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Electrophilicities and protein covalent binding of demethylation metabolites of colchicine Xiucai Guo, Dongju Lin, Weiwei Li, Kai Wang, Ying Peng, and Jiang Zheng Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00461 • Publication Date (Web): 04 Feb 2016 Downloaded from http://pubs.acs.org on February 16, 2016

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Electrophilicities and protein covalent binding of demethylation metabolites of colchicine †



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Xiucai Guo, Dongju Lin, Weiwei Li, Kai Wang, Ying Peng, * and Jiang Zheng * †

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School of Pharmacy, Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China ¶

Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101

Running title: Bioactivation of Colchicine

Corresponding Authors: Jiang Zheng, Ph. D Center for Developmental Therapeutics, Seattle Children's Research Institute, Seattle, Washington 98101 Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, Washington 98105 Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China Email: [email protected] Tel: 206-884-7651; Fax: 206-987-7660 Ying Peng, Ph. D. School of Pharmacy, Shenyang Pharmaceutical University, PO Box 21,103 Wenhua Road, Shenyang 110016, P. R. China Email: [email protected] Tel: +86-24-23986361; Fax: +86-24-23986510

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Abstract Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever.

The

administration of colchicine was found to cause liver injury in humans.

The

mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ.

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Introduction Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has widely been used in the treatment of gout and familial Mediterranean fever.1-3 It is also well known as an antimitotic agent by inhibiting microtubule polymerization through binding to tubulin.4, 5 Recently, the therapeutic applications of the drug have been extended to cover some other diseases, such as amyloidosis,6, 7 progressive systemic scleroderma,8 cirrhosis,9,

10

and Behcet's disease.11 Additionally, colchicine has a

suppressive effect on collagen synthesis and enhances collagenase activity.12,

13

Colchicine represses mitosis of cells in liver fibrosis and decreases functions of inflammation cells by its anti-inflammatory effect, and therefore it prevents fibrosis and lipid peroxidation.14, 15 Colchicine was reported to induce high incidence of liver injury in experimental animals,16 including fatty liver and necrosis as well as elevation of serum transaminase levels,17, 18 Clinically, the cases of haematological toxicity were found to be associated with colchicine exposure, and patients given colchicine were subjected to liver and renal failure,19,

20

The mechanisms of colchicine-induced

toxicities remain unknown. Colchicine has been shown to undergo extensive hepatic metabolism, and demethylation was the major route of metabolism.

Four

demethylation metabolites, including 1-, 2-, 3, and 10-demethylated colchicine, have been documented in microsomal incubations after exposure to colchicine.21, 22 P450 3A enzymes played a major role in this process.23 Biliary glucuronides and sulfates derived from 2- and 3-demethylated colchicine, along with a 1-demethylated -4-

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colchicine-derived glutathione conjugate, have been reported in rats administered with colchicine.24 The major objective of our study was to investigate the electrophilicities of 2-, 3-, and 10-demethylation colchicine metabolites and protein covalent binding derived from the demethylation metabolites.

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Materials and Methods Chemicals and Materials. Colchicine (purity>99%) and pronase E (purity>98%) were obtained from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). N-Acetyl cysteine (NAC), L-cysteine, and NADPH were purchased from Sigma-Aldrich Co. (St. Louis, MO). 2-demethylated colchicine (2-DMC) and 3demethylated colchicine (3-DMC) were purchased from Toronto Research Chemicals (Toronto, Canada). 10-demethylated colchicine (10-DMC) was acquired from TLC Pharmaceutical Standards Ltd. (Vaughan, Canada).

Human liver microsomes were

purchased from BD Gentest (Woburn, MA). All other chemicals and reagents were obtained from various sources and were of either analytical or high-performance LC grade.

Microsomal Incubations.

Rat liver microsomes were prepared according to the

description of our laboratory.25 Colchicine, 2-DMC, 3-DMC or 10-DMC (100 µM) was incubated with rat or human liver microsomes (1.0 mg protein/mL) supplemented with NAC or cysteine at a final concentration of 1.0 mM. The total incubation volume was 500 µL. The reaction was triggered by addition of NADPH (1.0 mM). The control group excluded NADPH. The reactions were terminated by adding and mixing with equal volume of ice-cold acetonitrile adequately after 60 min of the incubation at 37 °C. The resulting mixture was vortexed and centrifuged at 19,000 g for 10 min to remove precipitated protein. The resultant supernatants were injected into LC-MS/MS for analysis.

