In vitro biotransformation of pyrrolizidine alkaloids in different species: part II—identification and quantitative assessment of the metabolite profile of six structurally different pyrrolizidine alkaloids

Abstract

Pyrrolizidine alkaloids (PA) exert their toxic effects only after bioactivation. Although their toxicity has already been studied and metabolic pathways including important metabolites were described, the quantification of the latter revealed a large unknown portion of the metabolized PA. In this study, the qualitative and quantitative metabolite profiles of structurally different PAs in rat and human liver microsomes were investigated. Between five metabolites for europine and up to 48 metabolites for lasiocarpine were detected. Proposals for the chemical structure of each metabolite were derived based on fragmentation patterns using high-resolution mass spectrometry. The metabolite profiles of the diester PAs showed a relatively good agreement between both species. The metabolic reactions were summarized into three groups: dehydrogenation, oxygenation, and shortening of necic acid(s). While dehydrogenation of the necine base is considered as bioactivation, both other routes are considered as detoxification steps. The most abundant changes found for open chained diesters were dealkylations, while the major metabolic pathway for cyclic diesters was oxygenation especially at the nitrogen atom. In addition, all diester PAs formed several dehydrogenation products, via the insertion of a second double bond in the necine base, including the formation of glutathione conjugates. In rat liver microsomes, all investigated PAs formed dehydropyrrolizidine metabolites with the highest amount formed by lasiocarpine, whereas in human liver microsomes, these metabolites could only be detected for diesters. Our findings demonstrate that an extensive analysis of PA metabolism can provide the basis for a better understanding of PA toxicity and support future risk assessment.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment–A Workshop Report

“…While the formation of reactive metabolites is considered toxification, the other transformation steps are considered pathways leading to detoxification. While almost none of these bioactivated metabolites were detected in incubations of monoesters, all diesters investigated were metabolized to such products, with lasiocarpine forming the highest number and amount of such reactive metabolites [49].…”

“…In terms of PA structures, the highest levels were detected for 1,2 unsaturated retronecine or heliotridine diesters with angelic acid at C7 regardless of whether the diesters are open or cyclic [48]. For 6 selected PAs, a comprehensive metabolite profile study was performed, and between 5 metabolites for europine and up to 48 metabolites for lasiocarpine were detected [49]. For each metabolite, potential structures were proposed based on high-resolution mass spectrometry fragmentation patterns.…”

“…4 , the N -oxide forms of monoesters and cyclic diesters account for the highest proportion of PA exposure 47 . Subsequently, hepatic PA metabolism was investigated using rat and human liver microsomal preparations 48 , 49 . If the results were focused on metabolic transformation of the parent PA, there was a trend toward higher transformation rates in rat liver microsomes compared to humans, where especially for monoesters, no metabolic degradation was found.…”

Metabolism-mediated cytotoxicity and genotoxicity of pyrrolizidine alkaloids

“…Similarly, a study on metabolism of lasiocarpine in liver microsomes of human, pig, rat, mouse, rabbit, and sheep found no significant difference in all non-human cases, except that an additional metabolite generated from O-demethylation was identified with human liver microsomal incubation (Fashe et al 2015). Another study investigated the metabolite profiles of six structurally different PAs (including monoesters, openchain diesters and cyclic diesters PAs) in rat and human liver microsomes, and human liver microsomes demonstrated higher metabolic activation of these PAs compared with rat liver microsomes (Geburek et al 2020). On the other hand, hydrolysis, an essential pathway for PA detoxification, is featured by remarkable species difference.…”

“…Compared to macrocyclic-diester PAs, the C-oxidation on the necine base of open-chain diester PA (lasiocarpine) was much more prominent, which is in good correlation to the toxicity discovered from individual PA congeners, while negligible metabolites generated from C-oxidation on necine base were detected from monoester PAs, confirming that monoester PAs are the least toxic. The most abundant change found in open-chain diester PAs was hydrolysis, while the major metabolic pathway for cyclic diester PAs was N-oxidation (Geburek et al 2020). Collectively, the lipophilicity and steric hindrance of structurally different PAs may influence the accessibility of PAs to the active site of CYPs or confer resistance to ester bond cleavage, leading to the significant variation of metabolic activation rates of PAs.…”