Genotoxicity of selected pyrrolizidine alkaloids in human hepatoma cell lines HepG2 and Huh6

Naji Said Aboud Hadi; Ezgi Eyluel Bankoglu; Lea Schott; Eva Leopoldsberger; Vanessa Ramge; Olaf Kelber; Hartwig Sievers; Helga Stopper

doi:10.1016/j.mrgentox.2020.503305

Genotoxicity of selected pyrrolizidine alkaloids in human hepatoma cell lines HepG2 and Huh6

Mutat Res Genet Toxicol Environ Mutagen. 2021 Jan-Feb:861-862:503305. doi: 10.1016/j.mrgentox.2020.503305. Epub 2020 Dec 28.

Authors

Naji Said Aboud Hadi¹, Ezgi Eyluel Bankoglu², Lea Schott², Eva Leopoldsberger², Vanessa Ramge², Olaf Kelber³, Hartwig Sievers⁴, Helga Stopper⁵

Affiliations

¹ Institute of Pharmacology and Toxicology, University of Wuerzburg, Wuerzburg, Germany; School of Health and Human Sciences, Pwani University, Kilifi, Kenya.
² Institute of Pharmacology and Toxicology, University of Wuerzburg, Wuerzburg, Germany.
³ Steigerwald Arzneimittelwerk GmbH, Bayer Consumer Health, Darmstadt, Germany.
⁴ PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany.
⁵ Institute of Pharmacology and Toxicology, University of Wuerzburg, Wuerzburg, Germany. Electronic address: stopper@toxi.uni-wuerzburg.de.

PMID: 33551105
DOI: 10.1016/j.mrgentox.2020.503305

Abstract

Introduction: Pyrrolizidine alkaloids (PAs) are found in many plant species as secondary metabolites which affect humans via contaminated food sources, herbal medicines and dietary supplements. Hundreds of compounds belonging to PAs have been identified. PAs undergo hepatic metabolism, after which they can induce hepatotoxicity and carcinogenicity. Many aspects of their mechanism of carcinogenicity are still unclear and it is important for human risk assessment to investigate this class of compounds further.

Material and methods: Human hepatoma cells HepG2 were used to investigate the genotoxicity of different chemical structural classes of PAs, namely europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine and lasiocarpine, in the cytokinesis-block micronucleus (CBMN) assay. The different ester type PAs europine, seneciphylline, and lasiocarpine were also tested in human hepatoma Huh6 cells. Six different PAs were investigated in a crosslink comet assay in HepG2 cells.

Results: The maximal increase of micronucleus formation was for all PAs in the range of 1.64-2.0 fold. The lowest concentrations at which significant induction of micronuclei were found were 3.2 μM for lasiocarpine and riddelliine, 32 μM for retrorsine and echimidine, and 100 μM for seneciphylline, europine and lycopsamine. Significant induction of micronuclei by lasiocarpine, seneciphylline, and europine were achieved in Huh6 cells at similar concentrations. Reduced tail formation after hydrogen peroxide treatment was found in the crosslink comet assay for all diester type PAs, while an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration.

Conclusion: The widely available human hepatoma cell lines HepG2 and Huh6 were suitable for the assessment of PA-induced genotoxicity. Selected PAs confirmed previously published potency rankings in the micronucleus assay. In HepG2 cells, the crosslinking activity was related to the ester type, which is a first report of PA mediated effects in the comet assay.

Keywords: Crosslink comet assay; Genomic damage; HepG2 cells; Huh6 cells; Micronuclei; Pyrrolizidine alkaloids.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Carcinoma, Hepatocellular / drug therapy
Carcinoma, Hepatocellular / genetics
Carcinoma, Hepatocellular / pathology*
Cell Proliferation*
DNA Damage*
Humans
Liver Neoplasms / drug therapy
Liver Neoplasms / genetics
Liver Neoplasms / pathology*
Micronucleus Tests
Pyrrolizidine Alkaloids / pharmacology*
Tumor Cells, Cultured

Substances

Pyrrolizidine Alkaloids