Metabolism of an Insecticide Fenitrothion by Cunninghamella elegans ATCC36112

Article abstract of DOI:10.1021/acs.jafc.7b04273

In this study, the detailed metabolic pathways of fenitrothion (FNT), an organophosphorus insecticide by Cunninghamella elegans, were investigated. Approximately 81% of FNT was degraded within 5 days after treatment with concomitant accumulation of four m

Full text of DOI:10.1021/acs.jafc.7b04273

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Article
Metabolism of an Insecticide Fenitrothion
by Cunninghamella elegans ATCC36112
Yong-Zhe Zhu, Min Fu, In-Hong Jeong, Jeong-Han Kim, and chuan-jie zhang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04273 • Publication Date (Web): 16 Nov 2017
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Journal of Agricultural and Food Chemistry
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RESEARCH ARTICLE
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Agricultural and Environmental Chemistry
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Metabolism of an Insecticide Fenitrothion by Cunninghamella
elegans ATCC36112
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YongꢀZhe Zhu1, Min Fu1, InꢀHong Jeong2, JeongꢀHan Kim3, ChuanꢀJie Zhang4*
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1College of Chemistry and Pharmaceutical Science, Qingdao Agricultural University,
Changcheng Rd, Chengyang district, Qingdao, Shandong, 266ꢀ109, China
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2Division of Crop Protection, National Institute of Agricultural Science, Rural Development
Administration, Jeollabukꢀdo 55365, Republic of Korea
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3Department of Agricultural Biotechnology, Seoul National University, 599 Gwanakꢀro, Silimꢀ
dong, GwanakꢀGu, Seoul, 151ꢀ742, Republic of Korea
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4 Department of Plant Science, University of Connecticut, 1376 Storrs Road, Uꢀ4163, Storrs, CT
06269, United State
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Running title: Metabolism of fenitrothion by Cunninghamella elegans
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20 ABSTRACT: In this study, the detailed metabolic pathways of fenitrothion (FNT), an
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organophosphorus insecticide by Cunninghamella elegans was investigated. Approximately 81%
of FNT was degraded within 5 d after treatment with concomitant accumulation of four
metabolites (M1ꢀM4). The four metabolites were separated by HPLC and their structures were
identified by MS and/or NMR. M3 is confirmed to be an initial precursor of others and identified
as fenitrothionꢀoxon (FNTO). On the basis of their metabolic profiling, the possible metabolic
pathways involved in phase I and II metabolism of FNT by C. elegans was proposed. We also
found C. elegans was able to efficiently and rapidly degrade other organophosphorus pesticides
(OPs). Thus, these results will provide an insight into understanding of the fungal degradation of
FNT and the potential application for bioremediation of OPs. Furthermore, the ability of C.
elegans to mimic mammalian metabolism would help us elucidate the metabolic fates of organic
compounds occurring in mammalian liver cells and evaluate their toxicity and potential adverse
effects.
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34 KEYWORDS: Bioremediation; Cunninghamella elegans; fenitrothion; fenitrothionꢀoxon;
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fungal metabolism; organophosphorus insecticide;
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Abbreviations used: FNT, fenitrothion; FNTO, fenitrothionꢀoxon; HPLC, highꢀperformance
liquid chromatography; MS, mass spectrometry; MNP, 3ꢀmethylꢀ4ꢀnitrophenol; NMR, nuclear
magnetic resonance spectroscopy; OPs, organophosphorus pesticides.
