Identification and Formation Pattern of Metabolites of Cyazofamid by Soil Fungus Cunninghamella Elegans

Appl Biol Chem (2016) 59(1):9–14 Online ISSN 2468-0842 DOI 10.1007/s13765-015-0127-6 Print ISSN 2468-0834

ARTICLE

Identiﬁcation and formation pattern of metabolites of cyazofamid by soil fungus Cunninghamella elegans

Hyeri Lee1,2 . Eenhye Kim1 . Yongho Shin1 . Jong-Hwa Lee1 . Hor-Gil Hur3 . Jeong-Han Kim1

Received: 16 May 2015 / Accepted: 23 September 2015 / Published online: 2 October 2015 Ó The Korean Society for Applied Biological Chemistry 2015

Abstract This study was performed to investigate the Introduction formation of microbial metabolites from cyazofamid by the soil fungus Cunninghamella elegans. The incubation of Cyazofamid (4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimi- cyazofamid with C. elegans was conducted for 10 days. dazole-1-sulfonamide), a sulfonamide fungicide, has been Cyazofamid disappeared after 7 days of incubation, pro- used for the protection of several vegetables and fruits from ducing three metabolites. Metabolites identified by liq- various diseases, such as tomato late blight (Phytophthora uid chromatography–tandem mass spectrometry were infestans) and downy mildews (Pseudoperonospora 4-chloro-5-(4-(hydroxymethyl)phenyl)-imidazole-2-car- cubensis of cucumber), by inhibiting the Qi site (the ubi- bonitrile (CHCN), 4-(4-chloro-2-cyanoimidazole-5- quinone-reducing site) of the cytochrome bc1 in complex III yl)benzoic acid (CCBA) and 4-chloro-2-cyano-5-(4-(hy- (ubiquinol-cytochrome c reductase) of the mitochondrial droxymethyl)phenyl)N,N-dimethyl-1H-imidazole-1-sul- respiratory chain (Mitani et al. 2001; Tomlin 2009). This fonamide (CCHS). A new metabolite, CCHS, was further compound displays a relatively low toxicological profile in confirmed by 1H-13C HSQC (heteronuclear single-quantum mammals and in ecological effects (Tomlin 2009). correlation) using nuclear magnetic resonance. As a pos- In in vivo absorption, distribution, metabolism, and sible metabolic pathway, cyazofamid could be oxidized to excretion (ADME) studies of cyazofamid, the major route CCHS, degraded to CHCN and further oxidized to CCBA. of excretion for the low dose group (0.5 mg/kg) was urine, The metabolic system of C. elegans would be a powerful and in the case of the high-dose group (1000 mg/kg), the tool for predicting and identifying phase I metabolites that major route was feces, with 4.4–11.6 h of half-life in whole could be formed in mammalian systems. blood. The major metabolites in urine were 4-(4-chloro-2- cyanoimidazole-5-yl)benzoic acid (CCBA), 4-chloro-5-[b-

