Capsaicin is efficiently transformed by multiple cytochrome P450s from Capsicum fruit-feeding Helicoverpa armigera

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Capsaicin is efficiently transformed by multiple cytochrome P450s from Capsicum fruit-feeding Helicoverpa armigera Kai Tiana,b, Jiang Zhua,b, Mei Lia, Xinghui Qiua, a b

⁎

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Capsaicin Metabolism Cytochrome P450 CYP6B CYP9A Helicoverpa armigera

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the most abundant capsaicinoids found in hot peppers (Capsicum annum and Capsicum frutescens). It has been well documented that capsaicin plays an important role in the defense against the attack of herbivores or pathogens on Capsicum plants. A few insect herbivores such as Helicoverpa armigera and Helicoverpa assulta have been recorded to be capable of feeding on hot pepper fruits, suggesting that these insects evolve mechanisms against the toxicity of capsaicin. Although cytochrome P450meidated detoxification is considered to be an important mechanism by which cotton bollworms cope with capsaicin, experimental evidence is lacking. In this study, we compared the capacity of four H. armigera P450s (CYP6B6, CYP9A12, CYP9A14 and CYP9A17) in capsaicin metabolism, and the capsaicin metabolites were screened and tentatively identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). HPLC analyses showed that depletion rates of capsaicin were 21.9 ± 0.1, 11.9 ± 1.5, 16.3 ± 1.4 and 14.8 ± 0.2 min−1 for CYP6B6, CYP9A12, CYP9A14 and CYP9A17 respectively. The transformation of capsaicin was inhibited by the P450 inhibitor piperonyl butoxide. A total of seven products were detected, and hydroxylation (aromatic and aliphatic) and dehydrogenation were found to be two main pathways in capsaicin metabolism. In addition, capsaicin metabolism was enzyme selective: M1 (ω-hydroxylated N-macrocyclic metabolite) and M3 (ω-hydroxylated metabolite) were uniquely detected in the CYP6B6 catalytic reaction, while M4 (ω-n hydroxylated capsaicin), M5 (diene of capsaicin) and M6 (doubly oxidized metabolite of dehydrogenated capsaicin) were only detectable in CYP9A metabolisms. A capsaicin dimer (5, 5′-dicapsaicin) was found to be the major metabolite of CYP9A reactions, but the minor product produced by CYP6B6. An overall more similar behavior in capsaicin metabolism was observed among CYP9As than between CYP6B6 and CYP9As. Our data demonstrate that CYP6B6 and CYP9As have a potent capability to transform capsaicin, and individual P450 produce unique metabolite profile. These findings help us to understand the molecular basis of capsaicin adaptation in H. armigera.

1. Introduction Capsaicinoids are the principal pungent substances found in “hot” peppers (Capsicum annum and Capsicum frutescens). There are numerous naturally occurring capsaicinoid analogs, with capsaicin (8-methyl-Nvanillyl-6-nonenamide) being the most abundant. The absolute and relative content of capsaicin in hot peppers is subject to change, depending on cultivars, and environmental and developmental conditions (Tewksbury et al., 2006). Capsaicin has been well documented to play an important role in the defense against the attack of herbivores or pathogens on Capsicum plants (Ahn et al., 2011a; Tewksbury et al., 2008) Studies on the interactions between capsaicin and insects have ⁎

revealed the toxic effects of capsaicin on some insects. For example, capsaicin is able to inhibit the feeding of a ladybird beetle Henosepilachna vigintioctomaculata (Hori et al., 2011), and to retard larval growth of the spiny bollworm Earias insulana (Weissenberg et al., 1986). In addition, capsicum extracts were reported to have larvicidal activity to Anopheles stephensi and Culex quinquefasciatus (Madhumathy et al., 2007). So far, a few insect herbivores that are capable of feeding on hot pepper fruits have been recorded (Ahn et al., 2011b). The polyphagous cotton bollworm Helicoverpa armigera can tolerate a certain dose of capsaicin in its diet (Ahn et al., 2011a; Jia et al., 2012; Liu et al., 2004). Detoxification is considered to be an important mechanism by which cotton bollworms cope with capsaicin. For example, UDP-