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40 INTRODUCTION
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Organophosphorus pesticides (OPs) are widely used as insecticides for controlling a wide
range of pests. These pesticides act as acetylcholinesterase (AchE) inhibitor causing disruption
of nervous impulse transmission and can thus be hazardous as a result of runoff from areas of
application.1 OPs have been listed as highly toxic to mammal, and exhibited chemical stability
and resistance to biodegradation.2ꢀ4 Although recently developed pesticides are considered to be
much safer than those with a long history (e.g., organochlorine), unexpected health risks and
potential environmental hazard are still inevitable (e.g., carcinogenesis, pesticide residual,
environmental pollution).5
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Fenitrothion (FNT), O,Oꢀdimethyl Oꢀ4ꢀnitroꢀmꢀtolyl phosphorothioate, an organophosphorus
insecticide, has been extensively used in agriculture production to control a broad range of
insects. FNT is categorized as a moderately hazardous compound and listed as an alternative
insecticide for DDT, dieldrin, and endrin.6 As a consequence of increasing use of FNT, serious
environmental problems such as soil and water contamination have occurred as well as its acute
poisoning from accidental or suicidal ingestion.7 In some developing countries, aging and large
quantities of stored FNT has become a potential threat to human health and environmental
contamination.6 Additionally, in terms of food safety, FNT has been reported the most common
insecticide residue in Australian food products.8 Therefore, it is necessary to study the metabolic
fate of FNT and find a possible solution for this problem. Studies on biological and
nonbiological degradation of FNT have been conducted.9ꢀ10 Among those methods, microbial
degradation proves to be an effective and environmentally friendly.10
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Several bacterial species such as Pseudomonas sp., Bacillus sp. have been reported to degrade
a wide range of organophosphorus insecticides including FNT.11ꢀ14 However, there have been
only a few studies of fungalꢀassociated degradation of FNT.9 Unlike prokaryotes, eukaryotic
fungi have demonstrated diverse metabolic potential resulting in metabolites similar to those
produced in the mammalian enzyme system,15ꢀ20 which would help us reveal the metabolic fates
of organic compounds occurring in mammalian liver cells. C. elegans, a filamentous fungus, has
been extensively used as a microbial model system of mammalian metabolism for studying
biotransformation of drugs, pesticides, and environmental cleanup of pollutants.17,20ꢀ26 FNT has
been documented to be hydrolyzed and cometabolized by the fungus Trichoderma viride,9
however, little is known about the biodegradation fate of FNT by fungi. In this study, C. elegans
was used as a model of mammalian metabolism for studying metabolic fate of FNT and its
degradative potential for other OPs with the aim of using this application for bioremediation.
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74 MATERIALS AND METHODS
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Chemicals and reagents. FNT and the other OPs standards involved in this study were
purchased from Chem Service (West Chester, PA), Dr. Ehrenstorfer (Augsburg, Germany), and
Fluka (Buchs, Germany), with the highest available purity. HPLCꢀgrade acetone, acetonitrile,
and methanol were obtained from Honeywell (Morristown, NJ). LCꢀMSꢀgrade formic acid and
ammonium formate were from Fluka (Buchs, Germany). CD3OD and CDCl3 were from Merk
(Darmstadt, Germany), and anhydrous Na2SO4 and NaCl were from Junsei (Japan). Metaꢀ
choroperbenzoic acid (MCPBA) (<77%) was from SigmaꢀAldrich (Shanghai, China). All other
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chemicals and regents used in this study were of analytical grade. Potato dextrose broth (PDB)
and potato dextrose agar (PDA) were purchased from BD Korea (Seoul, Korea).
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Microorganism. Cunninghamella elegans ATCC36122 was obtained from American Type
Culture Collection (Manassas, VA). Fungal cultures were maintained on PDA at 27
on a
rotary shaker operating at 170 rpm, while the liquid cultures were maintained on PDB at 27
(170 rpm). In this study, all culture media were sterilized at 121 for 15 min prior to use. Seed
cultures were prepared on PDB at 27 (170 rpm) for 48 h prior to inoculation.
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Metabolism of FNT by C. elegans.
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Determination of standard curve for FNT. A stock standard solution of FNT (5000 mg/L) was
prepared with acetonitrile and stored in dark at ꢀ18 . A range of concentrations (0.1ꢀ100.0 mg/L)
were prepared by diluting a standard solution with acetonitrile for determining a standard curve
using an Agilent 1100 HPLC series (Agilent, Santa Clara, USA) equipped with a Halo C18
column (150 × 2.1 mm i.d., 2.7 µm; USA), using a mobile phase of water (A) and acetonitrile (B)
containing 0.1% formic acid at a flow rate of 1 mL/min which was programed as follows: 0ꢀ1
min, 10%B; 1ꢀ40 min, 10ꢀ90%B; 40ꢀ45 min, 90%B; 45ꢀ47 min, 90ꢀ10%B; 47ꢀ55 min, 10%B.