Keywords Cunninghamella elegans Á Cyazofamid Á (methylsulfinyl)-p-tolyl]imidazole-2-carbonitrile (CH3SO- Liquid chromatography–tandem mass spectrometry Á CCIM), and 4-chloro-5-[b-(methylsulfonyl)-p-tolyl]imida- Metabolite Á Nuclear magnetic resonance zole-2-carbonitrile (CH3SO2-CCIM), and that in bile was CCBA (Evaluation Report Cyazofamid 2004). It was & Jeong-Han Kim reported that in aerobic soil, cyazofamid degraded rapidly [email protected] (DT50 in soil: 3–5 days) into the major degradations, such as 4-chloro-5-p-tolylimidazole-2-carbonitrile (CCIM), 1 Department of Agricultural Biotechnology and Research 4-chloro-5-p-tolylimidazole-2-carboxamide (CCIM–AM), Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-741, Republic of Korea and 4-chloro-5-p-tolylimidazole-2-carboxylic acid (CTCA), which were covalently bound to organic matter 2 Environmental Measurement and Analysis Center, National Institute of Environmental Research, Incheon 404-708, (Evaluation Report Cyazofamid 2004; Pestcide Fact Sheet Republic of Korea Cyazofamid 2004). 3 Many pesticides including cyazofamid undergo meta- Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, bolism in microorganisms, soil, plants and mammals, and Gwangju 500-712, Republic of Korea 123 10 Appl Biol Chem (2016) 59(1):9–14 metabolism studies are very important for the understand- for 10 days. Control and blank incubations were made with ing of pesticide toxicity and safety (Choi et al. 2007; Lee sterilized culture medium or in the absence of cyazofamid, et al. 2014, 2012; Singh and Tandon 2015; Tandon and respectively. Each culture sample (10 mL) was extracted Singh 2015; Tseng et al. 2009). Metabolic reactions, which with ethyl acetate (20 mL 9 2) at 0, 1, 2, 3, 5, 7, and 10 days produce different classes of metabolites, consist of two after treatment. The extracts were combined and dried under major types of reactions: phase I and phase II reactions. pressure at 40 °C before being dissolved with 1 mL of Phase I reactions are primarily catalyzed by the cyto- acetonitrile. The extracts were then analyzed using an Agi- chrome P450 (CYP) group of enzymes to produce oxidized lent HPLC 1100 series (USA) equipped with a Kinetex C18 compounds, whereas phase II reactions produce conjugates column (2.1 mm i.d. 9 100 mm, 2.6 lm; PhenomenexÒ, with glucuronic acid, glucose, glutathione, and so on USA) at 40 °C. The mobile phase consisted of 0.1 % formic (Abass et al. 2014; Hodgson and Rose 2008). acid in water (A) and 0.1 % formic acid in acetonitrile (B). Soil fungus Cunninghamella species have the ability to The gradient condition used was as follows: 30 % B at 0 to biotransform a wide variety of xenobiotics, similar to those 2 min, 95 % B at 13–18 min, and 30 % B at 20–25 min. The in mammalian metabolism systems (Asha and Vidyavathi injection volume was 2 lL, and the UV detector wavelength 2009; Keum et al. 2009; Zhang et al. 1996). C. berthol- was 280 nm. letiae, C. elegans, and C. echinulata are common species (Asha and Vidyavathi 2009), and C. elegans is the most Metabolite identification useful fungus for mimicking mammalian metabolism of xenobiotics, including pesticides (Keum et al. 2009; The metabolites were identified using a Varian 500-MSn Pothuluri et al. 2000; Zhu et al. 2010). mass spectrometer (USA) equipped with an Agilent 1100 The present study was conducted to elucidate the HPLC and Luna C18 column (2.0 mm i.d. 9 150 mm, degradation pattern/pathway of cyazofamid and the for- 3.0 lm; PhenomenexÒ)at40 °C. The mobile phases and the mation of its metabolites during the incubation of cyazo- gradient condition were identical to the analytical HPLC famid with the soil fungus C. elegans. The metabolites condition. The sample was analyzed in ESI-positive mode were identified using liquid chromatography-tandem mass (needle voltage; 4000 V) from m/z 190 to m/z 350. The spectrometry (LC–MS/MS) and nuclear magnetic reso- drying gas temperature, drying gas pressure, and nebulizer nance (NMR). gas pressure were 350 °C, 30, and 40 psi, respectively. The Turbo Data Dependent Scanning (TurboDDSTM, Varian, USA) mode was used to obtain MS3 spectra of ion m/z 341. Materials and methods The proposed structures for the fragment ions of the ion m/z 341 were determined using Mass FrontierTM software Materials and microorganism (version 6.0, HighChem, Ltd., Slovakia). On HPLC, one metabolite peak (retention at 10.19 min) was fractionated Cyazofamid (98.4 %) was purchased from FlukaTM with a fraction collector FC 205 (Gilson, USA) after mul- (Buchs, Switzerland). All solvents (High performance liq- tiple injections of 25 lL. The separation column used was a uid chromatography (HPLC) grade) were obtained from CAPCELL PAK C18 UG120 column (4.6 mm Burdick and JacksonÒ (Korea), and sodium chloride was i.d. 9 250 mm, 5 lm; Shiseido, Japan). The mobile phase, purchased from Samchun Pure Chemical Co., Ltd. (Korea). gradient condition, and detector wavelength were identical Cunninghamella elegans ATCC36112 was provided by the with the analytical HPLC condition. 2D 1H-13C HSQC National Center for Toxicological Research of the U.S. (heteronuclear single-quantum correlation) NMR spectra FDA (USA). Potato dextrose agar (PDA) and broth (PDB) were recorded on a 400 MHz NMR spectrometer (Jeol were purchased from BD Korea, Ltd. (Korea). Fungal JNM-LA400, JEOL Ltd., Tokyo, Japan) in CDCl3 (99.8 %, cultures were typically maintained on PDA, whereas the Merck, Germany) at 292 K. Residual CHCl3 in CDCl3 was corresponding liquid culture was performed on PDB at used as a reference (d = 7.27). 27 °C and 200 rpm. To stabilize the fungal metabolic reaction system, the PDB seed culture was incubated for 2 days. Results and discussion

