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

Abstract

This study was performed to investigate the formation of microbial metabolites from cyazofamid by the soil fungus Cunninghamella elegans. The incubation of cyazofamid with C. elegans was conducted for 10 days. Cyazofamid disappeared after 7 days of incubation, producing three metabolites. Metabolites identified by liquid chromatography–tandem mass spectrometry were 4-chloro-5-(4-(hydroxymethyl)phenyl)-imidazole-2-carbonitrile (CHCN), 4-(4-chloro-2-cyanoimidazole-5-yl)benzoic acid (CCBA) and 4-chloro-2-cyano-5-(4-(hydroxymethyl)phenyl)N,N-dimethyl-1H-imidazole-1-sulfonamide (CCHS). A new metabolite, CCHS, was further confirmed by ¹H-¹³C HSQC (heteronuclear single-quantum correlation) using nuclear magnetic resonance. As a possible metabolic pathway, cyazofamid could be oxidized to CCHS, degraded to CHCN and further oxidized to CCBA. The metabolic system of C. elegans would be a powerful tool for predicting and identifying phase I metabolites that could be formed in mammalian systems.

Introduction

Cyazofamid (4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonamide), a sulfonamide fungicide, has been used for the protection of several vegetables and fruits from various diseases, such as tomato late blight (Phytophthora infestans) and downy mildews (Pseudoperonospora cubensis of cucumber), by inhibiting the Qi site (the ubiquinone-reducing site) of the cytochrome bc1 in complex III (ubiquinol-cytochrome c reductase) of the mitochondrial respiratory chain (Mitani et al. 2001; Tomlin 2009). This compound displays a relatively low toxicological profile in mammals and in ecological effects (Tomlin 2009).

In in vivo absorption, distribution, metabolism, and excretion (ADME) studies of cyazofamid, the major route of excretion for the low dose group (0.5 mg/kg) was urine, and in the case of the high-dose group (1000 mg/kg), the major route was feces, with 4.4–11.6 h of half-life in whole blood. The major metabolites in urine were 4-(4-chloro-2-cyanoimidazole-5-yl)benzoic acid (CCBA), 4-chloro-5-[β-(methylsulfinyl)-p-tolyl]imidazole-2-carbonitrile (CH₃SO-CCIM), and 4-chloro-5-[β-(methylsulfonyl)-p-tolyl]imidazole-2-carbonitrile (CH₃SO₂-CCIM), and that in bile was CCBA (Evaluation Report Cyazofamid 2004). It was reported that in aerobic soil, cyazofamid degraded rapidly (DT₅₀ in soil: 3–5 days) into the major degradations, such as 4-chloro-5-p-tolylimidazole-2-carbonitrile (CCIM), 4-chloro-5-p-tolylimidazole-2-carboxamide (CCIM–AM), and 4-chloro-5-p-tolylimidazole-2-carboxylic acid (CTCA), which were covalently bound to organic matter (Evaluation Report Cyazofamid 2004; Pestcide Fact Sheet Cyazofamid 2004).

Many pesticides including cyazofamid undergo metabolism in microorganisms, soil, plants and mammals, and metabolism studies are very important for the understanding of pesticide toxicity and safety (Choi et al. 2007; Lee et al. 2014, 2012; Singh and Tandon 2015; Tandon and Singh 2015; Tseng et al. 2009). Metabolic reactions, which produce different classes of metabolites, consist of two major types of reactions: phase I and phase II reactions. Phase I reactions are primarily catalyzed by the cytochrome P450 (CYP) group of enzymes to produce oxidized compounds, whereas phase II reactions produce conjugates with glucuronic acid, glucose, glutathione, and so on (Abass et al. 2014; Hodgson and Rose 2008).

Soil fungus Cunninghamella species have the ability to biotransform a wide variety of xenobiotics, similar to those in mammalian metabolism systems (Asha and Vidyavathi 2009; Keum et al. 2009; Zhang et al. 1996). C. bertholletiae, C. elegans, and C. echinulata are common species (Asha and Vidyavathi 2009), and C. elegans is the most useful fungus for mimicking mammalian metabolism of xenobiotics, including pesticides (Keum et al. 2009; Pothuluri et al. 2000; Zhu et al. 2010).

The present study was conducted to elucidate the degradation pattern/pathway of cyazofamid and the formation of its metabolites during the incubation of cyazofamid with the soil fungus C. elegans. The metabolites were identified using liquid chromatography-tandem mass spectrometry (LC–MS/MS) and nuclear magnetic resonance (NMR).

