PXR: Structure-specific activation by hepatotoxic pyrrolizidine alkaloids

https://doi.org/10.1016/j.cbi.2018.04.017Get rights and content

Highlights

  • PAs can activate the human nuclear receptor PXR.

  • PXR activation by Em leads to higher CYP3A4 mRNA level and enzyme activity.

  • Activation of PXR appears to be dependent on PA structure.

  • PXR is only activated by the open-chain PA diesters Em and Lc.

  • Combination effects of Em and Lc on PXR activation follow the assumption of dose addition.

Abstract

Pyrrolizidine alkaloids (PAs) comprise a large group of more than 660 secondary metabolites found in more than 6000 plant species worldwide. Acute PA intoxication induces severe liver damage. Chronic exposure to sub-lethal doses may cause cumulative damage or cancer. Nuclear receptor activation often constitutes a molecular event for xenobiotic-induced toxicity. However, so far nothing is known about potential interactions of PAs with nuclear receptors as a toxicological mode of action. Thus, in the present study PA-dependent activation of a comprehensive panel of nuclear receptors (PPARs, LXRα, RARα, RXRα, FXR, CAR, PXR, ERα/β) was investigated using GAL4/UAS-based transactivation reporter gene assays. To cover the most frequently occurring PA structure types (retronecine, heliotridine and otonecine type; as well as monoester, open-chain diester and cyclic diester) different PAs were analyzed for interaction with nuclear receptors.

Most of the nuclear receptors investigated were not affected by the tested PAs. However, significant activation was found for PXR, which was exclusively activated by the open-chain diesters, echimidine and lasiocarpine. Induction of the model PXR target gene CYP3A4 by PAs was verified at the mRNA, protein and enzyme activity level.

In conclusion, PXR activation and PXR-mediated induction of CYP3A4 expression by PAs seem to be structure-dependent. Data suggest that only open-chain diesters act as PXR agonists. This might imply that a PXR-mediated mode of action may contribute to the hepatotoxicity of PAs that is dependent on PA structure.

Introduction

Pyrrolizidine alkaloids (PAs) are a large group of heterocyclic secondary plant metabolites occurring worldwide in plant families like Boraginaceae, Compositae and Leguminosae. PAs are synthesized as defensive compounds against herbivores [1,2]. Over 660 structurally different PAs have already been identified in more than 6000 plant species as either ternary PAs or their N-oxide derivatives. PAs share a common chemical structure: they consist of a necine base predominantly being one of the four necine bases retronecine, heliotridine, otonecine, or platynecine and a necic acid moiety (for review see Ref. [3]). Hepatotoxic PAs possess a 1,2-unsaturated necine base, like retronecine-type, heliotridine-type, or otonecine-type PAs, while saturated platynecine-type PAs are considered to be non-toxic. One well described mechanism of PA toxicity is associated with the hepatic metabolic activation into a reactive pyrrole derivate (dehydropyrrolizidine ester) mainly catalyzed by CYP3A enzymes [[4], [5], [6], [7], [8]]. Several in vitro and in vivo studies revealed a metabolism-dependent genotoxic potential, as evidenced by the detection of DNA-adducts, DNA-DNA- and DNA-protein crosslinks, gene mutations, chromosomal damage such as sister chromatide exchanges, and chromosomal aberrations. Animal studies further demonstrated acute and chronic toxicity (liver necrosis, veno-occlusive disease, and pneumotoxicity) as well as embryotoxicity. The carcinogenic potential of PAs in rodent liver, lung, kidneys, and gastrointestinal tract has also been shown (for review see Ref. [3]). However, it remains still unclear whether an induction of molecular mechanism by the parent compound itself additionally occurs.

Humans can be exposed to PAs due to a contamination of foods like honey, cereals, and salads. Several studies also revealed high contents of PAs in commercially available herbal tea samples in Germany [9,10]. Other routes of exposure comprise pollen-containing food supplements, PA-containing remedies, or traditional Chinese medicine [11,12]. Orally ingested PAs are rapidly absorbed from the gastro-intestinal tract. In humans, acute intoxications with high doses of PAs result in liver pain, ascites due to occlusion of the hepatic veins, and an enlargement of the liver (hepatomegaly) which is associated with a high mortality. Subacute/subchronic intoxication with moderate doses causes veno-occlusive disease, an obstruction of the small venous blood channels [13]. Chronic exposure to lower doses is associated with hepatic cirrhosis, and effects on tumor development are hypothesized. In our previously published study [14] we performed a whole genome transcriptomics analysis in primary human hepatocytes after exposure to four structurally different PAs to identify their hepatic molecular mode of action. Analysis of the data set revealed strong regulation of gene expression by PAs which might be associated with a structure-dependent toxic potential of the different PAs.

Nuclear-receptor mediated toxicity is often a prominent molecular mode of action for xenobiotics due to the role of nuclear receptors in the regulation of diverse metabolic pathways [15]. To systematically elucidate whether nuclear receptors might contribute to the molecular mode of action of PA toxicity (beside the well characterized metabolic activation into reactive metabolites) we intended to analyze the direct interaction of PAs with nuclear receptors. In order to identify the induction potential of PAs parent compounds itself on nuclear receptor activation pathways independent of hepatic PA metabolism, we aimed to exclude any influence of PA metabolism by specifically chosing metabolic incompetent cell systems. To furthermore analyze potential structure-activity relationships, we initially used the same four structurally different PAs representing the main structural characteristics as in our previous study (for chemical structures see Fig. 1): heliotrine, echimidine, senecionine, and senkirkine. All four PAs belong to one of the three different PA types known to exhibit toxic properties. Heliotrine represents a monoester of the heliotridine-type PAs, whereas echimidine and senecionine belong to the retronecine type, representing an open-chain diester and a macrocyclic diester, respectively. Senkirkine represents also a macrocyclic diester but belongs to the otonecine-type PAs. Thus, the present study aimed to contribute to a better understanding of the structure-dependent hepatotoxic mode of action within the wide variety of structurally different PAs as well as the potency of non-metabolized PAs to induce toxicological-relevant nuclear receptor activity.

Section snippets

Chemicals

Heliotrine and trichodesmine were purchased from Latoxan (Portes lès Valence, France). All other PAs were obtained from Phytolab (Vestenbergsgreuth, Germany). Troglitazone, 3-methylcholanthrene, GW501516 and CITCO (6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime) were purchased from Enzo Life Sciences (Lörrach, Germany). GW7647 and calcitriol were obtained from Axon Medchem BV (Groningen, The Netherlands) and Biomol (Hamburg, Germany), respectively.

Activation of nuclear receptors by structural different PAs

Prior to analyses of functional molecular endpoints, initial cytotoxicity assays were conducted with HEK-293 and HepG2 cells in order to select non-toxic concentrations for further experimentation. All four initially selected PAs, i.e. heliotrine, echimidine, senecionine, and senkirkine, did not exert cytotoxicity up to concentrations of 250 μM in both cell lines (Supplemental Figs. S1 and S2). A maximal concentration of 100 μM for each PA was chosen for subsequent experiments to exclude

Discussion

Pyrrolizidine alkaloids (PAs) are heterocyclic secondary plant metabolites occurring worldwide in numerous plants. Due to food contamination, PAs can be taken up into the human body. As 1,2-unsaturated PAs are hepatotoxic, pneumotoxic, genotoxic and carcinogenic they pose a serious human health risk. Recently the EFSA panel on Contaminants in the Food Chain (CONTAM) established a new reference point (BMDL10) of 237 μg/kg body weight per day to assess the carcinogenic risk of PAs. However,

Funding

This work was funded by the German Research Foundation [grant number LA1177/12-1] and by the German Federal Institute for Risk Assessment [grant numbers 51-006, 1322-454 and 1322-591].

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

We would like to thank Regina Al-Hamwi, Maria Maares and Anja Friedrich for their excellent technical support.

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