Incubation and analysis of extracts Degradation of cyazofamid and formation of metabolites The culture medium with mycelia (10 mL) was added in fresh PDB (250 mL), supplemented with cyazofamid (1 mg The HPLC analysis of the culture extracts indicated that in 250 lL acetonitrile) and cultured at 27 °C and 200 rpm cyazofamid rapidly degraded to three metabolites 1, 2, and 123 Appl Biol Chem (2016) 59(1):9–14 11

(A) 120 (A) 350000 H+ 300000 Cl 325.1 100 Cyazofamid N 250000 C N 80 200000 N 60 O S O 150000 H3C Counts N

mAU 326.95 40 100000 H3C CH3 20 50000 (B) 350000 0 200234.05 220 240 260 280 300 320 340 300000 H+ Cl 0 5 10 15 20 25 250000 m/z N C N Retention time (min) 200000 N H 150000 236.04 HO Counts C 100000 H2 (B) 120 50000 100 CM1 50000 200 220 240 260 280 300 320 340 80 CM3 (C) 248.06 + 40000 Cl H N 60 m/z CM2 30000 C N

mAU N 40 H Cyazofamid O

Counts 20000 C 20 250.02 OH 0 10000 0 5 10 15 20 25 80000 (D) 200216.1 220 240 260 280 300 320 340 + Retention time (min) 60000 m/zCl H N C N 40000 N 341.0 Fig. 1 Formation of metabolite CM1, CM2, and CM3 from cyazo- 218.1

Counts HO O S O C famid when it was incubated with C. elegans for 5 days at 27 °C. N 20000 H2 H C CH 343.0 (A) Control incubation, (B) 5 days of incubation 3 3 0 200 220 240 260 280 300 320 340 1000 m/z

Fig. 3 LC-MS/MS spectra and structures of cyazofamid (A) and 800 metabolite CM1 (B), CM2 (C), and CM3 (D)

