Identification of quinone methide intermediate resulting from metabolic

Apr 2, 2019 - Many herbal medicines such as epimedium have been reported to cause adverse effects, and icaritin is the common aglycone of many ...
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Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Identification of Quinone Methide Intermediate Resulting from Metabolic Activation of Icaritin in Vitro and in Vivo Yan Chen,† Xiucai Guo,† Yufei Ma,† Xingjia Hu,† Ying Peng,*,† and Jiang Zheng*,†,‡ †

Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou 550004, P. R. China

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ABSTRACT: Many herbal medicines such as epimedium have been reported to cause adverse effects, and icaritin is the common aglycone of many glucosides in epimedium. Our present work aimed at the clarification of the metabolic activation of icaritin possibly responsible for the adverse effects of epimedium. A quinone methide metabolite (M1) was detected in icaritinfortified microsomal incubations. A glutathione (GSH) conjugate (M2) and N-acetyl-L-cysteine (NAC) conjugate (M3) derived from icaritin were observed in GSH/NAC-supplemented rat/human liver microsomal incubations. CYP3A family was the predominant enzyme catalyzing the bioactivation of icaritin. In conclusion, sufficient evidence indicates the metabolic activation of icaritin to quinone methide metabolite. “Herba Epimedium (Yin-Yang-Huo in Chinese)”, a traditional Chinese herbal medicine, is extensively used for the treatment of bone loss and cardiovascular diseases and is also employed to cure neurological- and sexual-function disorders.1 Prenyl flavonoids are main active ingredients occurring in folk medicine epimedium. Icaritin, the main component in epimedium, reportedly displayed strong antitumor activity2,3 and prevented osteoporosis diseases.4 With a growing consumption of herbal remedies including epimedium in clinical practice, more and more cases of clinically significant liver damage associated with traditional Chinese medicines have been reported such as Xianniu Jiangu particle, Zhuanggu Guanjie capsule, and Xianling Gubao capsule.5−8 Unfortunately, the mechanisms of epimedium-induced liver hepatotoxicity remain unknown. Toxicity of chemicals is closely related to their structures as well as that of their metabolic products. Some toxicities are considered to be caused by the destruction of biomacromolecules.9 Macromolecules, such as nucleic acids and proteins, with nucleophilic centers could be attacked by electrophilic species including parent compounds and their metabolites.10 Several representative toxic structures have been documented in structure−toxicity relationship studies.9,11 Several studies have illustrated that the toxicities of methyleugenol resulted from electrophilic intermediates after bioactivation, and the electrophilic species reacted with DNA bases and protein amino acid residues.12,13 Eugenol, generated by O-demethylation of methyleugenol, was oxidized to a quinone methide metabolite,14 © XXXX American Chemical Society

a highly reactive electrophilic species possibly responsible for cytotoxicity through reaction with nucleophiles of macromolecules.15,16 Icaritin is an allyl flavonol derivative with similar structure as eugenol (Figure S1). This led us to hypothesize that icaritin is subject to bioactivation to the corresponding quinone methide, which may respond to the negative effects of epimedium as reported. Given that MS fragmentation behaviors maintain consistency between metabolites and their parent compounds, the fragment ions of icaritin were determined prior to characterizing the structures of metabolites. Icaritin exhibited [M + H]+ ion of m/z 369 in positive mode. Fragment ions of icaritin were obtained as shown in Figure S1. The fragment ion of m/z 313 was considered to be the benzyl cation group generated by the cleavage of isopentene group. Metabolic activation study was started with icaritin-fortified rat/human liver microsomal incubations. Quinone methide metabolite M1 (ion transition m/z 367/311) was detected by HPLC−MS/MS (Figures 1B and S2). M1 was not detected in negative control group in which NADPH was excluded (Figure 1A), which suggested that the generation of the metabolite was P450-dependent. M1 (retention time 9.42 min) displayed its molecular ion at m/z 367, which was 2 Da less than that of icaritin, indicating that M1 was a metabolite resulting from loss of two hydrogens of icaritin. Received: December 25, 2018 Published: April 2, 2019 A

DOI: 10.1021/acs.chemrestox.8b00418 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Rapid Report

Figure 1. Characterization of oxidation metabolite M1. Extracted ion (m/z 367/311) chromatograms acquired from HPLC−MS/MS analysis of RLM incubations containing icaritin in the (A) absence or (B) presence of NADPH. (C) Fragment ion pattern of M1 formed in rat microsomal incubations. (D) Extracted ion (m/z 367/311) chromatogram acquired from HPLC/MS-MS analysis of synthetic M1. (E) Fragment ion pattern of synthetic M1.

