Active Transport of Hepatotoxic Pyrrolizidine Alkaloids in HepaRG Cells.

Anne-Margarethe Enge,Christoph Gottschalk,Florian Kaltner,Albert Braeuning,Stefanie Hessel-Pras

doi:10.3390/ijms22083821

Abstract

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na+/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics.

Highlights

Pyrrolizidine alkaloids are secondary plant metabolites that are estimated to be produced by more than 6000 plant species and to be present in about 3% of the world’s flowering plants [1,2]
A first step in assessing pyrrolizidine alkaloids (PAs) transport processes in HepaRG cells was to ascertain the general mode of action of transport
As passive permeability can mask active uptake mechanisms in hepatocytes, incubations at 4 ◦ C can be used as a first qualitative approach to evaluate active transport processes [40]

Summary

Introduction

Pyrrolizidine alkaloids are secondary plant metabolites that are estimated to be produced by more than 6000 plant species and to be present in about 3% of the world’s flowering plants [1,2]. Pyrrolizidine alkaloids are found as food contaminants in e.g., tea, honey, and herbs, as well as in feed, turning them into a relevant concern for risk assessment [3,4,5,6,7]. Pyrrolizidine alkaloids share a common basic structure. They are esters of 1-hydroxymethylpyrrolizidine, the necine base, that can be esterified at the hydroxyl groups at ring positions C-7 and/or C-9 with one or two aliphatic mono- or dicarboxylic acids, so-called necic acids.

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