In vitro metabolism of pyrrolizidine alkaloids – Metabolic degradation and GSH conjugate formation of different structure types

Geburek, Ina; Preiß-Weigert, Angelika; Lahrssen‐Wiederholt, Monika; Schrenk, Dieter; These, Anja

doi:10.1016/j.fct.2019.110868

Cited by 31 publications

(39 citation statements)

References 41 publications

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“…It remains to be elucidated if the lower sensitivity of human HepG2 C9 (CYP3A4) cells is due to species differences, e.g., in the pattern of metabolism including activation and detoxification pathways. Recently, no marked differences were found when human and rat liver microsomes were used to generate GSH adducts with a number of potent PAs (Geburek et al 2020). It was shown by Ruan et al (2014) that a variety of CYP enzymes can activate PAs in human liver microsomes to a variable extent.…”

Section: Discussionmentioning

confidence: 99%

“…The latter techniques and TLC separation (visualized with o-chloroanil in toluene) revealed a purity of about 97%. In addition, all PAs were analyzed for impurities by LC/MS/MS analysis as described previously (Geburek et al 2020). None of the PAs was contaminated by other congeners above their LODs except for heliotrine containing 0.5% heliosupine.…”

Section: Chemicals Media and Cellsmentioning

confidence: 99%

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Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells

et al. 2020

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1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration–response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration–response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.

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Section: Discussionmentioning

confidence: 99%

Section: Chemicals Media and Cellsmentioning

confidence: 99%

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells

et al. 2020

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“…Glutathione (GSH) acts as trapping agent for those reactive metabolites as, for instance, pyrrolic metabolites. Covalent GSH conjugates with dehydropyrrolizidine were identified as metabolites of senecionine and several other PAs, and considered as an indirect confirmation of the bioactivation pathway (Chen et al 2016 ; Geburek et al 2020 ; Huan et al 1998b ; Lin et al 1998 ; Ramsdell and Buhler 1987 ; Reed et al 1992 ). A study by Yan et al investigated 22 PAs as potential targets in GSH metabolism, and they identified glutathione S-transferase A1 (GSTA1) and glutathione peroxidase 1 (GPX1) as protein targets (Yan et al 2016 ).…”

Section: Introductionmentioning

confidence: 99%

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

2020

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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.

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“…Furthermore, the expression and activity of CYP2E1 in human have been reported to be 80% identical to that in rat (Martignoni et al 2006), and, therefore, rats may be an appropriate model to study CYP2E1-dependent metabolism in human (Zuber et al 2002). This is further supported by the fact that rats and human liver microsomes displayed qualitatively similar activation and detoxification activities in incubations with monocrotaline (Couet et al 1996;Geburek et al 2019). Metabolism of PAs is generally occurring via three pathways, namely hydrolysis, N-oxidation, and hydroxylation followed by dehydrogenation ( Fig.…”

Section: Introductionmentioning

confidence: 83%

Monocrotaline-induced liver toxicity in rat predicted by a combined in vitro physiologically based kinetic modeling approach

2020

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The aim of the present study was to use an in vitro-in silico approach to predict the in vivo acute liver toxicity of monocrotaline and to characterize the influence of its metabolism on its relative toxic potency compared to lasiocarpine and riddelliine. In the absence of data on acute liver toxicity of monocrotaline upon oral exposure, the predicted dose-response curve for acute liver toxicity in rats and the resulting benchmark dose lower and upper confidence limits for 10% effect (BMDL 10 and BMDU 10) were compared to data obtained in studies with intraperitoneal or subcutaneous dosing regimens. This indicated the predicted BMDL 10 value to be in line with the no-observed-adverse-effect levels (NOAELs) derived from availabe in vivo studies. The predicted BMDL 10-BMDU 10 of 1.1-4.9 mg/kg bw/day also matched the oral dose range of 1-3 mg PA/kg bw/ day at which adverse effects in human are reported. A comparison to the oral toxicity of the related pyrrolizidine alkaloids (PAs) lasiocarpine and riddelliine revealed that, although in the rat hepatocytes monocrotaline was less toxic than lasiocarpine and riddelliine, due to its relatively inefficient clearance, its in vivo acute liver toxicity was predicted to be comparable. It is concluded that the combined in vitro-PBK modeling approach can provide insight in monocrotaline-induced acute liver toxicity in rats, thereby filling existing gaps in the database on PA toxicity. Furthermore, the results reveal that the kinetic and metabolic properties of PAs can vary substantially and should be taken into account when considering differences in relative potency between different PAs. Keywords Monocrotaline • Acute toxicity • Liver • PBK modeling • Reverse dosimetry Abbreviations AIC Akaike information criterion ALT Alanine aminotransferase BMD 10 Benchmark dose resulting in a 10% effect above background level BMDL 10 Lower confidence limit of the BMD 10 BMDU 10 Upper confidence limit of the BMD 10 EFSA European Food Safety Authority GI Gastrointestinal GSH Glutathione HPC Hepatic parencymal cells HVOD Hepatic veno-occlusive disease Ka Absorption rate for uptake from the GI tract compartment into the liver PAs Pyrrolizidine alkaloids PBK Physiologically based kinetic POD Point of departure NOAEL No observed adverse effect level LOAEL Lowest observed adverse effect level pRPF Provisional relative potency factors 3Rs Replacement, reduction and refinement Electronic supplementary material The online version of this article (

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In vitro metabolism of pyrrolizidine alkaloids – Metabolic degradation and GSH conjugate formation of different structure types

Cited by 31 publications

References 41 publications

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells

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

Monocrotaline-induced liver toxicity in rat predicted by a combined in vitro physiologically based kinetic modeling approach

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