Materials and methods

Materials and microorganism

Cyazofamid (98.4 %) was purchased from Fluka™ (Buchs, Switzerland). All solvents (High performance liquid chromatography (HPLC) grade) were obtained from Burdick and Jackson^® (Korea), and sodium chloride was purchased from Samchun Pure Chemical Co., Ltd. (Korea). Cunninghamella elegans ATCC36112 was provided by the National Center for Toxicological Research of the U.S. FDA (USA). Potato dextrose agar (PDA) and broth (PDB) were purchased from BD Korea, Ltd. (Korea). Fungal cultures were typically maintained on PDA, whereas the corresponding liquid culture was performed on PDB at 27 °C and 200 rpm. To stabilize the fungal metabolic reaction system, the PDB seed culture was incubated for 2 days.

Incubation and analysis of extracts

The culture medium with mycelia (10 mL) was added in fresh PDB (250 mL), supplemented with cyazofamid (1 mg in 250 μL acetonitrile) and cultured at 27 °C and 200 rpm for 10 days. Control and blank incubations were made with sterilized culture medium or in the absence of cyazofamid, respectively. Each culture sample (10 mL) was extracted with ethyl acetate (20 mL × 2) at 0, 1, 2, 3, 5, 7, and 10 days after treatment. The extracts were combined and dried under pressure at 40 °C before being dissolved with 1 mL of acetonitrile. The extracts were then analyzed using an Agilent HPLC 1100 series (USA) equipped with a Kinetex C18 column (2.1 mm i.d. × 100 mm, 2.6 μm; Phenomenex^®, USA) at 40 °C. The mobile phase consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). The gradient condition used was as follows: 30 % B at 0 to 2 min, 95 % B at 13–18 min, and 30 % B at 20–25 min. The injection volume was 2 μL, and the UV detector wavelength was 280 nm.

Metabolite identification

The metabolites were identified using a Varian 500-MSⁿ mass spectrometer (USA) equipped with an Agilent 1100 HPLC and Luna C18 column (2.0 mm i.d. × 150 mm, 3.0 μm; Phenomenex^®) at 40 °C. The mobile phases and the gradient condition were identical to the analytical HPLC condition. The sample was analyzed in ESI-positive mode (needle voltage; 4000 V) from m/z 190 to m/z 350. The drying gas temperature, drying gas pressure, and nebulizer gas pressure were 350 °C, 30, and 40 psi, respectively. The Turbo Data Dependent Scanning (TurboDDS™, Varian, USA) mode was used to obtain MS³ spectra of ion m/z 341. The proposed structures for the fragment ions of the ion m/z 341 were determined using Mass Frontier™ software (version 6.0, HighChem, Ltd., Slovakia). On HPLC, one metabolite peak (retention at 10.19 min) was fractionated with a fraction collector FC 205 (Gilson, USA) after multiple injections of 25 μL. The separation column used was a CAPCELL PAK C18 UG120 column (4.6 mm i.d. × 250 mm, 5 μm; Shiseido, Japan). The mobile phase, gradient condition, and detector wavelength were identical with the analytical HPLC condition. 2D ¹H-¹³C HSQC (heteronuclear single-quantum correlation) NMR spectra were recorded on a 400 MHz NMR spectrometer (Jeol JNM-LA400, JEOL Ltd., Tokyo, Japan) in CDCl₃ (99.8 %, Merck, Germany) at 292 K. Residual CHCl₃ in CDCl₃ was used as a reference (δ = 7.27).

Results and discussion

Degradation of cyazofamid and formation of metabolites

The HPLC analysis of the culture extracts indicated that cyazofamid rapidly degraded to three metabolites 1, 2, and 3 (CM1, CM2, and CM3) within 10 days, whereas no appreciable degradation was observed in a sterilized control experiment (Fig. 1). Approximately 50 % of cyazofamid degraded on the first day, and after 7 days of incubation, all of the cyazofamid disappeared was exhausted. CM1 was detected from the first day and increased through the incubation period, whereas CM3 was also observed from the first day but started to decrease after 5 days. CM2 was formed by the third day and increased at a slow rate (Fig. 2).