Corresponding author. E-mail address: [email protected] (X. Qiu).

https://doi.org/10.1016/j.pestbp.2019.02.015 Received 16 September 2018; Received in revised form 18 February 2019; Accepted 24 February 2019 0048-3575/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Kai Tian, et al., Pesticide Biochemistry and Physiology, https://doi.org/10.1016/j.pestbp.2019.02.015

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3000 nmol of cytochrome c per min (Guengerich et al., 2009).

glycosyltransferases catalyze the conjugation of capsaicin with UDPglucose to form capsaicin β-glucoside, a metabolite found in the larval feces of three Helicoverpa species (Ahn et al., 2011b; Heidel-Fischer and Vogel, 2015). Cytochrome P450s (CYPs or P450s for short) comprise a superfamily of heme-thiolate proteins ubiquitously in organisms. CYPs have been acknowledged to metabolize numerous endogenous and exogenous compounds including plant allelochemicals (Feyereisen, 2012; Schuler, 2011). It has been documented that capsaicin can be metabolized by several mammal CYPs (Reilly et al., 2003; Reilly et al., 2013). In addition to metabolites arising from aromatic and alkyl hydroxylation, an unusual macrocyclic metabolite, a dehydrogenated alkyl diene, and a dehydrogenated imide metabolite were found to be generated by P450s (Reilly et al., 2003). Metabolites with O-demethylation and N-dehydrogenation were also observed (Reilly et al., 2003), and a dimer of capsaicin was identified (Reilly et al., 2013). However, until today there has been no report regarding capsaicin metabolism by insect P450s. There are about 114 P450s in the H. armigera genome (Pearce et al., 2017). The large number of P450s coexisted in a species challenge the functional characterization of individual P450. So far, which P450 is possibly involved in capsaicin metabolism in H. armigera remains unknown. The success in recombinant expression of four P450s belonging to CYP6 and CYP9 families from H. armigera (Tian et al., 2017; Liu et al., 2018) provides the possibility to ask whether these P450(s) contribute to capsaicin metabolism. The purpose of this study was to investigate the capacity of H. armigera P450s in metabolizing capsaicin using four E. coli produced CYP6B6, CYP9A12, CYP9A14 and CYP9A17. We also attempted to identify the major metabolites generated by these P450s in hope of providing insights into the likely metabolic fate of capsaicin in this Capsicum fruit-feeding insect.

2.2. Metabolism assays of capsaicin 10 mM capsaicin stock solution (in HPLC grade DMSO) was used for metabolism assays. The reaction mixture, in a final volume of 200 μL, consisted of 0.2 M Tris-HCl buffer at pH 7.4, 0.25 mM MgCl2, the NADPH-generation system (included 1 mM NADPH, 1 unit/ml G-6PDH, and 1 mM G-6-P), 150 nM P450 (in membrane fraction) and 100 μM substrates. Controls were set up in parallel by omitting the NADPH-generation system (NADPH-minus). Piperonyl butoxide (PBO) was used in the inhibition assays at a final concentration of 250 μM. The P450-containing membrane fractions were mixed with the NADPH-regeneration system for one minute (pre-incubation). Substrate in DMSO was used to initiate the reaction. After incubation at 30 °C, 250 rpm for 30 min, the reactions were quenched by the addition of 200 μL ice-cold acetonitrile, followed by vortexing for five minutes and shaking for 10 min. Then the samples were centrifuged at 12,000 g for 5 min, and filtrated through PTFE filters into vials for HPLC and LC-Q-TOF MS analysis. 2.3. High-performance liquid chromatography (HPLC) analysis HPLC analysis was performed on an Agilent 1260 series instrument with a diode array detector (DAD). 30 μL samples were separated on an Acclaim™ 120 C18 reverse column (150 mm*4.6 mm, 3 μm 120 Å, Thermo scientific) by using water (A) and acetonitrile (B) in a gradient program (0–1 min, 5% B; 1–41 min,100% B; 41–42 min, 100% B; 42–45 min, 5% B; 55–50 min, 5% B). Capsaicin and its metabolites were detected at 230 nm with column temperature at 28 °C. 2.4. Mass spectrometry of metabolites