The ultraviolet (UV) detection was performed at 280 nm.
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Metabolic reactions of FNT by C. elegans. Precultured mycelia (approximately 2 g) was added in
fresh PDB (250 mL), supplemented with 0.5 mL (2.5 mg) of FNT standard solution and cultured
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at 27
(170 rpm). Aliquots of culture medium (10 mL) were sampled periodically and
transferred into a 50 mL test tube with 5 g NaCl and extracted with acetonitrile (twice with 20
mL). The organic layer was combined and dried over anhydrous Na2SO4. After evaporating the
solvent, the extract was dissolved with 2 mL of acetonitrile, and filtered using a 0.2 µm
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polytetrafluoroethylene (PTFE) filter for HPLC analysis (analytical conditions as
aforementioned). Controls consisted of culture broth with microorganism (blank control) and
sterile medium with FNT (negative control). For sterilized control, cultures were sterilized at
121 for 15 min. All experiments were conducted in triplicate. The percent degradation of FNT
at each sampling time was calculated using equation [1]:
ꢂꢀ )
[1]
Percent degradation = (ꢀ
× 100%
ꢁ
ꢃ
ꢀ
ꢁ
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To describe the degradation kinetics of FNT, a firstꢀorder kinetic equation [2] was used to fit the
Ct data as a function of sampling time. T1/2 (halfꢀlife time) was determined using equation [3]:
Cꢄ = Cꢅ × eꢂꢆꢄ
[2]
ln2
[3]
Tꢇ/ꢈ
=
k
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where C0 is defined as initial FNT concentration at 2 h after treatment, and Ct represents the
concentration at time t (day), respectively, k is degradation rate constant (dꢀ1), and T1/2
corresponds to the time at which half of FNT has been degraded. HPLC analysis showed that 4
metabolites (M1ꢀM4) were produced by C. elegans.
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Isolation and identification of FNT metabolites by large scale culture. The identification of the 4
metabolites was carried out through large scale culture. Eight of the 1000 mL cultures were
extracted at 15 d after treatment (maximum metabolite formation), combined, and concentrated
as described previously. The 4 metabolites for a Varian 500ꢀMSn (Walnut creek, CA, USA)
analysis were fractionated from the extract through an Aglient 1100 HPLC equipped with a
semiꢀprep C18 (250 × 4.6 mm i.d., 10 µm, Phenomenex, USA) column. The same mobile phase
as aforementioned was used at a flow rate of 1 mL/min and programed as follows: 0ꢀ1 min,
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52%B; 1ꢀ15 min, 52ꢀ58% B; 15ꢀ16 min, 58ꢀ95%B; 16ꢀ22 min, 95%B; 22ꢀ23 min, 95ꢀ52%B; 23ꢀ
30 min, 52%B. Parameters set for MS analysis were as follows: scan type, positive and negative
ion modes (m/z 50–500); nebulizer gas pressure, 40 psi; drying gas pressure, 30 psi; drying
temperature, 350 ; capillary voltage, 46.9 V; needle voltage, 4000 (+) and 4000 (ꢀ) V; collision
induced dissociation voltage, 2.0 V. The Turbo Data Dependent Scanning (TurboDDSTM, Varian,
USA) mode was used to obtain MS2 spectra of those metabolites. The proposed structures for the
fragmentation ions were determined using Mass FrontierTM software (Version 6.0, HighChem,
Ltd., Slovakia). As sufficient amount of M1 was collected, 1HꢀNMR and 13CꢀNMR
measurements were further performed with a NMR spectrometer (Advance 400, Bruker) at 400
MHz.
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Synthesis of FNTO and its degradation by C. elegans. As FNTO was confirmed to be an initial
precursor of other metabolites, to reveal the metabolic relationship among those metabolites,
FNTO was synthesized by a previous described method27 and subjected to degrade by C. elegans.