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Protein modification and digestion. Colchicine was mixed with rat or human liver microsomes (1.0 mg protein/mL, final volume: 500 µL), and the reaction was initiated by addition of NADPH (1.0 mM). After 30 min incubation at 37 °C, the protein samples were denatured by heating in a water bath at 80 °C for 10 min, followed by centrifugation at 19,000 g for 10 min. The resulting pallets were suspended in 50 mM ammonium bicarbonate (pH 8.0), vortexed, and centrifuged at 19,000 g for 10 min. The resulting pallets were harvested. The protein washing was repeated for three times. The resultant washed protein samples were re-suspended in 200 µL of 50 mM ammonium bicarbonate, mixed with DTT (5.0 mM), and incubated at 60 °C for 1 h. The DTT-treated proteins were digested with pronase E (2.0 mg/mL) in the presence of 5.0 mM CaCl2 with continuous incubation at 37 °C for 15 h.26-28 The digested protein samples were centrifuged at 19,000 g for 10 min, and the supernatants were subjected to LC-MS/MS analysis.

LC-MS/MS Analysis.

Mass spectrometric analyses were performed on an AB

SCIEX Instruments 4000 Q-Trap™ (Applied Biosystems, Foster City, CA) equipped with an ekspert ultraLC 100 system. The chromatographic separation was achieved on an Ultimate® XB-C18 column (3 µm, 2.1×100 mm; Welch Materials, Inc., Shanghai, China).

Mobile phases were composed of 0.1% (v/v) formic acid in

acetonitrile (solvent A) and 0.1% (v/v) formic acid in water (solvent B). The gradient elution for analyses of NAC conjugates started from 10% solvent A and maintained for 2 min, and increased to 90% solvent A linearly in 6 min, maintained for 1 min, and decreased to 10% solvent A in 1 min, maintained for 2 min to equilibrate the -7-

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column. The gradient elution for separation of DMCs started from 10% solvent A and maintained for 2 min, and increased to 20% solvent A in 1 min, increased to 25% solvent A in 7 min, increased to 100% solvent A in 1 min and maintained for 4 min, decreased to 10% solvent A in 1 min and maintained for 2 min. The flow rate was 0.4 mL/min, and the column temperature was maintained at 25 °C. LC-MS/MS analyses were performed on 2 µL aliquots of samples. Multiple-reaction monitoring (MRM) scans were performed.

The parameters of ion pairs (collision energy, CE;

declustering potential, DP; collision cell exit potential, CXP) were m/z 400 → 358 (30, 90, 10; for colchicine), 386 → 344 (29, 88, 8; for DMCs), and 547 → 418 (26, 125, 9; for NAC conjugates), and 533 → 404 (26, 125, 9; for NAC conjugates). detection was achieved in positive ion mode.

The

Ion spray voltage and source

temperature were set at 5,500 V and 650 °C. Curtain gas, ion source gas 1, and ion source gas 2 were set at 20, 50, and 50 psi, respectively. The information-dependent acquisition method was used to trigger the enhanced product ion (EPI) scans by analyzing MRM.

The EPI scan was run in positive mode at a scan range for

productions from m/z 50 to 600. The collision energy was set at 40 ± 15 eV. Digested protein samples were analyzed on an AB SCIEX Instruments 5500 QTrap (Applied Biosystems, Foster City, CA) equipped with an Agilent 1260 Series Rapid Resolution LC system. The same column described above was employed. Likewise, mobile phase A was acetonitrile with 0.1% (v/v) formic acid (solvent A), and mobile phase B was water with 0.1% (v/v) formic acid (solvent B). The mobile phases consisted of linear gradients of solvent A and solvent B. The gradient elution -8-

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started from 10% solvent A and maintained for 2 min, and increased to 90% A linearly in 6 min, maintained for 4 min, and decreased to 10% in 1 min, maintained for 2 min to equilibrate the column. The LC flow rate was 0.4 mL/min. The injection volume was 5 µL. The parameters of ion pairs (collision energy, CE; declustering potential, DP; collision cell exit potential, CXP) for colchicine-derived cysteine conjugates were m/z 505 → 418 (26, 125, 9). The other parameters of LC-MS were in keeping with the same conditions described above except the curtain gas, which was set at 35 psi. The data were analyzed by Applied Biosystems/SCIEX Analyst software (version 1.6.2). LC-MS/MS analyses were also conducted on an Agilent 1200 Series Rapid Resolution LC system equipped with a hybrid quadrupole time-of-flight (Q-TOF) MS system (microQ-TOF; Bruker Corporation, Billerica, MA). The same column and gradient system described above were applied.

