1,2-unsaturated pyrrolizidine alkaloids (PA) are plant metabolites predominantly occurring in the plant families Asteraceae and Boraginaceae. Acute and chronic PA poisoning causes severe hepatotoxicity. So far, the molecular mechanisms of PA toxicity are not well understood. To analyze its mode of action, primary human hepatocytes were exposed to a non-cytotoxic dose of 100 µM of four structurally different PA: echimidine, heliotrine, senecionine, senkirkine. Changes in mRNA expression were analyzed by a whole genome microarray. Employing cut-off values with a |fold change| of 2 and a q-value of 0.01, data analysis revealed numerous changes in gene expression. In total, 4556, 1806, 3406 and 8623 genes were regulated by echimidine, heliotrine, senecione and senkirkine, respectively. 1304 genes were identified as commonly regulated. PA affected pathways related to cell cycle regulation, cell death and cancer development. The transcription factors TP53, MYC, NFκB and NUPR1 were predicted to be activated upon PA treatment. Furthermore, gene expression data showed a considerable interference with lipid metabolism and bile acid flow. The associated transcription factors FXR, LXR, SREBF1/2, and PPARα/γ/δ were predicted to be inhibited. In conclusion, though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure.
Overall design: Primary human hepatocytes (Clonetics® Normal Human Hepatocytes from Lonza, Walkersville, USA) were treated for 24 h with 100 µM echimidine, heliotrine, senecionine, senkirkine or solvent control (1 % acetonitrile) in triplicate. Prior to RNA isolation, cells were washed three times with pre-warmed PBS. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. For genome-wide microarray analysis quality and concentration of RNA was determined using Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA). Affymetrix GeneChip hybridization (HG-U133 Plus 2.0) was conducted with 250 ng RNA according to the manufacturer’s instructions.
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