A solution of 400 mmol MCPBA dissolved in 10 mL methylene chloride was prepared and then
added dropwise to a roundꢀbottled flask containing 200 mmol FNT in an ice bath and then stirred
for 20 mins. The reaction solution was transferred to a separatory funnel, washed with Na2CO3
and saturated NaCl solution (three times). The organic phases were combined and dried with N2.
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The extract was analyzed by HPLC/MS and H and 13C NMR accordingly. The study of
degradation of FNTO by C. elegans was performed as aforementioned for FNT.
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Biodegradation of other OPs by C. elegans. The degradative potential of C. elegans to other
OPs was also investigated. Individual stock standard solutions of the twenty nine OPs (1000
mg/L) were prepared with acetonitrile and diluted to a range of concentrations (0.5ꢀ100.0 mg/L)
for each individual to determine a standard curve using an Agilent HPLC 1100 series equipped
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with a Kinetex C18 column (100 × 2.1 mm i.d., 2.6 µm; Phenomenex, USA), using the same
mobile phase as aforementioned at a flow rate of 0.2 mL/min and programmed as follows: 0ꢀ1
min, 5%B; 1ꢀ40 min, 5ꢀ95% B; 40ꢀ45 min, 95%B; 45ꢀ47 min, 95ꢀ5%B; 47ꢀ55 min, 5%B. UV
detection wavelengths, retention times, and standard curve regression equations of 29 OPs were
shown in Table S1. Linear relationships (R2> 0.99) between peak area and pesticide
concentrations were found within the concentration range of 0.1ꢀ50.0 mg/L with a limit detection
(LOD; S/N> 3) of 0.5 mg/L. Recovery experiments performed at two spike levels (0.2 and 1.0
mg) demonstrated that recoveries of those OPs ranged between 76.7 and 112.3% with relative
standard deviation (RSD) values lower than 10.2% (Table S2). Procedures for biodegradation of
29 OPs by C. elegans were same as that of FNT. Cultures extraction were conducted at 2 h, 4 d,
and 7 d after treatment and the residues were determined by HPLC. The percent degradation was
calculated based on equation [1]. All experiments were conducted in triplicate.
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158 RESULTS AND DISCUSSION
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Metabolism of FNT by C. elegans. Standard regression equation (y= 35.9xꢀ5.3) of FNT
demonstrated a strong linear relationship (R2= 1) between peak area and concentrations within
the concentration range of 0.1ꢀ100.0 mg/L. LOD (S/N> 3) was calculated 0.01 mg/L.
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Regarding metabolism of FNT by C. elegans, HPLC analysis of the culture extracts revealed
that four metabolites M1, M2, M3, and M4 were detected at 17.7, 19.3, 21.6, and 25.5 minutes,
respectively, whereas blank and negative controls had no peaks corresponding to any of those
metabolites (Figure 1). Accumulation of metabolites was accompanied by concomitant decrease
of FNT (Figure 2A and 2B). Among those metabolites, M2 and M3 were detected at 1 d after
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treatment and gradually increased after that, however, the accumulation of M3 reached a
maximum at 9 d after treatment and decreased gradually afterward, indicating that M3 is an
initial precursor of other metabolites (Figure 2B). M1 and M4 were detected at 4 d after
treatment. The accumulation of M1 continuously increased and became the most abundant
metabolite until the end of experiment, whereas M4 increased slowly and was detected at low
concentrations (Figure 2B). FNT was detected approximately 81% degraded in the culture at 5 d
after treatment and 91.5% at 7 d after treatment, whereas only 3% of FNT was naturally
degraded in the negative control at 7 d. At 15 d after treatment, it was almost undetectable and
the proportions for those metabolites (M1ꢀM4) in culture were 58.7%, 15.1%, 20.8%, and 4.8%,
respectively. The kinetic model determined for degradation of FNT is Ct= 3220.3 × eꢀ0.37t with
R2= 0.997, and the T1/2 is 1.88 d. The identification of the four metabolites (M1ꢀM4) was
performed through large scale culture.