The mass spectrum data were

acquired in positive ion mode. The mass spectrometric parameters were optimized as follows: end plate offset, -500 V; capillary voltage, -4,500 V; nebulizer gas pressure, 1.2 bar; dry gas, high-purity nitrogen (N2); dry gas flow rate, 8.0 liters per minute; and gas temperature, 180°C. The spectra were acquired at 2 seconds per spectrum in the range of m/z 50-1500. The data were analyzed by Bruker Daltonics Data Analysis 3.4 software.

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Results Identification of colchicine demethylation metabolites in microsomes. Colchicine was incubated in rat liver microsomes, and four demethylation metabolites (M1-M4) were detected (Fig. 1B).

No such metabolites were detected in the microsomal

incubation system in the absence of NADPH (Fig. 1A). The four metabolites were eluted out at retention times of 5.7 (M1), 6.0 (M2), 7.9 (M3), and 11.3 min (M4), respectively. M1, M2, and M4 were assigned as 2-DMC (Fig. 1C), 3-DMC (Fig. 1D), and 10-DMC (Fig. 1E), based on their chromatographic and mass spectral behaviors in comparison with those of the corresponding authentic standards. M3 was proposed to be 1-DMC by exclusion, although the authentic standard of the demethylated colchicine was lacking. Colchicine was incubated in rat liver microsomes supplemented with NAC as a trapping agent. Two NAC conjugates, including M5 (Rt = 5.1 min) and M6 (Rt = 5.5 min) were detected by the mass spectrometry with ion transition m/z 547 → 418 (Fig. 2B). No such metabolites were detected in the microsomal incubation system in the absence of NADPH (Fig. 2A), indicating that metabolism was mediated in the formation of M5 and M6. The tandem mass spectrometric (MS/MS) spectra of M5 and M6 were obtained by MRM-EPI scanning (ion transition m/z 547 → 418). M5 and M6 had their [M+H]+ ions at m/z 547. The MS/MS spectrum of M5 provided fingerprint fragment ions at m/z 505, 418, 359, 331, and 303 (Fig. 3A), and MS/MS spectrum of M6 provided fingerprint fragment ions at m/z 505, 418, 359, 328, and 303 (Fig. 3C). The spectra showed the indicative characteristic fragmentations associated - 10 -

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with the cleavage of the NAC moiety, and the product ion at m/z 418 was corresponded to the characteristic neutral loss of 129 Da for NAC conjugates. The mixture was also analyzed by LC/Q-TOF MS. M5 and M6 showed their protonated molecule ion [M+H]+ at m/z 547.1764 and m/z 547.1757 in positive ion mode, which matches the elemental composition of [M+H]+ C26H31N2O9S (m/z 547.1745) (Table. 1). To further characterize M5 and M6, we conducted microsomal incubations with 2-DMC, 3-DMC, or 10-DMC instead of colchicine, followed by LC-MS/MS analysis. In the incubation of 10-DMC, two peaks were observed with the same retention time of M5 and M6 (Fig. 2C), and the mass spectra of the two metabolites were identical to those of M5 and M6 (Fig. 3B and 3D). No such metabolites were detected in the incubation of 2-DMC or 3-DMC (data not shown). This indicates that M5 and M6 originated from10-DMC. The two NAC conjugates were also detected in human liver microsomes (Fig. S1). Two more metabolites (M7 and M8) with retention times at 4.1 and 4.6 min were detected by MRM scanning with ion transition m/z 533 → 404 (Fig. 4B). No such conjugates were found in the microsomal incubation system in the absence of NADPH (Fig. 4A), indicating that metabolism was involved in the formation of M7 and M8.