Fig. 1

Formation of metabolite CM1, CM2, and CM3 from cyazofamid when it was incubated with C. elegans for 5 days at 27 °C. (A) Control incubation, (B) 5 days of incubation

Fig. 2

Degradation of cyazofamid and formation of metabolites (CM1, CM2, and CM3) from cyazofamid when cyazofamid (4 μg/L) were incubated with C. elegans for 10 days at 27 °C

Metabolite identification

To identify the metabolites of cyazofamid, the incubation mixture of C. elegans was analyzed by LC–MS/MS (Fig. 3). The two metabolites, CM1 and CM2, gave [M + H]⁺ at m/z 234 and 248, respectively. On comparing with molecular weight, CM1 must be CHCN and CM2 must be CCBA, which were observed and identified from in vivo (rats) metabolism studies of cyazofamid (Evaluation Report Cyazofamid 2004). CM3 gave [M + H]⁺ at m/z 341 with [M + H+2]⁺ at m/z 343 (intensity of 341: 343 = 3:1). These results suggest that CM3 contains a 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 weight of 16 indicates the insertion of one oxygen atom into the cyazofamid molecule to produce CM3 by oxidation of an N-methyl group or a tolyl group. In the Data Dependent Scanning mode on the ion trap MS, the critical MS² fragment ion m/z 296 from [M + H]⁺ m/z 341 and a MS³ fragment ion m/z 232 from the MS² fragment ion m/z 296 (Figs. 4, 5) indicated that the oxidation of the tolyl group (CH₃ → CH₂OH) produced CM3. If CM3 is an N-hydroxy derivative, there must be an MS² fragment ion m/z 280 from [M + H]⁺ m/z 341 instead of the MS² fragment ion m/z 296. This result was supported by a study on the microsomal metabolism with diuron, which demonstrated that the oxidation of the N-methyl group yielding the N-hydroxy derivative [–N(CH₃)CH₂OH] was not observed due to its instability (Suzuki and Casida 1981).

Fig. 3

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

Fig. 4

Ion tree report for MS³ fragmentation of CM3 by Turbo data dependent scanning (TurboDDS™)

Fig. 5

MS³ fragmentation scheme of CM3 (m/z 341)

To confirm the structure of CM3, its peak on HPLC was isolated via fractionation for further investigation with NMR. ¹H-¹³C HSQC (Fig. 6) revealed that three protons and ¹³C of C-7 in cyazofamid provided a ¹H singlet at 2.44 ppm and a ¹³C singlet at 21.53 ppm, respectively. However, in CM3, the ¹H and ¹³C peaks shifted to a ¹H singlet at 4.77 ppm and a ¹³C singlet at 64.54 ppm, respectively, suggesting that an electro-negative oxygen must be attached at C-7, as predicted by LC–MS/MS data. These results from LC–MS/MS and ¹H-¹³C HSQC clearly indicated CH₃ of cyazofamid oxidized to CH₂OH, yielding CM3. Slade and Casida (1970) also observed the oxidation of the tolyl group of landrin in a study on mice liver microsome metabolism. Recently, the rapid oxidation of the ring methyl group in the gemfibrozil to hydroxymethyl group by C. elegans was reported (Kang et al. 2009). The identified metabolite in this study, CM3 (4-chloro-2-cyano-5-(4-(hydroxymethyl)phenyl)N,N-dimethyl-1H-imidazole-1-sulfonamide; CCHS), has not been reported in any other studies to date.

Fig. 6

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

Microorganisms, such as Cunninghamella, can be used as reliable alternatives to in vitro models for drug metabolism studies (Asha and Vidyavathi 2009; Rydevik et al. 2013), and C. elegans metabolism experiments have generated major mammalian metabolites from various drugs and other metabolites with a high yield and low cost, for example, amoxapine (Moody et al. 2000), mirtazapine (Moody et al. 2002), flurbiprofen (Amadio et al. 2010), and gemfibrozil (Kang et al. 2009). From pesticides, such as methoxychlor (Keum et al. 2009), cyprodinil (Schocken et al. 1997), vinclozolin (Pothuluri et al. 2000), and isoproturon (Hangler et al. 2007), phase I metabolites that could be formed in mammalian systems were identified in incubation with C. elegans.

In conclusion, for the possible metabolic pathway (Fig. 7), cyazofamid was first oxidized to CCHS in a C. elegans metabolic system, and then, it was degraded to CHCN and further oxidized to CCBA, as indicated by their structural relationship and the formation pattern of those three metabolites as well as from the report of Slade and Casida (1970), which observed further oxidation of landrin-alcohol to landrin-acid.

Fig. 7

Proposed metabolic pathway of cyazofamid by C. elegans

CCIM from in vivo (rats) metabolism studies of cyazofamid (Evaluation Report Cyazofamid 2004) was not observed in present study, probably because it is a simple hydrolysis product of cyazofamid in urine and bile. The C. elegans metabolic reaction provided the critical evidence to elucidate the metabolites of the first stage of oxidation reactions, CCHS, and two degradation metabolites. The metabolic system of C. elegans will be a powerful tool for predicting and identifying phase I metabolites that could be formed in mammalian systems.