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Mass spectrum of those metabolites showed FNT and M3 were detected in a positive ion
mode and M1, M2, and M4 were detected in a negative ion mode (Figure 3), respectively. MS2
fragmentation ions of those metabolites are summarized in Figure 4. M3 showed its molecular
ion at m/z 262 ([M + H]+) (100), which was 16 Da lower than that of FNT, indicating that an S
atom in FNT may be substituted by an O atom (P=S → P=O) (Figure 3A and 4). To verify this
hypothesis, FNTO was synthesized with 96.4% purity and identified by NMR and HPLC/MS.
1
The H and 13C NMR spectrum of synthesized FNTO matched with its structure (Table 1).
HPLC/MS analysis showed FNTO was detected at 22.1 minute and its molecular ion was at m/z
262 ([M + H]+) (100), which are consistent with the results obtained for M3 (Figure 5),
demonstrating that M3 is FNTO (O,Oꢀdimethyl Oꢀ(3ꢀmethylꢀ4ꢀnitrophenyl) phosphate).
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In the case of M1, it displayed a molecular ion at m/z 232 ([M ꢀ H]ꢀ) (100), which was 29 Da
lower than that of M3, indicating that two Oꢀmethyl groups in M3 may be hydroxylated to
produce M1 (ꢀOCH3 → ꢀOH) (Figure 3B and 4). Additionally, the fragment ion at m/z 152 ([M ꢀ
1
H]ꢀ) (40) corresponds to 3ꢀmethylꢀ4ꢀnitrophenol (Figure 3B and 4).6ꢀ7,10,28ꢀ29 The H NMR and
13C NMR spectra of M1 were obtained with purified samples to further determine its chemical
1
structure. The H NMR spectrum of M1 showed the presence of 3 aromatic ring protons (7.42,
7.46, and 7.18 ppm) and 3 carbohydrate protons with one methyl group (2.63 ppm). No signal
was detected for hydroxy group proton, which may be due to hydrogen–deuterium exchange
(CD3OD as solvent). The 13C NMR spectrum indicated the presence of 6 aromatic carbons
(154.2, 149.3, 144.8, 126.9, 121.7, and 119.8 ppm) and one methyl group (20.9 ppm) (Table 1).
On the basis of these results, M1 was identified 3ꢀmethylꢀ4ꢀnitrophenyl dihydrogen phosphate.
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With regard to M2, it gave a molecular ion value of m/z 262 [M ꢀ H]ꢀ (100) in mass spectrum
(Figure 3C). A decrease in molecular weight of 14 in comparison with FNT which probably
corresponds to a ꢀCH2. The fragment ion at m/z 152 ([M ꢀ H]ꢀ) (23) corresponding to 3ꢀmethylꢀ4ꢀ
nitrophenol was observed (Figure 3C and 4). 6ꢀ7,10,28ꢀ29 These results indicated that one of two Oꢀ
methyl groups in FNT was hydroxylated (ꢀOCH3 → ꢀOH), thus yielding M2. It was tentatively
identified Oꢀmethyl Oꢀ(3ꢀmethylꢀ4ꢀnitrophenyl) hydrogen phosphorothioate.
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As for M4, its structural identification by NMR was infeasible due to the samll amout isolated.
The mass spectrum of M4 exhibited a molecular ion at m/z 372 (100), which was significantly
greater than that of FNT, indicating that a conjunction reaction may have occurred (Figure 3D).
MS2 showed that M4 gave fragment ion m/z at 168 ([M ꢀ H]ꢀ) (45), which probably corresponds
to hydroxylated 3ꢀmethylꢀ4ꢀnitrophenol. On the basis of these results and literature data,29 M4
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was tentatively identified as glucuronideꢀ3ꢀ(hydroxymethyl)ꢀ4ꢀnitrophenol (Fig. 4). The
molecular ion at m/z 372 was speculated as [M + HCN]ꢀ.30ꢀ31
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As M3 (FNTO) was found to be an initial precursor of other metabolites, FNTO was
synthesized and its metabolism by C. elegans was further studied to reveal the metabolic
relationship among those metabolites.