MS/MS spectra of M7 and M8 were obtained by MRM-EPI scanning (ion

transition m/z 533 → 404). MS/MS spectrum of M7 provided fingerprint fragment ions at m/z 491, 404, 362, and 312 (Fig. 5A), and MS/MS spectrum of M8 revealed fingerprint fragment ions at m/z 491, 404, 345, 317, and 289 (Fig. 5D). Product ion m/z 404 was corresponded to the characteristic neutral loss of 129 Da for NAC - 11 -

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conjugates associated with the cleavage of the NAC moiety. The molecular ions of M7 and M8 were found to be m/z 533 that is m/z 28 less than that of the molecular ion of colchicine. This suggests that formation of M7 and M8 resulted from the loss of two methyl groups from colchicine and addition of a molecule of NAC. Microsomal incubations of 2-DMC or 3-DMC produced a metabolite with the same retention time and mass spectrum as those of M7 (Fig. 4C, 4D, 5B, and 5C). This further indicates that M7 was formed after loss of two methyl groups. Specifically, its formation resulted from O-demethylation of 2-DMC or 3-DMC. The incubations in the absence of NADPH failed to produce such metabolite. In microsomal incubation of 10-DMC supplemented with NAC, a peak was detected with the same retention time and MS/MS spectrum as those of M8 (Fig. 4E and 5E). No such peak was detected in the microsomal incubation system in the absence of NADPH (date not shown). Thus, 10DMC was subjected to metabolism to M8.

Protein covalent binding of DMCs.

Colchicine was incubated with rat liver

microsomes, and the resulting microsomal proteins were completely digested by proteinase. Two colchicine-derived cysteine adducts, namely CA-1 and CA-2, were detected by LC-MS/MS from the proteolytic digestion sample. The two adducts monitored by MRM scanning with ion transition m/z 505 → 418 shared the same retention time (6.8 and 7.4 min) as two cysteine conjugates generated in microsomal incubation of colchicine complemented with cysteine (Fig. 6B and 6C). No such adducts were detected in the control group in which NADPH was excluded in the microsomal incubations (Fig. 6A).

To determine the origin of the two cysteine - 12 -

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adducts, 10-DMC was incubated with cysteine, followed by LC-MS/MS analysis. The reaction produced two products that showed the same mass spectrometric and chromatographic behaviors as those of CA-1 and CA-2 (Fig. 6D). This indicates that the two cysteine conjugates were derived from 10-DMC.

Similar protein covalent

binding study was conducted using human liver microsomes, and CA-1 was detected (Fig. S2).

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Discussion Colchicine contains four O-methyl groups.

Oxidative demethylation was

reportedly the major metabolism pathway,21, 22 and P450 3A subfamily was found to be the principal enzymes responsible for the phase I reaction.23 As expected, four Odemethylated colchicine were detected by LC-MS/MS in microsomal incubations of colchicine, namely 1-, 2-, 3-, and 10-DMC. 2-DMC and 3-DMC showed a similar polarity by sharing similar retention time (5.7 min for 2-DMC and 6.0 min for 3DMC). Interestingly, 10-DMC was eluted out at 11.3 min, far behind 2- and 3-DMCs and even behind parent compound colchicine (Rt = 9.0 min, data not shown). It is most likely that the intramolecular hydrogen bond between 10-hydroxy group and the nearby ketone group of the heptatomic aromatic ring made the molecule less hydrophilic. A total of four NAC-derived conjugates (M5-M8) were detected by LC-MS/MS in microsomal incubations of colchicine supplemented with NAC. The mass spectra of the four metabolites showed the characteristic neutral loss of 129 Da derived from NAC moiety. NAC conjugates M5 and M6 were detected in the incubation of NAC with 10-DMC but not in that of 2-DMC or 3-DMC. Not only does this suggest that M5 and M6 were derived from 10-DMC, but also this indicates that 10-hydroxyl group of 10-DMC played a crucial role in the production of M5 and M6. As shown in Scheme 1, there are three carbon-carbon double bonds in 10-DMC’s heptatomic aromatic ring where three carbons (C8, C11, and C12) are available for the reactions with the sulfhydryl group of NAC. The reaction of the sulfhydryl group of NAC with - 14 -

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C8 or C11 produces the corresponding catechols that can be spontaneously oxidized to M5/M6 (Scheme 1A and 1B). However, the product from the reaction of NAC with 10-DMC at C12 is actually a α-diketone that is relatively stable against auto-oxidation (Scheme 1C). Besides, C4 is unlikely a favorite carbon to react with NAC, since C4 is unavailable for electron delocalization with the ketone group. This may explain why only two conjugates were detected in the incubation of 10-DMC with NAC. M7 and M8 are considered to be NAC conjugates derived from the reactive intermediates formed through two sequential processes of O-demethylation of colchicine.