Scheme 1. Proposed Biotransformation Pathways of Icaritin

Da), 3CO (84 Da) from ion at m/z 311, respectively. This provides further proof for the generation of dehydrogenation metabolite of icaritin designated as M1. Chemical synthesis was conducted to verify the structure of M1. Quinone methide 1 (yield: 76.3%, Scheme 1) was synthesized through oxidation of icaritin using 2,3-dichloro-

The fragment ion pattern of M1 determined by MRM-IDA-EPI scanning demonstrated characteristic fragment ions of m/z 311, 283, 255, and 227. The fragment at m/z 311, equivalent to m/z 313 produced from icaritin itself, was generated by the cleavage of the isopentene group of M1, and ions at m/z 283, 255, and 227 were formed by the elimination of 1CO (28 Da), 2CO (56 B

DOI: 10.1021/acs.chemrestox.8b00418 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

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Figure 2. Characterization of GSH conjugate M2. Extracted ion (m/z 674/367 M2) chromatograms acquired from HPLC−MS/MS analysis of incubations containing RLMs, icaritin, and GSH in the (A) absence or (B) presence of NADPH. (C) Fragment ion pattern of M2 acquired from rat microsomal incubations.

Figure 3. Characterization of NAC conjugate M3. Extracted ion (m/z 530/367 M3) chromatograms acquired from HPLC−MS/MS analysis of incubations containing RLMs, icaritin, and NAC in the (A) absence or (B) presence of NADPH. (C) Fragment ion pattern of M3 acquired from HPLC−MS/MS analysis of RLM incubations.

7.04 (2H, d, J = 8.6 Hz, H-3′, H-5′), 3.80 (3H, s, OCH3). Protons at 7.95, 6.08, and 5.14 ppm corresponded to the individual protons at H-6, H-1′′, and H-2′′. Proton resonances at 8.01, 7.04, 3.80, 1.54, 1.57 ppm were responsible for the protons at H-2′/6′, H-3′/5′, OCH3, H-4′′, and H-5′′. Except for proton resonance of H-1′′, all the proton resonances were similar to that of icaritin reported.17

5,6-dicyano-p-benzoquinone (DDQ) based on our published procedure.14 The retention time and fragment pattern of the synthetic product were identical to that of M1 generated in microsomal incubations (Figures 1D,E). The product was purified and characterized by NMR. 1H NMR spectrum data (DMSO-d6, 600 MHz): δ10.56 (1H, s, OH), 7.95 (1H, s, H-6), 6.08 (1H, s, H-1′′), 5.14 (1H, t, J = 6.7 Hz, H-2′′), 1.57 (3H, s, H-4′′), 1.54 (3H, s, H-5′′), 8.01 (2H, d, J = 8.6 Hz, H-2′, H-6′), C

DOI: 10.1021/acs.chemrestox.8b00418 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Rapid Report

molecule ion and product ions led us to propose that M3 was a NAC conjugate derived from M1. Compounds 2 and 3 were synthesized via reaction of compound 1 with GSH and NAC, respectively. In terms of the retention time, protonated molecular ions, and MS fragmentation patterns of the resultant products (Figures S3D, S3E, S4D, and S4E), all of these were similar to those of M2 and M3 detected in the microsomal incubation studies. Hence, compound 2 was assigned as the conjugate derived from GSH, which was attached at C-1′′ of icaritin. Because of the instability issue, we were unable to obtain sufficient amount of compound 2 for NMR. NMR analysis of compound 3 was carried out after purification. 1H NMR spectrum data (DMSO-d6, 600 MHz): δ8.09 (2H,t, J = 9.6 Hz, H-2′, H-6′), 7.09−7.00 (2H, m, H-3′, H5′), 6.02 (1H, s, H-6), 5.73 (1H, d, J = 4.0 Hz, H-2′′), 5.22−4.24 (1H, m, cysteine-α), 4.19 (1H, s, H-1′′), 3.84 (3H, s, OCH3), 3.06 (1H, d, J = 7.1 Hz, cysteine -β), 2.94 (1H, dd, J = 12.9, 5.3 Hz, cysteine -β), 1.85−1.79 (6H, m, H-5′′; acetyl group), 1.23 (3H, s, H-4′′). Three aromatic proton resonances at 8.09, 7.09, and 6.02 ppm were responsible for the protons at H-2′/6′, H-3′/ 5′, and H-6, respectively, and protons at 1.23 and 1.85 ppm corresponded to H-4′′ and H-5′′/acetonyl, respectively. The observed three proton resonances at 5.22, 3.06, and 2.94 ppm are inferred as α- and β-carbon protons of cysteine. The proton resonances at 4.19 and 5.73 ppm were responsible for the protons at H-1′′ and H-2′′. This allowed us to draw a conclusion that compound 3 was a NAC conjugate and the conjugation took place at C-1′′ of icaritin. To explore the metabolic activation of icaritin in vivo, urinary excretion of phase II metabolites was determined by MRM-EPI in rats after an intragastric administration of icaritin. As expected, the corresponding NAC-conjugate was found in urine, and no such metabolite was observed in the urine of animals without icaritin treatment (Figures S4). This may explain that icaritin was oxidized to quinone methide 1 (M1) that then reacted with GSH by Michael addition, and the resulting GSH conjugate (M2) was sequentially biotransformed to M3, which was excreted in urine (Scheme 1). The observed GSH and NAC conjugates in vivo and in vitro imply that the reactive metabolite might covalently bind to proteins or other biological molecules, which could initiate the reported liver injury. In addition, sulfation is a metabolic activation process leading to the formation of highly reactive electrophilc species that are both mutagenic and carcinogenic.18 Icaritin might be subjected to hydroxylation to form benzyl alcohol which might undergo sulfation to give 1′−OH icaritin sulfate that further reacted with nucleophilic species by SN1/SN2 reaction. For exploring the effects of sulfotransferases on the metabolic activation of icaritin, rat liver cytosol and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) were incorporated in microsomal incubation mixtures containing the hydroxylation metabolite formed in situ. Vehicle in place of PAPS was included in control incubations. No significant difference in the formation of M2 was observed between PAPS- and vehicle-fortified incubations (data not shown). This indicates that sulfation pathway may not participate in the metabolic activation of icaritin. Recombinant human P450 incubation experiments were performed to determine which P450 enzyme(s) was/were mainly responsible for the bioactivation of icaritin. Nine P450 enzymes (CYPs 1A2, 2B6, 2C9, 2A6, 2C19, 2D6, 2E1, 3A4, and 3A5) were individually incubated with icaritin in the presence of NADPH and GSH, and the resulting incubation mixtures were