2. Material and methods The Agilent 1200-6520 LC-Q-TOF MS spectrum running in positive ionization mode in the range of m/z 100–1000 was applied for metabolites structural analysis. Samples (5 μL) were separated by a ZORBAX Eclipse Plus C18 (4.6*150 mm 5 μm, Agilent, USA) column at a flow rate of 0.5 mL/min by using water (A) and acetonitrile (B) in a gradient program (0–1 min, 5% B; 1–40 min, 95% B; 40–51 min, 95% B; 51–53 min, 5% B; 53–57 min, 5% B). The instrument was running with a capillary voltage at 3500 V, drying-gas flow rate at 10 L per minute, drying-gas temperature at 350 °C, and nebulizer pressure at 60 psi. For MS/MS analysis, the voltages of fragmentor and collision were set at 120 V and 15 V respectively. Because analytical standards are not available, quantitative analysis of the putatively identified metabolite was achieved using peak area generated from the extracted ion chromatogram (EIC) of each metabolite and calibrated by an internal standard (xanthotoxin, spiked at 20 μM). Difference in ionization efficiency was not considered.

Capsaicin (99.2%), xanthotoxin (99%), glucose-6-phosphate dehydrogenase and 5-aminolevulinic acid hydrochloride were purchased from Sigma-Aldrich Chemical Corp. (Shanghai, China). β-NADPH (βnicotinamide adenine dinucleotide phosphate), glucose-6-phosphate, IPTG (isopropyl β-D-thiogalactoside) were purchased from Aladdin Chemical Co., Ltd. (Shanghai, China). HPLC grade acetonitrile and HPLC grade DMSO were purchased from Anpel Chemical Co., Ltd. (Shanghai, China). 2.1. Heterogeneous co-expression and membrane preparation The coding cDNA of four H. armigera P450 genes (HaCYP6B6, HaCYP9A12, HaCYP9A14, and HaCYP9A17) was N-terminal modified using the 17α-strategy respectively (Barnes et al., 1991), and each 17αHaP450 was ligated into pCWori+ plasmid via NdeI and KpnI to create pCW-HaP450 plasmid (Liu et al., 2018). The construction of pACYCHaCPR was described in Tian et al. (2017). Each recombinant pCWHaCYP was co-transformed with pACYC-HaCPR into competent E. coli BL21 cells using a protocol described in our previous paper (Tian et al., 2017). The cells were grown in 100 mL modified terrific broth medium inoculated with 1 mL overnight cultures. When the optical density at 600 nm reached 0.7 to 1.0, the culture was cooled to 23 °C, and 0.5 mM 5-aminolevulinic acid and 1 mM IPTG were supplemented. After incubation for 40 h with orbital shaking at 180 rpm, the cells were harvested. Cell membranes were prepared and stored as described previously (Tian et al., 2017). Protein concentrations were determined by the method of Bradford (1976). P450 contents were measured by the method established by Omura and Sato (1964). The HaCPR was assayed by its NADPH–cytochrome c reduction activity according to the method described in Guengerich et al. (2009), and reported as nmol of NADPH–cytochrome P450 reductase using a conversion factor that 1 nmol of NADPH–cytochrome P450 reductase will reduce about

3. Result 3.1. Heterologous co-expression of CYP9A and HaCPR Under the conditions described in this study, approximate 0.28 to 0.70 nmol cytochrome P450s and 3.0 to 12 nmol HaCPR per mg protein were detected in the membrane fraction (Table 1). These preparations were used for metabolism experiments. 3.2. Depletion rates of capsaicin by four H. armigera P450s Capsaicin metabolism was monitored by both substrate disappearance and metabolite appearance. The metabolic rates for individual P450s were determined only by the depletion of capsaicin using HPLC peak area and a standard curve of capsaicin because analytical standards for the metabolites are not available. After incubation 2

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and 14.8 ± 0.2 min−1 for CYP6B6, CYP9A12, CYP9A14 and CYP9A17 respectively. HPLC analysis revealed the appearance of multiple new peaks in the NADPH-plus P450 reactions compared with their NADPH-minus controls. At least six metabolites (P1-P6) were identified in the NADPHplus incubations and the formation of these metabolites was inhibited by the addition of 250 μM PBO in the reactions (Fig. 2). In addition, the metabolites P1 and P3 were detected only in the CYP6B6 reaction, while P4 and P5 were significantly present in the CYP9A reactions. P2 and P6 were the shared metabolites for all the four tested CYPs (Fig. 3).