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Metabolism of FNTO by C. elegans. HPLC analysis of the culture extracts showed FNTO was
rapidly degraded to two metabolites O1 and O2 within 3 d after treatment by C. elegans (Figure
6), whereas no significant degradation was observed in negative control (data not shown). As a
small amount of FNT (approximately 4%) was contained in synthesized FNTO, a small increase
in FNTO was observed at 1 d after treatment, indicating that FNT was transformed to FNTO
within 1 d (Figure 2C). This is consistent with the time of appearance of M3 in FNT treated
culture (Figure 2B). At 3 d after treatment, O1 and O2 were detected at 17.1 and 19.0 minutes by
HPLC, respectively, and gradually increased afterward (Figure 2D and 6). At 7 d after treatment,
approximately 50% FNTO was degraded. Approximately 22% incomplete degraded FNTO
persisted in the culture until the end of the experiment and the proportions for O1 and O2 were
53.1% and 27.4%, respectively. The kinetic model determined for degradation of FNTO is Ct=
2491.5 × eꢀ0.116t with R2= 0.994. The T1/2 of FNTO is 5.97 d which is longer than that of FNT
(1.88 d), which may be due to its high toxicity compared with FNT.2
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O1 and O2 were detected in a positive and negative ion mode, respectively (Figure 6). O1
showed its molecular ion at m/z 278 ([M + H]+) (34), which was 17 Da higher than that of
FNTO, indicating that a methyl group in FNT may be alcoholized to produce O1 (ꢀCH3 → ꢀ
CH2OH). Moreover, O1 also gave a fragment ion m/z at 260 ([M ꢀ H]+) (100). On the basis of
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these results, O1 was tentatively identified O,Oꢀdimethyl Oꢀ[3ꢀ(Hydroxymethyl)ꢀ4ꢀnitrophenyl]
phosphate (Figure 7).
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O2 displayed a molecular ion at m/z 232 ([M ꢀ H]ꢀ) (100) and fragment ion at m/z 152 ([M ꢀ
H]ꢀ) (45), which coincided with M1 (Figure 4 and 6). HPLC analysis showed retention time of
O2 was 19.0 minutes, which was comparable to that of 17.7 minutes for M1. These results
demonstrated that O2 is identical to M1.
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In synthesized FNTO treated cultures, O2 was detected at 3 d after treatment, while M1 was
detected at 4 d after treatment in FNT treated cultures. Considering that unsynthesized FNT in
FNTO treated cultures were transformed to FNTO at 1 d after treatment, it could be concluded
that M1 was directly derived from M3 in FNT treated cultures (Fig. 2B and 2D). At 15 d after
treatment, the residue of FNTO in synthesized FNTO treated cultures was 462 mAU*min (peak
area), which was slightly lower than that of 567 mAU*min (peak area) for M3 in FNT treated
cultures. There results demonstrated that FNT was continuously transformed to FNTO (M3)
within 15 d, and FNTO (M3) was further transformed to O2 (M1). According to comparative
analysis of the metabolites derived from those two pesticides, no trace amount of O1 was
detected in FNT treated cultures. We speculated that the presence and accumulation of M2 in
culture may inhibit the formation of O1, and this may also explain why there is no available data
published on the metabolite O1. Additionally, M2 was found at the initial step in FNT treated
cultures, in contrast, FNTO treated cultures had no trace amount of M2 detected due to the
presence of low amount (4%) of FNT. If M2 did form, however, it did so in amounts too small to
be detected under our analytical conditions.
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On the basis of these results, the metabolic pathway was proposed (Figure 7). Two major
degradation pathways are proposed: the first pathway occurs with the substitution of an S atom
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by an O atom, and the second pathway involves the hydroxylation of the organophosphate band.