Figure 4. Role of individual human recombinant P450 enzymes was investigated from incubations. The catalytic capabilities of the enzymes were evaluated by monitoring the formation of M2 after normalization according to the relative content of the corresponding P450 enzyme in human liver microsomes. Data represent the mean ± SD (n = 3).

GSH and NAC were employed as trapping agents to characterize the reactive metabolite that may be responsible for the reported liver injury of epimedium.5−8 Both GSH conjugate M2 and NAC conjugate M3 were observed in rat microsomal incubations of icaritin in the presence of GSH/NAC (Figures 2B and 3B). As expected, M2 and M3 were not detected in microsomal incubations where NADPH was excluded (Figures 2A and 3A). Moreover, the same GSH conjugate was detected in human liver microsomal incubations (Figure S3B). M2 (retention time = 7.88 min) was characterized by monitoring molecular/product ion pair m/z 674/367. MS/MS spectrum of M2 was acquired through EPI scanning, which displayed the indicative fragment ions associated with the loss of the GSH moiety from the conjugate (Figures 2C and S3C). Loss of glycine portion (75 Da) and γ-glutamyl portion (129 Da) from [M + H]+ of m/z 674 resulted in product ions at m/z 599 and 545, respectively, and ion m/z at 528 detected was postulated to be derived from the cleavage of C−N bond of cysteine residue. The primary fragment ion for M2 was m/z 401 that was possibly formed by the cleavage of the C−S bond on GSH moiety (α-carbon of the cysteine) with sulfur retained on the benzyl site, and the observed ions at m/z 367 and 313 were in agreement with that of the parent compound. On the basis of the mass spectrometric data, M2 was characterized as a GSH adduct with conjugation at benzylidenyl site produced by oxidation of icaritin on ring A. M3 (ion transition: m/z 530/367) with retention time of 8.24 min was detected in rat liver microsomal incubations containing icaritin and NAC (Figure 3B). MS fragment pattern was determined to further characterize the structure of the NAC conjugate (Figure 3C). Molecular ion of M3 ([M + H]+ at m/z 530) was 161 Da higher than that of icaritin, which suggested an addition of a molecule of NAC to icaritin. MS/MS spectrum of M3 revealed the characteristic fragment ion at m/z 401 by MRM-EPI scanning, which indicated the neutral loss of 129 Da resulting from the cleavage of the sulfur−carbon bond of NAC moiety. This suggested the presence of the benzylic thioether motif in the NAC conjugate. The product ion at m/z 339 was considered to result from the elimination of CO from icaritin. The product ion at m/z 367 possibly originated from the loss of intact NAC moiety, and ions at m/z 297 and 313 were the characteristic fragments of icaritin. The observed protonated D

DOI: 10.1021/acs.chemrestox.8b00418 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

