Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells

Abstract

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells.

Potency ranking of pyrrolizidine alkaloids in metabolically competent human liver cancer cells and primary human hepatocytes using a genotoxicity test battery

“…In the liver, PAs not only damage hepatocytes, but also proximate liver sinusoidal endothelial cells (LSEC), which was attributable to the efflux of reactive metabolites from the hepatocytes and uptake into LSEC (Hessel-Pras et al 2020 ). This has recently also been demonstrated using a co-culture model of HepG2 liver cells and metabolically deficient HeLa cells (Hadi et al 2023 ). It is further noteworthy that PAs can also be transported to other organs such as the lungs, where they can cause toxicity (He et al 2021a ; Song et al 2020 ).…”

Covalent Interactions of Anthocyanins with Proteins: Activity-Based Protein Profiling of Cyanidin-3-O-glucoside

“…Based on our previous work on anti-insulin resistance activity, we first revealed the protein profiling of C3G in the whole proteome of HepG2 cells and found its covalent binding to some target proteins. Due to the good metabolic regulatory activity of anthocyanins, we further verified the protein covalent binding ability in HeLa cells with an equally active metabolism . These results will promote the chemical development of anthocyanin structures.…”

“…Due to the good metabolic regulatory activity of anthocyanins, we further verified the protein covalent binding ability in HeLa cells with an equally active metabolism. 28 These results will promote the chemical development of anthocyanin structures. In this work, the clickable alkynyl group was introduced into the A ring of C3G to avoid affecting the activity, 29,30 and the modification of the two phenolic hydroxyl groups was referred to Wang et al 31 As shown in Figure 1A, the covalent binding probe C3G-Probe and the noncovalent photoaffinity probe C3G-Diazirine were obtained based on the synthetic route we developed previously.…”

Pyrrolizidine Alkaloids—Pros and Cons for Pharmaceutical and Medical Applications

“…The PA-induced disturbances in mitosis may contribute to the formation of micronuclei. Europine, riddelliine, and lasiocarpine induced micronuclei in HepG2 cells [89].…”