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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.

Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals. (C) 2015 Elsevier Inc. All rights reserved.

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) is mediated mainly by cytochrome P₄₅₀ monooxygenases (CYP) CYP1A1, 1A2 and 1B1. Several PAH are known to induce these CYP via aryl hydrocarbon receptor (AhR) signaling. Recently, it was shown that the PAH benzo[a]pyrene (BaP) can induce CYP3A4 as well. The induction was suggested to be mediated by the pregnane X receptor (PXR) rather than AhR. Metabolism by CYP3A4 is only known for dihydrodiol metabolites of PAH but not for their parent compounds. In the present study, a CYP3A4 reporter gene assay, requiring the overexpression of PXR, was used to investigate whether the PAH parent compounds BaP, benzo[c]phenanthrene (BcP) and dibenzo[a,l]pyrene (DBalP) as well as their corresponding phase I metabolites, the respective dihydrodiols and diol epoxides, can induce CYP3A4 promoter activity. BaP, BcP and their dihydrodiols were found to significantly activate the CYP3A4 promoter. Moreover, activation of PXR by all four compounds was detected by using a PXR transactivation assay, supporting that PXR mediates CYP3A4 induction by PAH. Taken together, these results show that both PAH parent compounds as well as their phase I metabolites induce CYP3A4 promoter via the transcription factor PXR.

1,2-Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds. These substances are found in several plants such as Asteraceae and Boraginaceae families. Acute PA poisoning by food contamination causes severe damage to liver; long-term, sub-lethal doses may cause cumulative damage or cancer. A previous whole genome μ-array analysis of PA in primary human hepatocytes revealed potential interactions of PA with certain nuclear receptors acting or working as transcription factors. Employing reporter gene assays it was analyzed whether PA can activate or inhibit nuclear receptors such as RARα, RXRα, LXRα, FXR, PPARα, PPARγ, PPARδ, PXR, ERα and ERβ. As PXR and VDR mediate CYP3A4 promoter activity a potential induction or inhibition of CYP3A4 promoter activity was additionally investigated. To cover the most frequently occurring PA structures (retronecine, heliotridine and otonecine type as well as monoester, diester and cyclic diester) the four PA senecionine, heliotrine, echimidine and senkirkine were selected as representative PA. Heliotrine was found to inhibit the transactivation of LXRα, RXRα, PPARα, PPARγ, PPARδ and PXR. Senecionine activated ERα and β. Echimidine was the only PA that activated PXR. Consequently, it also exclusively induced CYP3A4 promoter activity. Neither interactions nor transactivation could be observed for senkirkine. In conclusion, the prediction from the μ-array data suggesting an interaction of PA with different nuclear receptors could be confirmed. However, the interactions were found to be heterogeneous between the structurally different PA. This suggests a specific structure–activity relationship of PA concerning their interaction with nuclear receptors.