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Structure-dependent relative toxic potencies of selected pyrrolizidine alkaloids

  • Pyrrolizidine alkaloids are naturally occurring secondary plant metabolites mainly found in plant families of Asteraceae, Boraginaceae, and Fabaceae. Chemically, PAs consist of a pyrrolizidine core bearing hydroxyl groups, the so-called necine base, and mono- or dicarboxylic necine acids bound to the pyrrolizidine core via ester linkages. 1,2-unsaturated PAs are hepatotoxic, genotoxic, and carcinogenic due to the highly reactive pyrrolic metabolites formed by cytochrome P450 monooxygenases (CYPs) primarily in the liver. The presence of PAs as frequent contaminants in the wide variety of food and feed products would be a concern for public health. Due to the inadequate data, the risk assessment of PAs was mainly approached using the two most toxic potent congeners, i.e., lasiocarpine and riddelliine. However, the toxic potencies of individual PA congeners differentiated widely between the congeners probably related to their structural features. The risk of PA-containing products is indeed overestimated, and a comprehensive risk assessment should take these differences into account. After analyzing the data of many PAs, Merz and Schrenk derived interim Relative Potency (iREP) factors to present the differences in their toxicity between the sub-groups concerning their structural features. But since this concept was derived from an inadequate database, it was found that the relative toxicity of individual congeners cannot be entirely reliably evaluated. My work aimed to achieve more comprehensive congener-specific in vitro toxicological data and estimate the structure-related characteristics for refining this concept. For this purpose, ten congeners, lasiocarpine, monocrotaline, retrorsine, senecionine, seneciphylline, echimidine, europine, heliotrine, indicine, and lycopsamine, were determined in a series of in vitro test systems with different endpoints to quantify their cytotoxicity, genotoxicity, and mutagenicity. Cytotoxicity was assessed using the Alamar blue assay. A clear structure dependence could be demonstrated in primary rat hepatocytes and HepG2 (CYP3A4) cells. On the contrary, in HepG2 cells, none of the selected PAs exhibited cytotoxic effects, probably due to the lack of CYPs. The role of CYP450 enzymes in metabolic activation was further confirmed using an inhibition assay and the activity of CYP450 enzymes was measured by a kinetic assay analyzing 7-benzyloxyresorufin-O-dealkylation (BROD). Furthermore, utilizing a glutathione-reductase-DTNB recycling assay indicated that glutathione might not play a critical role in PA-induced cytotoxicity. A micronucleus test was used for determining the PA-induced clastogenic genotoxicity. All selected PA congeners exhibited a concentration-dependent manner in the HepG2 (CYP3A4) cells. The relative potencies of PA congeners estimated from Alamar blue assay and micronucleus assay are generally consistent with the following ranking: lasiocarpine > senecionine > seneciphylline ≥ retrorsine > heliotrine (?) echimidine ≥ europine ≈ indicine ≈ lycopsamine ≈ monocrotaline. Compared to the iREP reported by Merz and Schrenk, monocrotaline exhibited considerably lower toxic potency. However, echimidine was more toxic than expected. On the other hand, mutagenicity was measured in Ames fluctuation assay with Salmonella typhimurium strains TA98 and TA100. None of the selected PA congeners up to 300 µM showed mutagenic effects despite metabolic activation with S9-mix.

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Author:Lan Gao
URN:urn:nbn:de:hbz:386-kluedo-68313
DOI:https://doi.org/10.26204/KLUEDO/6831
Advisor:Dieter Schrenk, Jörg Fahrer
Document Type:Doctoral Thesis
Language of publication:English
Publication Date:2022/05/23
Year of Publication:2022
Publishing Institute:Technische Universität Kaiserslautern
Granting Institute:Technische Universität Kaiserslautern
Acceptance Date of the Thesis:2022/05/05
Date of the Publication (Server):2022/06/01
GND-Keyword:pyrrolizidine alkaloids; relative toxic potencies; genotoxicity; cytotoxicity
Number of page:3, XIII, 130
Faculties / Organisational entities:Fachbereich Chemie
DDC-Cassification:5 Naturwissenschaften und Mathematik / 540 Chemie
Collections:Universitätsbibliothek
Licence (German):Creative Commons 4.0 - Namensnennung, nicht kommerziell, keine Bearbeitung (CC BY-NC-ND 4.0)