Table 1 The contents of cytochrome P450 and NADPH cytochrome P450 reductase in the membrane fraction of E. coli co-transformed with pCW-HaCYP and pACYCHaCPR plasmids.

CYP6B6 CYP9A12 CYP9A14 CYP9A17

P450 (nmol/mg protein)

HaCPR (nmol/mg protein)

0.7 ± 0.14 0.28 ± 0.05 0.35 ± 0.12 0.60 ± 0.06

11.82 ± 1.24 5.10 ± 1.21 2.96 ± 0.30 4.54 ± 0.32

3.3. Metabolite identification by LC-Q-TOF From the total ion chromatogram (TIC) of LC-Q-TOF analysis (Fig. 3), seven significant metabolite peaks (M1-M7) were detected: one at m/z 320 (M1), two at m/z 304 (M2 and M5), two at m/z 322 (M3 and M4), one at m/z 336 (M6), and one at m/z 609 (M7). The analytes showing a base peak m/z value of 304, 320, 322, 336 and 609 were consistent with dehydrogenation (m/z 304), dehydrogenation and hydroxylation (m/z 320), hydroxylation (m/z 322), double hydroxylation and dehydrogenation (m/z 336), and dimer of capsaicin (m/z 609). The relative amounts of the seven metabolites were P450 dependent. M1 and M3 were only formed by CYP6B6, while M2 and M7 were detected in all the CYP6B6 and CYP9A incubations. M4, M5 and M6 were commonly observed in the three CYP9As (Fig. 3). Based on the relative abundance on LC-Q-TOF, the major product was M2 for CYP6B6, M4 for CYP9A12, M7 for CYP9A14 and CYP9A17, respectively (Fig. 4). The sites of attack in capsaicin reactions were tentatively identified based on previous published findings (Reilly et al., 2003; Reilly et al., 2013). The most abundant fragment ion at m/z 137, corresponding to an unchanged vanillyl moiety, was observed for M1, M2, M3, M4 and M5. This result indicated that these metabolites were products formed

Fig. 1. Depletion of capsaicin by four Helicoverpa armigera P450s. Reactions are performed by incubating 100 μM capsaicin with 150 nM P450 at 30 °C for 30 min.

for 30 min, 98.3%, 53.6%, 73.4% and 66.4% of 20 nmol capsaicin were transformed by 30 pmol of CYP6B6, CYP9A12, CYP9A14 and CYP9A17, respectively. By comparison, CYP6B6 showed the highest activity to deplete capsaicin (Fig. 1). The average depletion rates of capsaicin (mean ± standard error) were 21.9 ± 0.1, 11.9 ± 1.5, 16.3 ± 1.4

Fig. 2. HPLC chromatogram of capsaicin metabolism reactions. In vitro metabolism studies (with NADPH, without NADPH, with both NADPH and 250 μM PBO) are performed by incubating 100 μM capsaicin with 150 nM P450 at 30 °C for 30 min. 3

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Fig. 3. The total ion chromatogram (TIC) of the metabolism reactions detected on LC-Q-TOF/MS.