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(5) Zhang, Y. L., Liao, X., Liu, F. M., Wang, G. Q., and Xie, Y. M. (2017) Systematic review on safety of xianling gubao capsule. Zhongguo Zhong Yao Za Zhi 42, 2845−2856. (6) Du, Q., Wang, Z., Yun, N., Huang, Y., Xu, Q., and Wang, B. H. (2017) Literature Analysis of 185 Cases of ADR Induced by Xianling Gubao Capsule. China Pharmacy 28, 3785−3787. (7) Wang, X., Xu, L., and Wang, M. (2007) Hepatotoxicity caused by commonly-used Chinese medicinal herbs and compound preparation. Journal of Capital Medical University 28, 220−224. (8) Chen, Y. F., and Cai, H. (1999) Investigation of liver damage associated with traditional Chinese medicines. Adverse Drug Reactions Journal 1, 27−32. (9) Chi, M. N., Peng, Y., and Zheng, J. (2016) Characterization of glutathione conjugates derived from reactive metabolites of bakuchiol. Chem.-Biol. Interact. 244, 178−186. (10) Rietjens, I. M., Cohen, S. M., Fukushima, S., Gooderham, N. J., Hecht, S., Marnett, L. J., Smith, G. I., Adams, T. B., Bastaki, M., Harman, C. G., and Taylor, S. V. (2014) Impact of structural and metabolic variations on the toxicity and carcinogenicity of hydroxy- and alkoxysubstituted allyl- and propenylbenzenes. Chem. Res. Toxicol. 27, 1092− 1103. (11) Baillie, T. A. (2006) Future of toxicology metabolic activation and drug design: challenges and opportunities in chemical toxicology. Chem. Res. Toxicol. 19, 889−893. (12) Burkey, J. L., Sauer, J. M., McQueen, C. A., and Sipes, I. G. (2000) Cytotoxicity and genotoxicity of methyleugenol and related congeners-a mechanism of activation for methyleugenol. Mutat. Res., Fundam. Mol. Mech. Mutagen. 453, 25−33. (13) Ellis, J. K., Carmichael, P. L., and Gooderham, N. J. (2006) DNA adduct levels in the liver of the F344 rat treated with the natural flavor methyl eugenol. Toxicology 226, 73−74. (14) Yao, H., Peng, Y., and Zheng, J. (2016) Identification of glutathione and related cysteine conjugates derived from reactive metabolites of methyleugenol in rats. Chem.-Biol. Interact. 253, 143− 152. (15) Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000) Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135−160. (16) Graham, D. G., Tiffany, S. M., Bell, W. R., and Gutknecht, W. F. (1978) Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro. Mol. Pharmacol. 14, 644−653. (17) Dell’Agli, M., Galli, G. V., Dal Cero, E., Belluti, F., Matera, R., Zironi, E., Pagliuca, G., and Bosisio, E. (2008) Potent inhibition of human phosphodiesterase-5 by icariin derivatives. J. Nat. Prod. 71, 1513−1517. (18) Gamage, N., Barnett, A., Hempel, N., Duggleby, R. G., Windmill, K. F., Martin, J. L., and McManus, M. E. (2006) Human sulfotransferases and their role in chemical metabolism. Toxicol. Sci. 90, 5−22.

analyzed by HPLC−MS/MS to determine the formation of M2. CYPs 3A5 and 3A4 were found to be the primary enzymes participating in the generation of M2, followed by CYPs 2D6, 2B6, and 1A2 (Figure 4). The results illustrated that the metabolic activation of icaritin was mediated by multiple P450 enzymes. In conclusion, icaritin was oxidized to a quinone methide intermediate by cytochrome P450s, and the intermediate was reactive to thiols. CYPs 3A5 and 3A4 exhibited the highest activity for the metabolic activation of icaritin. The findings enable us to better understand the biochemical mechanisms of liver injury induced by epimedium.

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* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00418.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-24-23986361. Fax: +86-24-23986510. *E-mail: [email protected]. Phone: +86-24-23986361. Fax: +86-24-23986510. ORCID

Jiang Zheng: 0000-0002-0340-0275 Author Contributions

J.Z. and Y.P. contributed equally to this work. Funding

This work was supported in part by the National Natural Science Foundation of China (Nos. 81430086, 81773813, and 81830104). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS MRM, multiple-reaction monitoring; RLMs, rat liver microsomes; GSH, glutathione; HLMs, human liver microsomes; NAC, N-Acetyl-L-cysteine; EPI, enhanced product ion scan mode; NADPH, β-nicotinamide adenine dinucleotide 2′phosphate reduced tetra sodium salt; DDQ, 2,3-dichloro-5,6dicyano-p-benzoquinone; ESI, electrospray ionization; IDA, information-dependent acquisition.

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REFERENCES

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DOI: 10.1021/acs.chemrestox.8b00418 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX