Lack of Metabolic Activation and Predominant Formation of an Excreted Metabolite of Nontoxic Platynecine-Type Pyrrolizidine Alkaloids

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.

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In vitro biotransformation of pyrrolizidine alkaloids in different species. Part I: Microsomal degradation

Archives of Toxicology ◽

10.1007/s00204-017-2114-7 ◽

2017 ◽

Vol 92 (3) ◽

pp. 1089-1097 ◽

Cited By ~ 11

Author(s):

Franziska Kolrep ◽

Jorge Numata ◽

Carsten Kneuer ◽

Angelika Preiss-Weigert ◽

Monika Lahrssen-Wiederholt ◽

...

Keyword(s):

Pyrrolizidine Alkaloids ◽

Metabolic Activation ◽

Strong Relationship ◽

In Vitro Degradation ◽

Chronic Effects ◽

Toxic Metabolites ◽

Conversion Rates ◽

Metabolic Degradation ◽

Acute And Chronic Effects

Abstract Pyrrolizidine alkaloids (PA) are secondary metabolites of certain flowering plants. The ingestion of PAs may result in acute and chronic effects in man and livestock with hepatotoxicity, mutagenicity, and carcinogenicity being identified as predominant effects. Several hundred PAs sharing the diol pyrrolizidine as a core structure are formed by plants. Although many congeners may cause adverse effects, differences in the toxic potency have been detected in animal tests. It is generally accepted that PAs themselves are biologically and toxicologically inactive and require metabolic activation. Consequently, a strong relationship between activating metabolism and toxicity can be expected. Concerning PA susceptibility, marked differences between species were reported with a comparatively high susceptibility in horses, while goat and sheep seem to be almost resistant. Therefore, we investigated the in vitro degradation rate of four frequently occurring PAs by liver enzymes present in S9 fractions from human, pig, cow, horse, rat, rabbit, goat, and sheep liver. Unexpectedly, almost no metabolic degradation of any PA was observed for susceptible species such as human, pig, horse, or cow. If the formation of toxic metabolites represents a crucial bioactivation step, the found inverse conversion rates of PAs compared to the known susceptibility require further investigation.

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Metabolic Activation of Pyrrolizidine Alkaloids: Insights into the Structural and Enzymatic Basis

Chemical Research in Toxicology ◽

10.1021/tx500071q ◽

2014 ◽

Vol 27 (6) ◽

pp. 1030-1039 ◽

Cited By ~ 79

Author(s):

Jianqing Ruan ◽

Mengbi Yang ◽

Peter Fu ◽

Yang Ye ◽

Ge Lin

Keyword(s):

Pyrrolizidine Alkaloids ◽

Metabolic Activation

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Pyrrolizidine Alkaloids—Genotoxicity, Metabolism Enzymes, Metabolic Activation, and Mechanisms

Drug Metabolism Reviews ◽

10.1081/dmr-120028426 ◽

2004 ◽

Vol 36 (1) ◽

pp. 1-55 ◽

Cited By ~ 317

Author(s):

Peter P. Fu ◽

Qingsu Xia ◽

Ge Lin ◽

Ming W. Chou

Keyword(s):

Pyrrolizidine Alkaloids ◽

Metabolic Activation ◽

Metabolism Enzymes

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Toxic Prediction of Pyrrolizidine Alkaloids and Structure-Dependent Induction of Apoptosis in HepaRG Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/8822304 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Pimiao Zheng ◽

Yuliang Xu ◽

Zhenhui Ren ◽

Zile Wang ◽

Sihan Wang ◽

...

Keyword(s):

In Silico ◽

Pyrrolizidine Alkaloids ◽

Cell Injury ◽

Metabolic Activation ◽

Screening Assay ◽

Discovery Studio ◽

Heparg Cells ◽

M Phase ◽

Apoptosis Inducer ◽

Induced Apoptosis

Pyrrolizidine alkaloids (PAs) are common phytotoxins and could cause liver genotoxicity/carcinogenicity following metabolic activation. However, the toxicity of different structures remains unclear due to the wide variety of PAs. In this study, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of 40 PAs were analyzed, and their toxicity was predicted by Komputer Assisted Technology (TOPKAT) using Discovery Studio software. The in silico results showed that all PAs except retronecine had good intestinal absorption, and all PAs were predicted to have different toxicity ranges. To verify the predictive results, 4 PAs were selected to investigate cell injury and possible mechanisms of the differentiation in HepaRG cells, including retronecine type of twelve-membered cyclic diester (retrorsine), eleven-membered cyclic diester (monocrotaline), noncyclic diester (retronecine), and platynecine type (platyphylline). After 24 h exposure, retronecine-type PAs exhibited concentration-dependent cytotoxicity. The high-content screening assay showed that cell oxidative stress, mitochondrial damage, endoplasmic reticulum stress, and the concentration of calcium ions increased, and neutral lipid metabolism was changed notably in HepaRG cells. Induced apoptosis by PAs was indicated by cell cycle arrest in the G2/M phase, disrupting the mitochondrial membrane potential. Overall, our study revealed structure-dependent cytotoxicity and apoptosis after PA exposure, suggesting that the prediction results of in silico have certain reference values for compound toxicity. A 1,2-membered cyclic diester seems to be a more potent apoptosis inducer than other PAs.

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