M7 was detected in microsomal incubations of 2-DMC or 3-DMC

supplemented with NAC but not in that of 10-DMC, and the formation of M7 was found to be NADPH-dependent. It is likely that O-demethylations of 2-DMC at C3 and 3-DMC at C2 virtually produced the same catechol that was sequentially oxidized to the corresponding o-quinone. The reaction of the resulting quinone with NAC offered M7 (Scheme 1D). M8 was detected in the incubation of 10-DMC (M4) supplemented with NAC, but not in those of 2- or 3-DMC. Likewise, M5 and M6 were found to result from 10-DMC (Fig. 2 and Fig. 3). We propose that the formation of M8 resulted from either O-demethylation of 10-DMC and sequential reaction with NAC or direct O-demethylation of M5 or M6 (Scheme 2). M8 likely originated from M5, since the two conjugates shared similar mass spectral pattern (Fig. 3A and 5D). The reactivity of the demethylated colchicine to NAC is evident.

Protein

covalent binding by these reactive metabolites was determined by microsomal incubation of colchicine, followed by examining modification of cysteine residues - 15 -

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within microsomal proteins. Proteolytic digestion of colchicine-exposed microsomal proteins offered two cysteine adducts, including CA-1 and CA-2 (Scheme 3). The cysteine adducts were derived from 10-DMC, which was confirmed by a direct reaction of 10-DMC with cysteine. Interestingly, no cysteine adducts derived from the other DMCs were detected in the proteolytic digestion sample, although 10-DMC was not the major demethylation metabolite of colchicine. Additionally, the cysteine modification was found to depend on NADPH, indicating that metabolism was involved in the protein covalent binding.

Protein modification is an important

mechanism of toxic action. Involvement of reactive metabolites of many pro-toxins in protein modification has been well documented.29, 30 The observed protein adduction by 10-DMC provided the insight into the interactions of electrophilic metabolites of colchicine with proteins. The correlation of colchicine-induced hepatotoxicity with the formation of the reactive metabolites of colchicine and consequent protein adduction needs to be investigated. In conclusion, metabolic O-demethylation of colchicine produced four demethylation metabolites, including 1-, 2-, 3-, and 10-DMC.

These four

demethylated colchicine all showed electrophilicities to NAC. A quinone metabolite was generated after two sequential O-demethylation reactions. 10-DMC was found to modify cysteine residues of microsomal proteins after exposure to colchicine. The findings facilitate the understanding of mechanisms of hepatotoxicity induced by colchicine.

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Funding sources This work was supported in part by the National Natural Science Foundation of China [Grant 81373471 and 81430086].

Supporting Information Available Incubations of colchicine with cysteine in rat liver microsomes, metabolism and protein covalent binding of colchicine in human liver microsomes were supplied as supporting information. This material is available free of charge via the internet at http://pubs.acs.org.

Acknowledgement We would like to thank Mr. Robert Zheng for his assistance in manuscript preparation.

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Abbreviations 1-DMC, 1-demethylated colchicine; 2-DMC, 2-demethylated colchicine; 3-DMC, 3demethylated colchicine; 10-DMC, 10-demethylated colchicine; CE, collision energy; CXP, cell exit potential; DP, declustering potential; EP, entrance potential; EPI, enhanced product ion; NAC, N-Acetyl cysteine; IDA, information-dependent acquisition; MRM, multiple-reaction monitoring; and NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt.