c

p e a k a re a

1 .5 1 0 7 1 .2 1 0 7 6 .0 1 0 6

c

a

b

4 .0 1 0 6

a

a

b

2 .0 1 0 6 0 M1

respectively (Reilly et al., 2003). The observation that M2 was eluted at 20.3 min and M5 at 28.1 min made us propose that M2 was an Nmacrocyclic structure, while M5 was a linear structure with dehydrogenation of the alkyl chain at the terminal methyl position (diene of capsaicin). M3 (m/z 322.2005) and M4 (m/z 322.2003) were identified to be alkyl hydroxylated metabolites of capsaicin due to the presence of fragment ion m/z 137 (corresponding to an unmodified vanilloid ring), and product ions (m/z 169 and m/z 186) (corresponding to an increase of 16 amu from capsaicin's characteristic fragment ions m/z 153 and 170) (Fig. 5C1, C2, D1 and D2). The appearance of fragment ion m/z 292 was likely the result of the loss of terminal CH2O (Δm = 2.3 mDa) from m/z 322. These observations suggested that M3 was ω-hydroxylated metabolite of capsaicin. The m/z 266.1361 of M4 was more consistent with a fragment ion of hydroxylated capsaicin with a loss of –C4H7 (|Δm| = 2.0 mDa) than a structure of hydroxylated capsaicin with a loss of –C3H4O (|Δm| = 39.0 mDa), indicating that the hydroxylation position was more likely at some ω-n (ω-4, or − 5, or − 6) site, than at the penultimate (ω-1) carbon of the alkyl side chain (Fig. 5F1 and 5F2). Two peaks M6 (m/z 336.1797) and M7 (m/z 609.3888) were eluted later than capsaicin. The absence of m/z 137 suggested that capsaicin was modified at the vanilloid portion. The mass of M6 was 30 amu larger than that of capsaicin, suggesting that M6 was likely to be a doubly oxidized metabolite of dehydrogenated capsaicin. The detection of precursor ion with an m/z value of 609, coupled with the presence of fragment ions at m/z 180, 273, and 440 (Fig. 5G1 and G2), strongly indicated that M7 was 5,5′-dicapsaicin.

C YP9A12 C YP9A14 C YP9A17 CYP6B6

a a

M2

b

M3

M4

d

M5

M6

M7

Fig. 4. The abundance of metabolites produced by CYP6B6, CYP9A12, CYP9A14 and CYP9A17. Data are peak areas calibrated by the internal standard xanthotoxin. Difference in ionization efficiency is not considered due to the unavailability of analytical standards for the metabolites. Significant difference in peak area of each metabolite among P450s is marked by different letter after one-way ANOVA analysis and least significant difference (LSD) test.

by reaction at the alkyl chain. Based on the MS spectra (Fig. 5 and Table 2), we proposed that M1 was a ω-hydroxylated N-macrocyclic metabolite of capsaicin. Higher abundance of m/z 302 than m/z 320 indicated that M1 was easy to lose H2O when experiencing ionization (Fig. 5A1). The ion m/z 166 was corresponding to fragmentation of the dehydrated macrocyclic portion in the MS/MS spectrum (Fig. 5A2). The mass of M2 (304.1896) and M5 (304.1902) were 2.0168 and 2.0162 amu less than that of capsaicin (Figs. 5B1 and 5E1), indicating that they were dehydrogenated metabolites of capsaicin. The presence of the fragment ions m/z 137, 180, 168 and 151 suggested that dehydrogenation occurred at the alkyl portion of capsaicin (Figs. 5B2 and 5E2). The ions m/z 168 and 151 were resulted from the loss of the benzylic portion of M2 and loss of water from the macrocycle,

4. Discussion In this study, we characterized the metabolism of capsaicin by four P450s of H. armigera. HPLC analysis revealed an efficient depletion rate of capsaicin and the appearance of multiple metabolites (Fig. 2). The highest and the lowest transformation was observed in CYP6B6 and 4

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Fig. 5. MS and MS/MS spectra of the metabolites of capsaicin metabolism by individual Helicoverpa armigera P450s. Cap stands for capsaicin.

5

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Table 2 Summary of LC-MS/MS properties of the metabolites. Metabolite

m/z

Retention time (min)

Ions in MS/MS

M1 M2 M3 M4 M5 M6 M7

320 304 322 322 304 336 609

16.6 20.3 21.1 23.6 28.1 31.5 38.2

137.0589, 137.0591, 137.0592, 137.0600, 137.0592, 153.1259, 180.1399,

166.1214, 151.1103, 151.1053, 151.1123, 151.1031, 170.1527, 273.1118,

184.1311, 168.1376, 169.1227, 168.1397, 168.1375, 167.0340, 440.2423

196.1294 180.1342, 231.0835 186.1471, 198.1401, 292.1930 186.1502, 266.1361 231.0943 184.0600

Fig. 6. Proposed structures of the detected capsaicin metabolites. The P450s involved in the formation of corresponding metabolites are indicated.