The reaction of oxidation to form the oxon derivatives is quite common in phosphorothioate
pesticides such as parathion,32 cyanox,33 and bromophos.34 FNTO (M3) has been the most
common metabolite found in plants, animals, and microorganisms during the degradation of
FNT.2,9,29 In the presence of C. elegans, FNT was first biotransformed to FNTO (M3) by
oxidation of the P=S moiety, accumulated and became the initial precursor of other metabolites,
whereas M2 was formed from the hydrolysis of FNT (Oꢀmethyl group) (Figure 7). M2 has been
found in soil, microbial metabolism, and photoreaction in previous studies.9,29 Due to the
presence of thiomethyl, the rearrangement of the methoxy (thionoꢀthiolo) could result in
formation of an isomer of M2.29,35 Furthermore, our findings demonstrated that M1 (O2) and O1
were directly formed via M3 (FNTO) by the hydroxylation reaction. This provided
supplementary evidence to further elucidate the detailed degradation process of FNT by C.
elegans (Figure 7). To the best of our knowledge, M1 is rarely reported, however, it is the most
abundant metabolite of FNT by C. elegans in the present study. O1 resulted from the oxidation
of methyl group to hydroxymethyl. Formation of hydroxymethyl via oxidation of methyl by C.
elegans has been reported by several authors.23,36 The identified O1 in this study has not been
reported in any other studies to date. Moreover, although 3ꢀmethylꢀ4ꢀnitrophenol (MNP) was
reported as a major metabolite of FNT in previous studies,6ꢀ7, 9ꢀ10,28,37 it was not detected in the
present study. Instead, the corresponding sugar conjugate M4 was detected. Conjugation of
phenol with sugar is a common process during metabolic degradation steps.38 Several glucosides
have been reported for several pesticides such as clomazone,39 oxamy,40 and diphenamide.41 In
the present study, according to the identification and formation process of M4, it can be
concluded that 3ꢀhydroxymethylꢀ4ꢀnitrophenol which conjugates glucuronic acid was derived
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from 3ꢀmethylꢀ4ꢀnitrophenol (MNP) (Figure 3D, 4 and 7). In terms of structure, MNP may be
derived from all metabolites by hydroxylation reaction in a certain period of time (Figure 7, as
dash lines indicated).
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The metabolism of FNT in C. elegans was different from those of soil and plants. For
example, in soil, fenitrothion was degraded primarily by reduction of the nitro group to aminoꢀ
fenitrothion with lower redox, which was further degraded to demethyl amino.42 Malhat et al.28
reported soil extracts of FNT showed MNP was the major degrade of FNT in soil. Regarding the
metabolic pathways of FNT in and on plants, although oxidative desulfuration and conjugation
were involved as those confirmed in the current study, the oxidation of methyl group and
demethylation of the Oꢀmethyl group were not found.29 The primary degradation of FNT was
MNP with a trace amount of demethyl fenitrooxon, 3ꢀmethylꢀ4ꢀnitroanisole, and 1,2ꢀdihydroxyꢀ
4ꢀmethylꢀ5ꢀnitroꢀbenzene.43 In contrast, 3ꢀmethylꢀ4ꢀnitrophenyl dihydrogen was the most
abundant metabolite of FNT by C. elegans. In FNT treated cultures of Burkholderia sp., the
oxidative nitrite groupꢀremoving pathway was found, resulting in the formation of
methylhydroquinone,44 however, this pathway was not found in C. elegans in the current study.
In addition, an azo derivative was detected during MNP biodegradation by Aspergillus niger,6
however, this has not been reported in C. elegans. Other studies have shown that C. elegans can
metabolize a wide variety of xenobiotics as those reported in human and animals studies through
Nꢀoxidation and Nꢀdeethylation,17,20 demethylation,25 hydroxylation.22ꢀ23,36 Cytochrome P450
was considered to be responsible for those metabolisms (phase I metabolism).21,45 The two major
degradation pathways of FNT by C. elegans in the current study were also found in other
animals such as rats,46 female goats,47 and birds.48 Furthermore, aromatic hydroxylation during
the metabolism of FNT in birds,48 which has not reported in plant and soil, was also detected in
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C. elegans. Recently, flavinꢀdependent monooxygenase was also found to be involved in certain
parts of metabolism in C. elegans.22 In addition to phase I metabolism, C. elegans was also
involved in almost all of phase II metabolisms, such as sulfation, glucosylation, and glucuronide
formation.26,49ꢀ50 The ability of C. elegans to mimic mammalian metabolism and to perform
novel biotransformations would help us reveal the metabolic fates of organic compounds
occurring in mammalian liver cells instead of using live organisms. This microbial system also
provides an alternative method for the production of metabolites in large quantities.