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References (1) Wallace, S. L. (1974) Colchicine. Semin. Arthritis Rheum. 3, 369-381. (2) Roberts W. N., Liang, M. H., Stern, S. H. (1987) Colchicine in acute gout. Reassessment of risks and benefits. JAMA 257, 1920-1922. (3) Cerquaglia, C., Diaco, M., Nucera, G., La Regina, M., Montalto, M., Manna, R. (2005) Pharmacological and clinical basis of treatment of familial Mediterranean fever (FMF) with colchicine or analogues: an update. Curr. Drug Targets Inflamm. Allergy 4, 117-124. (4) Morejohn, L. C., Fosket, D. E. (1991) The biochemistry of compounds with anti-microtubuleactivity in plant cells. Pharmacol. Ther. 51, 217-230. (5) Fakih, M., Yagoda, A., Replogle, T., Lehr, J. E., and Pienta, K. J. (1995) Inhibition of prostate cancer growth by estramustine and colchicine. Prostate 26, 310315. (6) Ravid, M., Robson, M., Kedar, I. (1997) Prolonged colchicine treatment in four patients with amyloidosis. Ann. Intern. Med. 87, 568-570. (7) Levy, M., Spino, M., Read, S. E. (1991) Colchicine: A state of the art review. Pharmacotherapy 11, 196-211. (8) Alarcon-Segovia, D., Ramos-Niembro, F., Ibanez de Kasep, I., Alcacer, J., Tamayo, R. P. (1979) Long term evaluation of colchicine in the treatment of scleroderma. J. Rheumatol. 6, 705-712. (9) Kaplan, M. M., Alling, D. W., Zimmerman, H. J., Wolfe, H. J., Sepersky, R. A., Hirsch, G. S., Elta, G. H., Glick, K. A., Eagen, K. A. (1986) A prospective trial of colchicine for primary biliary cirrhosis. N. Engl. J. Med. 315, 1448-1454. (10) Kershenobich, D., Vargas, F., Garcia-Tsao, G., Tamayo, R. P., Gent, M., Rojkind, M. (1988) Colchicine in the treatment of cirrhosis of the liver. N. Engl. J. Med. 318, 1709-1713. (11) Tafi, L., Matucci-Cerinic, M., Falcini, F., Valentini, G., Bartolozzi, G. (1987) Colchicine treatment of Behcet’s disease in children. Arthritis Rheum. 30, 1435.

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(12) Leighton, J. A., Bay, M. K., Maldonado, A. L., Johnson, R. F., Schenker, S., Speeg, K. V. (1990) The effect of liver dysfunction on colchicine pharmacokinetics in the rat. Hepatology 11, 210-215. (13) Mourelle, M., Villalon, C., Amezcua, J. L. (1988) Protective effect of colchicine on acute liver damage induced by carbon tetrachloride. J. Hepatol. 6, 337342. (14) Leighton, J. A., Bay, M. K., Maldonado, A. L., Schenker, S., Speeg, K. V. (1991) Colchicine clearance is impaired in alcoholic cirrhosis. Hepatology 14, 10131015. (15) Meniño, M. J., Cutrín, C., Parafita, M. A. (1993) Effect of colchicine on lactate production by isolated hepatocytes in rats treated with carbon tetrachloride and ethanol. Drug Alcohol Depend. 32, 181-185. (16) Stein, O., Stein, Y. (1973) Colchicine-induced inhibition of very low density lipoprotein release by rat liver in vivo. Biochim. Biophys. Acta 306, 142-147. (17) Dubin, M., Maurice, M., Feldmann, G., Erlinger, S. (1980) Influence of colchicine and phalloidin on bile secretion and hepatic ultrastructure in the rat. Possible interaction between microtubules and microfilaments. Gastroenterology 79, 646-654. (18) Rao, C. V., Mehendale, H. M. (1991) Effect of colchicine on hepatobiliary function in CCl4 treated rats. Biochem. Pharmacol. 42, 2323-2332. (19) Dickinson, M., Juneja, S. (2009) Haematological toxicity of colchicine. Br. J. Haematol. 146, 465. (20) Borrás-Blasco, J., Enriquez, R., Sirvent, A. E., Amoros, F., Navarro-Ruiz, A., Reyes, A. (2005) Acute renal failure associated with an accidental overdose of colchicine. Int. J. Clin. Pharmacol. Ther. 43, 480-484. (21) Hunter, A. L., Klaassen, C. D. (1975) Biliary excretion of colchicine. J. Pharmacol. Exp. Ther. 192, 605-617. (22) Sabouraud, A., Rochdi, M., Urtizberea, M., Christen, M. O., Achtert, G., Scherrmann, J. M. (1992) Pharmacokinetics of colchicine: a review of experimental and clinical data. Z. Gastroenterol. 30 Suppl 1, 35-39. - 20 -