600 Metabolite identiﬁcation

400 To identify the metabolites of cyazofamid, the incubation

Peak area (mUV) mixture of C. elegans was analyzed by LC–MS/MS 200 (Fig. 3). The two metabolites, CM1 and CM2, gave [M ? H]? at m/z 234 and 248, respectively. On comparing 0 with molecular weight, CM1 must be CHCN and CM2 012345678910 must be CCBA, which were observed and identified from Incubation time (days) in vivo (rats) metabolism studies of cyazofamid (Evalua- Cyazofamid CM1 CM2 CM3 Control of cyazofamid tion Report Cyazofamid 2004). CM3 gave [M ? H]? at m/ z 341 with [M ? H?2]? at m/z 343 (intensity of 341: Fig. 2 Degradation of cyazofamid and formation of metabolites (CM1, CM2, and CM3) from cyazofamid when cyazofamid (4 lg/L) 343 = 3:1). These results suggest that CM3 contains a were incubated with C. elegans for 10 days at 27 °C chlorine atom in the molecule because cyazofamid also shows [M ? H]? at m/z 325 and [M ? H?2]? at m/z 327 (intensity of 325: 327 = 3:1). An increase in the molecular 3 (CM1, CM2, and CM3) within 10 days, whereas no weight of 16 indicates the insertion of one oxygen atom appreciable degradation was observed in a sterilized con- into the cyazofamid molecule to produce CM3 by oxida- trol experiment (Fig. 1). Approximately 50 % of cyazo- tion of an N-methyl group or a tolyl group. In the Data famid degraded on the first day, and after 7 days of Dependent Scanning mode on the ion trap MS, the critical 2 ? incubation, all of the cyazofamid disappeared was MS fragment ion m/z 296 from [M ? H] m/z 341 and a 3 2 exhausted. CM1 was detected from the first day and MS fragment ion m/z 232 from the MS fragment ion m/z increased through the incubation period, whereas CM3 was 296 (Figs. 4, 5) indicated that the oxidation of the tolyl also observed from the first day but started to decrease after group (CH3 ? CH2OH) produced CM3. If CM3 is an N- 2 5 days. CM2 was formed by the third day and increased at hydroxy derivative, there must be an MS fragment ion m/z ? 2 a slow rate (Fig. 2). 280 from [M ? H] m/z 341 instead of the MS fragment

123 12 Appl Biol Chem (2016) 59(1):9–14

Fig. 4 Ion tree report for MS3 fragmentation of CM3 by Turbo data dependent scanning (TurboDDSTM) ion m/z 296. This result was supported by a study on the These results from LC–MS/MS and 1H-13C HSQC clearly microsomal metabolism with diuron, which demonstrated indicated CH3 of cyazofamid oxidized to CH2OH, yielding that the oxidation of the N-methyl group yielding the N- CM3. Slade and Casida (1970) also observed the oxidation hydroxy derivative [–N(CH3)CH2OH] was not observed of the tolyl group of landrin in a study on mice liver due to its instability (Suzuki and Casida 1981). microsome metabolism. Recently, the rapid oxidation of To confirm the structure of CM3, its peak on HPLC was the ring methyl group in the gemfibrozil to hydroxymethyl isolated via fractionation for further investigation with group by C. elegans was reported (Kang et al. 2009). The NMR. 1H-13C HSQC (Fig. 6) revealed that three protons identified metabolite in this study, CM3 (4-chloro-2-cyano- and 13C of C-7 in cyazofamid provided a 1H singlet at 5-(4-(hydroxymethyl)phenyl)N,N-dimethyl-1H-imidazole- 2.44 ppm and a 13C singlet at 21.53 ppm, respectively. 1-sulfonamide; CCHS), has not been reported in any other However, in CM3, the 1H and 13C peaks shifted to a 1H studies to date. singlet at 4.77 ppm and a 13C singlet at 64.54 ppm, Microorganisms, such as Cunninghamella, can be used respectively, suggesting that an electro-negative oxygen as reliable alternatives to in vitro models for drug meta- must be attached at C-7, as predicted by LC–MS/MS data. bolism studies (Asha and Vidyavathi 2009; Rydevik et al.

