Short Communication Microsomal Formation of a Pyrrolic Alcohol Glutathione Conjugate of Clivorine Firm Evidence for the Formation of a Pyrrolic Metabolite of an Otonecine-Type Pyrrolizidine Alkaloid

The formation of the pyrrolic alcohol glutathione (GSH) conjugates of two different types of pyrrolizidine alkaloids (PAs), i.e. clivorine (an otonecine-type PA) and retrorsine (a retronecine-type PA), was investigated with rat microsomes in the presence of GSH. Two GSH conjugates identified as metabolites of retrorsine were the pyrrolic alcohol conjugated with one [7-GSH-dehydroretronecine (DHP)] or two (7,9-diGSH-DHP) molecules of GSH. diGSH-DHP, the less abundant of the two conjugates, had not been previously identified as a metabolite of PAs. In the case of clivorine, 7-GSHDHP was identified. This is the first unequivocal identification of a pyrrolic metabolite of an otonecine-type PA. Consequently, this study provides the strongest evidence obtained to date to support the hypothesis, suggested >25 years ago, that the mechanism of hepatotoxicity induced by otonecine-type PAs involves key metabolic steps similar to those for retronecine-type PAs. PAs occur naturally in a wide variety of plant species. Many PAs are hepatotoxins, causing irreversible liver damage. They represent a serious health problem to grazing livestock and also to humans via contamination of foods, herbal teas, and herbal medicines (Mattocks, 1968, 1986; McLean, 1970; Huxtable, 1980; White et al., 1973). Hepatotoxic PAs are classified into two types, namely the retronecine type [or heliotridine, a 7(S)-isomer of retronecine, type] and the otonecine type (fig. 1). For the former type, the mechanism of hepatotoxicity has been extensively investigated (Mattocks, 1968, 1986; Huxtable, 1989; Mattocks and White, 1971; White and Mattocks, 1971) and is considered to involve formation of reactive pyrrolic metabolites, the dehydro-PAs. For example, retrorsine, a potent hepatotoxic retronecine-type PA, is oxidized by hepatic cytochrome P450 monooxygenases to an unstable carbinolamine intermediate, which decomposes spontaneously to dehydroretrorsine, an electrophilic pyrrolic ester (fig. 1) (Mattocks, 1968, 1986; Huxtable, 1989; Mattocks and White, 1971; White and Mattocks, 1971). The pyrrolic ester then either rapidly forms covalent adducts with cellular macromolecules in the liver to produce hepatotoxicity or undergoes further biotransformations to generate less toxic or nontoxic metabolites, such as a stable pyrrolic alcohol (DHP) and DHP-GSH conjugates (fig. 1) (Mattocks, 1968, 1986; Huxtable, 1989; Mattocks and White, 1971; White and Mattocks, 1971). For otonecine-type PAs, it was originally suggested that hepatotoxicity also arises via pyrrolic esters formed by a similar route, after initial N-demethylation (Mattocks, 1986; Mattocks and White, 1971). However, there have been no reports on the identification of such toxic metabolites of this type of PA. In the present study of clivorine, a pyrrolic alcohol GSH conjugate, 7(R)-GSH-DHP, was identified in incubations of this otonecine-type PA with rat liver microsomes in the presence of GSH. This finding provides the firmest evidence obtained to date for the mechanism of otonecine-type PAinduced hepatotoxicity suggested .25 years ago (Mattocks, 1986; Mattocks and White, 1971). Materials and Methods Chemicals. Retrorsine, monocrotaline, GSH, and all other chemicals and solvents (purity, .99%) were purchased from Sigma Chemical Co. (St. Louis, MO). Clivorine was isolated from Ligularia hodgsonii Hook (Klásek et al., 1967). The GSH conjugates 7-GSH-DHP and 7(R),9-diGSH-DHP were synthesized using previously described methods (Mattocks et al., 1989, 1991). The identities of both isolated and synthesized compounds were confirmed by UV, IR, NMR, and MS analyses, and the purity of clivorine was determined to be .99% by TLC, HPLC, and NMR spectroscopy. Microsomal Preparation and Incubation. Male Sprague-Dawley rats (body weight, 200–250 g) were pretreated with phenobarbitone for 3 days (80 mg/kg, ip). Microsomes were prepared by a standard procedure (Williams et al., 1989), and protein content was determined using a modification of the method of Lowry et al. (1951). A typical microsomal incubation mixture (10 ml), in potassium phosphate buffer (100 mM, pH 7.4), contained liver microsomes (2 mg/ml protein), 0.25 mM clivorine or 0.32 mM retrorsine, 1 mM NADP, 1 mM NADH, 10 mM glucose-6-phosphate, 10 units glucose-6phosphate dehydrogenase, and 2.0 mM GSH. Incubations were performed at 37°C for 60 min. The resultant mixtures were centrifuged at 105,000g for 30 min at 4°C. Aliquots of the supernatant were directly analyzed by HPLC/MS. HPLC/MS Analysis. The analyses were carried out with a Finnigan TSQ 7000 mass spectrometer equipped with an electrospray interface (Finnigan MAT, San Jose, CA), using a PRP-1 reverse-phase HPLC column (5 mm, 150 mm 3 4 mm i.d.; Hamilton Co., Reno, NV). The mobile phase consisted of 1% (v/v) acetic acid (solvent A) and acetonitrile (solvent B). The gradient elution was as follows: 0–5 min, 100% A; 5–35 min, 100% A to 75% A, (linear); This work was supported by the Research Grant Council of Hong Kong (Earmarked Research Grant CUHK 415/95M). 1 Abbreviations used are: PA, pyrrolizidine alkaloid; DHP, dehydroretronecine [7(R)-hydroxy-1-hydroxymethyl-6,7-dihydro-5H-pyrrolizidine]; GSH, glutathione. Send reprint requests to: Dr. Ge Lin, Department of Pharmacology, The Chinese University of Hong Kong, Shatin, Hong Kong. 0090-9556/98/2602-0181–184$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 26, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 181 at A PE T Jornals on A uust 7, 2017 dm d.aspurnals.org D ow nladed from