title: Comparison of metabolism-mediated effects of pyrrolizidine alkaloids in a HepG2/C3A cell-S9 co-incubation system and quantification of their glutathione conjugates.
abstract: 1. Toxicity of pyrrolizidine alkaloids (PAs) largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrrolic derivatives. These then spontaneously release the esterifying acids to generate carbonium ions that form covalent adducts with cellular nucleophiles to exhibit toxicity. 2. In our investigation, metabolism-mediated toxicity of monocrotaline, retrorsine, lycopsamine, echimidine (retronecine-type PAs), heliotrine (a heliotridine-type PA) and senkirkine (an otonecine-type PA) was studied using an in vitro co-incubation assay. 3. Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver S9 fraction and the toxicity was assessed as lowered mitochondrial activity. 4. Bioactivation potential was measured by incubating PAs with rat liver S9 fraction, NADPH and GSH in a cell free system. Pyrrolic metabolites generated were entrapped as glutathione conjugates (7-GSH-DHP and 7,9-di-GSHDHP) which were quantified using LC-MS-MS analysis. 5. Our results indicated that PAs were metabolized by rat liver S9 fraction into reactive pyrrolic derivatives which were toxic to HepG2/C3A cells. This approach can be used to determine and compare bioactivation potential and metabolism-mediated toxicity of various PAs.
authors.affiliation: Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA. hemlata.tamta@fda.hhs.gov
authors.forename: Hemlata
authors.initials: H
authors.lastname: Tamta
authors.forename: Rahul S
authors.initials: RS
authors.lastname: Pawar
authors.forename: Wayne G
authors.initials: WG
authors.lastname: Wamer
authors.forename: Erich
authors.initials: E
authors.lastname: Grundel
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Krynitsky
authors.forename: Jeanne I
authors.initials: JI
authors.lastname: Rader
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002474:Cell-Free System; D002853:Chromatography, Liquid; D018920:Coculture Techniques; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D011763:Pyrrolizidine Alkaloids / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
chemical_list: D011763:Pyrrolizidine Alkaloids; D005978:Glutathione