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(23) Tateishi, T., Soucek, P., Caraco, Y., Guengerich, F. P., Wood, A. J. (1997) Colchicine biotransformation by human liver microsomes. Identification of CYP3A4 as the major isoform responsible for colchicine demethylation. Biochem. Pharmacol. 53, 111-116. (24) Xu, L., Adams, B., Jeliazkova-Mecheva, V. V., Trimble, L., Kwei, G., Harsch, A. (2008) Identification of novel metabolites of colchicine in rat bile facilitated by enhanced online radiometric detection. Drug Metab. Dispos. 36, 731-739. (25) Lin, G., Tang, J., Liu, X. Q., Jiang, Y., Zheng, J. (2007) Deacetylclivorine: a gender-selective metabolite of clivorine formed in female Sprague-Dawley rat liver microsomes. Drug Metab. Dispos. 35, 607-613. (26) Lin, H., Zhang, H., Jushchyshyn, M., Hollenberg, P. F. (2010) Covalent modification of Thr302 in cytochrome P450 2B1 by the mechanism-based inactivator 4-tert-butylphenylacetylene. J. Pharmacol. Exp. Ther. 333, 663-669. (27) Aloysius, H., Tong, V. W., Yabut, J., Bradley, S. A., Shang, J., Zou, Y., Tschirret-Guth, R. A. (2012) Metabolic activation and major protein target of a1benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonist. Chem. Res. Toxicol. 25, 1412-1422. (28) Wang, K., Li, W. W., Chen, J. M., Peng, Y., Zheng, J. (2015) Detection of cysteine- and lysine-based protein adductions by reactive metabolites of 2,5dimethylfuran. Anal. Chim. Acta 896, 93-101. (29) Phillips, M. B., Sullivan, M. M., Villalta, P. W., Peterson, L. A. (2014) Covalent modification of cytochrome c by reactive metabolites of furan. Chem. Res. Toxicol. 27, 129-135. (30) Moro, S., Chipman, J. K., Antczak, P., Turan, N., Dekant, W., Falciani, F., Mally, A. (2012) Identification and pathway mapping of furan target proteins reveal mitochondrial energy production and redox regulation as critical targets of furan toxicity. Toxicol. Sci. 126, 336-352. (31) Khojasteh, S. C., Hartley, D. P., Ford, K. A., Uppal, H., Oishi, S., Nelson, S. D. (2012) Characterization of rat liver proteins adducted by reactive metabolites of menthofuran. Chem. Res. Toxicol. 25, 2301-2309. - 21 -

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(32) Lin, D. J., Li, C. Y., Peng, Y., Gao, H. Y., Zheng, J. (2014) Cytochrome P450–Mediated Metabolic Activation of Diosbulbin B. Drug Metab. Dispos. 42, 1727-1736. (33) Labib, S., Boujraf, S., Berdai, A., and Harandou, M. (2014) Fatal colchicine intoxication. Saudi Journal of Anaesthesia 8, 394-395.

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Table 1. Summary of metabolite mass spectrometric data obtain from LC/QTOF MS analysis of rat microsomal incubations with colchicine in the presence of NADPH and NAC.

Description

Calculated

Formula

Measured

Absolute

Relative

Mass

Mass

Error

Error

+

mDa

ppm

547.1764

1.95

2.88

547.1757

1.32

1.74

[M+H] M5 M6

C26H30N2O9S1H

547.1745

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Figure Legends Figure 1. Extracted ion (m/z 386 → 344) chromatograms obtained from LC-MS/MS analysis of colchicine-fortified microsomal incubations in the absence (A) and presence (B) of NADPH. C: Extracted ion (m/z 386 → 344) chromatogram obtained from LC-MS/MS analysis of authentic 2-DMC. D: Chromatogram of authentic 3DMC. E: Chromatogram of authentic 10-DMC.

Figure 2. Extracted ion (m/z 547 → 418) chromatograms obtained from LC-MS/MS analysis of microsomal incubations containing colchicine and NAC in the absence (A) and presence (B) of NADPH. C: Extracted ion (m/z 547 → 418) chromatogram obtained from LC-MS/MS analysis of microsomal incubation containing 10-DMC and NAC.