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Biodegradation of other OPs by C. elegans. C. elegans was also capable of biodegradation of
other common used OPs including insecticide, herbicide, and fungicide (Table S2). At 4 d after
treatment, the percent degradation for those pesticides ranged between 23.1ꢀ94.5%, with 21
pesticides exhibiting degradation greater than 50%. At 7 d after treatment, the percent
degradation was greater than 70%, with 27 pesticides exhibiting the degradation greater than
80%. Five pesticides including azinphosꢀmethyl, phosmet, pirimiphosꢀmethyl, pirimiphosꢀethyl,
and pyrazophos were not detectable due to the response value of these pesticides lower than
LODs. C. elegans exhibited a wider degradation spectrum and stronger degradation ability to
OPs compared with several bacterial degradations reported in previous studies.11,13,51ꢀ52 The
results indicated the potential of this fungus to be used in OPs pollution remediation.
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In conclusion, this study reported a detailed metabolism of FNT by C. elegans by phase I
(oxidation and hydrolysis) and II (conjugation) metabolisms through several different pathways,
including those common in soil, plants, and animals. In addition to FNT, C. elegans was also
capable of biodegradation of 29 common used OPs. The findings of this study will provide
detailed insights regarding the fungal metabolism of FNT and complement the fungal
degradation of OPs. Its potential for bioremediation of OPs residual contamination may be useful
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for future remediation efforts. Additionally, this microbial system would serve as a model of
mammalian metabolism to elucidate the metabolic fates of organic compounds, evaluate their
toxicity and potential adverse effects. Burkholderia sp. strain NF100 has been found to carry a
plasmid encoding a FNTꢀhydrolyzing enzyme in FNT metabolism.45 Whether the metabolism of
FNT by C. elegans in the present study also involves plasmid encoding relevant enzymes needs
to be investigated in future studies.
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■ AUTHOR INFORMATION
Corresponding Author
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*Phone: +1ꢀ860ꢀ486ꢀ2924; Fax: +1ꢀ860ꢀ486ꢀ0682; Email: chuanjiezhang@snu.ac.kr
Acknowledgements
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The authors are thankful to Jonathan D. Mahoney for revision of the manuscript. This study was
carried out with the support of "Research Program for Agricultural Science & Technology
Development (Project No. PJ012094)", National Institute of Agricultural Sciences, Rural
Development Administration, Republic of Korea.
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Notes
The authors declare no competing financial interest.
YongꢀZhe Zhu and Min Fu contributed equally to this work.
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Figure captions
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Fig. 1. HPLC profiles of FNT and its metabolites produced by C. elegans. (A) blank control; (B)
negative control; (C) at day 0; (D) at 7 days.
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Fig. 2. Degradation and accumulation patterns of FNT and its metabolites (A, B) and FNTO (C,
D).
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Fig. 3. Mass spectra of four metabolites M3 (A), M1 (B), M2 (C), and M4+HCN (D) of FNT by
C. elegans. M3 was detected under ESI (+) mode and other three metabolites were detected
under ESI (ꢀ) mode, respectively.
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Fig. 4. MS2 fragmentation scheme of FNT and its metabolites (M1ꢀM4).
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Fig. 5. HPLC/MS information comparison between metabolite M3 (A) and synthesized FNTO
(B).
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Fig. 6. HPLC elution profiling of two metabolites (O1 and O2) of FNTO by C. elegans at 7 days
after treatment and their mass spectra. O1 and O2 were detected under ESI (+) and ESI (ꢀ) mode,
respectively.
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Fig. 7. Proposed metabolic pathway of fenitrothion by C. elegans. Abbreviations: s, soil; ph,
photolysis; and mi, microorganism.
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