHase specifically transform the neonicotinoid insecticide ACE to IM1-2. IM-1-2 is unstable due to chemical hydrolysis and produces the metabolites ACE-NH, ACE-NH2, and IM-1-4. The present findings will improve our understanding of the environmental fate of ACE and the corresponding enzymatic mechanisms.
Figure 7. Proposed metabolic degradation pathways for ACE by E. meliloti CGMCC 7333 and further degradation of IM-1-2.
CGMCC 7333 NHase overexpressing E. coli, and the purified CGMCC 7333 NHase with half-lives of 63.0 h, 19.7 min, and 30.1 min, respectively. LC-MS and NMR analyses indicated that CGMCC 7333 NHase mediated the hydration of ACE to metabolite IM-1-2. IM-1-2 is unstable in phosphate-buffered solutions with a half-life of 5.5 days, and this chemical degradation resulted in cleavage of the N-carbamoylimine group to an ACE-NH derivate. ACE-NH was then converted to ACE-NH2, which was subsequently cleaved to IM-1-4. The proposed pathway of ACE metabolism by E. meliloti CGMCC 7333 and chemical degradation of IM-1-2 are presented in Figure 7. Apparently, ACE degradation via the IM-1-2 pathway mediated by NHase and the chemical degradation of IM-1-2 resulted in the rapid degradation fo ACE in soil. Metabolites of neonicotinoid insecticides have been shown to contribute to the toxicity of these insecticides.7 For example, olefin metabolite of IMI was noted to be >10 times more toxic than the parent IMI,35,36 whereas ACE metabolites, including IM-2-1, IC-0, IM-O (6-chloro-3- pyridiylmethanol), IM-1-4, and IM-1-3, were found to exhibit lower toxicity than ACE.17,19,37 In our present study, we further proved that one of the major metabolites of ACE degradation, IM-1-2, also showed a dramatic reduction in toxicity (Table 2). Therefore, the rapid biotransformation and detoxification of the metabolites leading to the cyano-group-containing ACE is less toxic than the nitro-group-containing IMI against certain insects.37 However, the selectivity of neonicotinoids to insect nAChR could be attributed to the pharmacophore group nitroimine (NNO 2 ) and cyanoimine (NCN), because they have a much higher affinity for insects, when compared with vertebrate nAChRs, and the loss of a nitro or cyano group to the imine metabolite (NH) can completely
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AUTHOR INFORMATION
Corresponding Authors
*(L.-J.Z.) E-mail:
[email protected]. *(Y.-J.D.) E-mail:
[email protected] Funding
This research was financed by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, National Science Foundation of China (31370143), the Professional and Public Project of National Environmental Protection (201009033), and the Academic Natural Science Foundation of Jiangsu Province (14KJA180004). Notes
The authors declare no competing financial interest. 9963
dx.doi.org/10.1021/jf503557t | J. Agric. Food Chem. 2014, 62, 9957−9964
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Article
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