Figure 3. MS/MS spectra of M5 generated in microsomal incubations of colchicine (A) and 10-DMC (B). MS/MS spectra of M6 generated in microsomal incubations of colchicine (C) and 10-DMC (D).

Figure 4. Extracted ion (m/z 533 → 404) chromatograms obtained from LC-MS/MS analysis of colchicine-fortified microsomal incubations in the absence (A) or presence (B) of NADPH. Extracted ion (m/z 533 → 404) chromatogram obtained from LCMS/MS analysis of microsomal incubations with 2-DMC (C), 3-DMC (D), or 10DMC (E) in the presence of NADPH and NAC.

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Figure 5. MS/MS spectra of M7 generated in microsomal incubations of colchicine (A), 2-DMC (B), or 3-DMC (C).

MS/MS spectra of M8 generated in microsomal

incubations of colchicine (D) or 10-DMC (E).

Figure 6. Extracted ion (m/z 505 → 418) chromatograms obtained from LC-MS/MS analysis of proteolytic digestion of rat liver microsomes after exposure to colchicine in the absence (A) or presence (B) of NADPH.

Extracted ion (m/z 505 → 418)

chromatogram obtained from LC-MS/MS analysis of incubations of colchicine (C) or 10-DMC (D) with cysteine.

Scheme Legends

Scheme 1. Proposed metabolic pathways for the formation of M5/M6 and M7.

Scheme 2. Proposed metabolic pathway of colchicine.

Scheme 3. Proposed protein covalent binding pathway of colchicine.

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Figure 1. Extracted ion (m/z 386 → 344) chromatograms obtained from LC-MS/MS analysis of colchicinefortified microsomal incubations in the absence (A) and presence (B) of NADPH. C: Extracted ion (m/z 386 → 344) chromatogram obtained from LC-MS/MS analysis of authentic 2-DMC. D: Chromatogram of authentic 3-DMC. E: Chromatogram of authentic 10-DMC. 66x50mm (300 x 300 DPI)

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Figure 2. Extracted ion (m/z 547 → 418) chromatograms obtained from LC-MS/MS analysis of microsomal incubations containing colchicine and NAC in the absence (A) and presence (B) of NADPH. C: Extracted ion (m/z 547 → 418) chromatogram obtained from LC-MS/MS analysis of microsomal incubation containing 10DMC and NAC. 62x72mm (300 x 300 DPI)

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Figure 3. MS/MS spectra of M5 generated in microsomal incubations of colchicine (A) and 10-DMC (B). MS/MS spectra of M6 generated in microsomal incubations of colchicine (C) and 10-DMC (D). 127x51mm (300 x 300 DPI)

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Figure 4. Extracted ion (m/z 533 → 404) chromatograms obtained from LC-MS/MS analysis of colchicinefortified microsomal incubations in the absence (A) or presence (B) of NADPH. Extracted ion (m/z 533 → 404) chromatogram obtained from LC-MS/MS analysis of microsomal incubations with 2-DMC (C), 3-DMC (D), or 10-DMC (E) in the presence of NADPH and NAC. 63x86mm (300 x 300 DPI)

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Figure 5. MS/MS spectra of M7 generated in microsomal incubations of colchicine (A), 2-DMC (B), or 3-DMC (C). MS/MS spectra of M8 generated in microsomal incubations of colchicine (D) or 10-DMC (E). 66x95mm (300 x 300 DPI)

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Figure 6. Extracted ion (m/z 505 → 418) chromatograms obtained from LC-MS/MS analysis of proteolytic digestion of rat liver microsomes after exposure to colchicine in the absence (A) or presence (B) of NADPH. Extracted ion (m/z 505 → 418) chromatogram obtained from LC-MS/MS analysis of incubations of colchicine (C) or 10-DMC (D) with cysteine. 61x99mm (300 x 300 DPI)

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Scheme 1. Proposed metabolic pathways for the formation of M5/M6 and M7. 140x80mm (300 x 300 DPI)

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Scheme 2. Proposed metabolic pathway of colchicine. 114x101mm (300 x 300 DPI)

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Scheme 3. Proposed protein covalent binding pathway of colchicine. 139x83mm (300 x 300 DPI)

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