123 Appl Biol Chem (2016) 59(1):9–14 13

+ Cl Cl H Cl N N N Cl N Cl C N C N C N C N N N N N N C N HO O S O HO O S O H O O S O N C 2 C HO O S O N C N N C O S O H2 H C CH H2 H2 H C CH H N 3 3 H3C CH3 3 3 H C CH N 3 3 H C CH MW 340 m/ z 341 m/ z 341 m/ z 339 3 3 m/ z 311

Cl Cl Cl Cl N N N C N C N C N N N N N O O HO O S O HO O S O HO O S O HO S C NH C C C H H2 2 H3C CH3 H2 H2 m/ z 296 m/ z 244 m/ z 341 m/ z 296

Cl N Cl C N N N HO O O C S H2 m/ z 232 m/ z 216

Fig. 5 MS3 fragmentation scheme of CM3 (m/z 341)

Fig. 6 1H-13C HSQC spectra of cyazofamid (A) and CM3 (B)

2013), and C. elegans metabolism experiments have gen- In conclusion, for the possible metabolic pathway erated major mammalian metabolites from various drugs (Fig. 7), cyazofamid was first oxidized to CCHS in a C. and other metabolites with a high yield and low cost, for elegans metabolic system, and then, it was degraded to example, amoxapine (Moody et al. 2000), mirtazapine CHCN and further oxidized to CCBA, as indicated by their (Moody et al. 2002), flurbiprofen (Amadio et al. 2010), and structural relationship and the formation pattern of those gemfibrozil (Kang et al. 2009). From pesticides, such as three metabolites as well as from the report of Slade and methoxychlor (Keum et al. 2009), cyprodinil (Schocken Casida (1970), which observed further oxidation of lan- et al. 1997), vinclozolin (Pothuluri et al. 2000), and iso- drin-alcohol to landrin-acid. proturon (Hangler et al. 2007), phase I metabolites that CCIM from in vivo (rats) metabolism studies of cya- could be formed in mammalian systems were identified in zofamid (Evaluation Report Cyazofamid 2004) was not incubation with C. elegans. observed in present study, probably because it is a simple

123 14 Appl Biol Chem (2016) 59(1):9–14

Cl Cl Cl N N N Cl Cl N N C N C N C N N N C N C N N N H N O S O HO O S O HO H H H3C C C O O N H N C C H3C CH3 2 H3C CH3 H2 H OH Cyazofamid CCHS (CM3) CHCN (CM1) CCBA (CM2)

