title: Identification of a new reactive metabolite of pyrrolizidine alkaloid retrorsine: (3H-pyrrolizin-7-yl)methanol.
abstract: Pyrrolizidine alkaloids (PAs) such as retrorsine are common food contaminants that are known to be bioactivated by cytochrome P450 enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites known as dehydropyrrolizidine alkaloids (DHPs). We compared how both electrochemical (EC) and human liver microsomal (HLM) oxidation of retrorsine could produce short-lived intermediate metabolites; we also characterized a toxicologically important metabolite, (3H-pyrrolizin-7-yl)methanol. The EC cell was coupled online or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas the HLM oxidation was performed in 100 mM potassium phosphate (pH 7.4) in the presence of NADPH at 37 °C. The EC cell oxidation of retrorsine produced 12 metabolites, including dehydroretrorsine (m/z 350, [M + H(+)]), which was degraded to a new reactive metabolite at m/z 136 ([M + H(+)]). The molecular structure of this small metabolite was determined using high-resolution mass spectrometry and NMR spectroscopy followed by chemical synthesis. In addition, we also identified another minor but reactive metabolite at m/z 136, an isomer of (3H-pyrrolizin-7-yl)methanol. Both (3H-pyrrolizin-7-yl)methanol and its minor isomer were also observed after HLM oxidation of retrorsine and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the presence of reduced glutathione (GSH), each isomer formed identical GSH conjugates at m/z 441 and m/z 730 in the negative ESI-MS. Because (3H-pyrrolizine-7-yl)methanol) and its minor isomer subsequently reacted with GSH, it is concluded that (3H-pyrrolizin-7-yl)methanol may be a common toxic metabolite arising from PAs.
authors.affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland , P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Muluneh M
authors.initials: MM
authors.lastname: Fashe
authors.forename: Risto O
authors.initials: RO
authors.lastname: Juvonen
authors.forename: Aleksanteri
authors.initials: A
authors.lastname: Petsalo
authors.forename: Minna
authors.initials: M
authors.lastname: Rahnasto-Rilla
authors.forename: Seppo
authors.initials: S
authors.lastname: Auriola
authors.forename: Pasi
authors.initials: P
authors.lastname: Soininen
authors.forename: Jouko
authors.initials: J
authors.lastname: Vepsäläinen
authors.forename: Markku
authors.initials: M
authors.lastname: Pasanen
mesh_terms: D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D055664:Electrochemical Techniques; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011763:Pyrrolizidine Alkaloids / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: C000599131:(3H-pyrrolizin-7-yl)methanol; D011763:Pyrrolizidine Alkaloids; C003300:retrorsine