P450 enzymes (Reilly et al., 2003; Reilly et al., 2013). These findings demonstrate that there exist some common metabolic pathways in capsaicin metabolism between insects and mammals. Comparative analysis of capsaicin metabolism reveals marked differences in the metabolite profiles between CYP6B6 and CYP9As (Fig. 2 and Fig. 3). For example, CYP6B6 mainly produced the N-macrocyclic metabolite (M2) (Fig. 4). Macro-cyclization is deduced to occur after dehydrogenation of the tertiary carbon, but the catalytic mechanism remains unclear (Reilly et al., 2003). Notably, M1 and M3 were uniquely detected in CYP6B6, while M4, M5, M6 were only detectable in CYP9A reactions (Fig. 3 and Fig. 4). 5, 5′-Dicapsaicin (M7) was a major metabolite in the CYP9A reactions, while it was a minor product produced by CYP6B6 (Fig. 3 and Fig. 4). Overall, a similar behavior in capsaicin metabolism was observed among CYP9As (Fig. 2 and Fig. 3). However, although the three CYP9As had similar metabolite types, the amount of M4 produced by CYP9A14 was much less than CYP9A12 and CYP9A17 (Fig. 4). These results indicate that capsaicin metabolism is enzyme selective. This observation is in keeping with other reports, for

CYP9A12 incubations respectively, whereas CYP9A14 and CYP9A17 had a similar depletion capacity (Fig. 1). The metabolism of capsaicin was inhibited by PBO (Fig. 2), suggesting that capsaicin is catalyzed by the specific P450. To our knowledge, this study represents the first report to illustrate that capsaicin can be catalyzed by insect P450s at the individual molecule level. P450 enzymes are well known for catalyzing the hydroxylation of substrate. Among the seven metabolites of capsaicin tentatively identified by LC-Q-TOF MS analysis in this study, three hydroxylated products (M1, M3, M4) were identified. Moreover, three dehydrogenated metabolites (M1, M2 and M5) were detected, suggesting that dehydrogenation is also a major mechanism in capsaicin metabolism in addition to hydroxylation. The capsaicin dimer (5, 5′-dicapsaicin, M7) was found to be produced by the four P450s (Fig. 3). However, no Odemethylated metabolite previously reported (Halme et al., 2016; Reilly et al., 2003) was detected in this study. Among these metabolites, M2, M3, and M5 were reported to be generated by animal lung and liver microsomes, and M7 by human liver microsomes and several human 6

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example, CYP2C9 preferentially catalyze alkyl dehydrogenation, whereas CYP2E1 catalyzes ω-hydroxylation (Reilly and Yost, 2005). The difference in capsaicin metabolism among individual P450s is theoretically defined by the structure of each P450. The structural basis of differential catalysis of capsaicin is worthy of further investigations. The ability of P450 enzymes to transform capsaicinoids has been recognized to be an important determinant of the pharmacology and toxicology of these compounds (Reilly and Yost, 2005). The P450-dependent metabolism of capsaicin has been documented to serve as a protective mechanism against cell death in lung and liver cells (Reilly et al., 2003). Our results showing that capsaicin can be transformed by multiple P450s strongly indicate that H. armigera is well armed to cope with capsaicin in its host plants. The distinct metabolite profiles observed for individual P450s (Fig. 6) suggest that each P450 plays a different role in capsaicin tolerance in this insect. However, further study is required to understand the in vivo metabolic fate of capsaicin, and to assess the contribution of these P450s to capsaicin adaptation for the feeding larvae. In conclusion, metabolism studies revealed that capsaicin could be efficiently transformed by the four recombinant H. armigera P450s. Seven metabolites of capsaicin, including hydroxylated and dehydrogenated metabolites and capsaicin dimer, were putatively identified (Fig. 6). Our data demonstrate that CYP6B6 and CYP9As have differential metabolite profile and depletion capacity in capsaicin metabolism. The biological and ecological significance of capsaicin biotransformation will need further studies.

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