Fig. 7 Proposed metabolic pathway of cyazofamid by C. elegans hydrolysis product of cyazofamid in urine and bile. The C. Mitani S, Araki S, Takii Y, Ohshima T, Matsuo N, Miyoshi H (2001) elegans metabolic reaction provided the critical evidence to The biochemical mode of action of the novel selective fungicide cyazofamid: specific inhibition of mitochondrial complex III in elucidate the metabolites of the first stage of oxidation Phythium spinosum. Pestic Biochem Phys 71:107–115 reactions, CCHS, and two degradation metabolites. The Moody JD, Zhang DL, Heinze TM, Cerniglia CE (2000) Transfor- metabolic system of C. elegans will be a powerful tool for mation of amoxapine by Cunninghamella elegans. Appl Environ predicting and identifying phase I metabolites that could be Microb 66:3646–3649 Moody JD, Freeman JP, Fu PP, Cerniglia CE (2002) Biotransforma- formed in mammalian systems. tion of mirtazapine by Cunninghamella elegans. Drug Metab Dispos 30:1274–1279 Pestcide Fact Sheet Cyazofamid (2004). United States Environmental Protection Agency. http://www.epa.gov/pesticides/chem_search/ References reg_actions/registration/fs_PC-085651_01-Sep-04.pdf Pothuluri JV, Freeman JP, Heinze TM, Beger RD, Cerniglia CE (2000) Biotransformation of vinclozolin by the fungus Cunning- Abass K, Reponen P, Mattila S, Rautio A, Pelkonen O (2014) Human hamella elegans. J Agr Food Chem 48:6138–6148 variation and CYP enzyme contribution in benfuracarb metabo- Rydevik A, Thevis M, Krug O, Bondesson U, Hedeland M (2013) lism in human in vitro hepatic models. Toxicol Lett 224:300–309 The fungus Cunninghamella elegans can produce human and Amadio J, Gordon K, Murphy CD (2010) Biotransformation of equine metabolites of selective androgen receptor modulators flurbiprofen by Cunninghamella species. Appl Environ Micro- (SARMs). Xenobiotica 43:409–420 biol 76:6299–6303 Schocken MJ, Mao J, Schabacker DJ (1997) Microbial transforma- Asha S, Vidyavathi M (2009) Cunninghamella - A microbial model tions of the fungicide cyprodinil (CGA-219417). J Agric Food for drug metabolism studies: a review. Biotechnol Adv 27:16–29 Chem 45:3647–3651 Choi JH, El-Aty AMA, Park YS, Cho SK, Shim JH (2007) The Singh N, Tandon S (2015) Dissipation kinetics and leaching of assessment of carbendazim, cyazofamid, diethofencarb and cyazofamid fungicide in texturally different agricultural soils. Int pyrimethanil residue levels in P-ginseng (C.A. Meyer) by HPLC. J Environ Sci Technol 12:2475–2484 Bull Korean Chem Soc 28:369–372 Slade M, Casida JE (1970) Metabolic fate of 3,4,5- and 2,3,5- Evaluation Report Cyazofamid (2004) Food safety commission trimethylphenyl methylcarbamates, the major constituents in pesticides experts committee. https://www.fsc.go.jp/english/eva Landrin insecticide. J Agric Food Chem 18:467–474 luationreports/pesticide/cyazofamid_fullreport.pdf Suzuki T, Casida JE (1981) Metabolites of diuron, linuron, and Hangler M, Jensen B, Ronhede S, Sorensen SR (2007) Inducible methazole formed by liver microsomal-enzymes and spinach hydroxylation and demethylation of the herbicide isoproturon by plants. J Agric Food Chem 29:1027–1033 Cunninghamella elegans. FEMS Microbiol Lett 268:254–260 Tandon S, Singh N (2015) Dissipation kinetics of cyazofamid in Hodgson E, Rose RL (2008) Metabolic interactions of agrochemicals water. J Liq Chromatogr Relat Technol 38:993–996 in humans. Pest Manag Sci 64:617–621 Tomlin C (2009) The pesticide manual: a world compendium, 15th Kang SI, Kang SY, Kanaly RA, Lee E, Lim Y, Hur HG (2009) Rapid edn. British Crop Protection Council, Alton oxidation of ring methyl groups is the primary mechanism of Tseng SH et al (2009) Analysis of 81 pesticides and metabolite biotransformation of gemfibrozil by the fungus Cunninghamella residues in fruits and vegetables by diatomaceous earth column elegans. Arch Microbiol 191:509–517 extraction and LC/MS/MS determination. J Food Drug Anal Keum YS, Lee YH, Kim JH (2009) Metabolism of methoxychlor by 17:319–332 Cunninghamella elegans ATCC36112. J Agric Food Chem Zhang DL, Evans FE, Freeman JP, Yang YF, Deck J, Cerniglia CE 57:7931–7937 (1996) Formation of mammalian metabolites of cyclobenzaprine Lee H et al (2012) Establishment of analytical method for cyazofamid by the fungus, Cunninghamella elegans. Chem Biol Interact residue in apple, mandarin, Korean cabbage, green pepper, 102:79–92 potato and soybean. J Korean Soc Appl Biol Chem 55:241–247 Zhu YZ, Keum YS, Yang L, Lee H, Park H, Kim JH (2010) Lee H, Kim E, Lee JH, Sung JH, Choi H, Kim JH (2014) Analysis of Metabolism of a fungicide mepanipyrim by soil fungus Cun- cyazofamid and its metabolite in the environmental and crop ninghamella elegans ATCC36112. J Agric Food Chem samples using LC-MS/MS. Bull Environ Contam Toxicol 58:12379–12384 93:586–590

123