Assessment of pyrrolizidine alkaloid-induced toxicity in an in vitro screening model

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are a group of heterocyclic phytotoxins present in a wide range of plants. The consumption of PA-containing medicinal herbs or PA-contaminated foodstuffs has long been reported to cause human hepatotoxicity. However, the degrees of hepatotoxicity of different PAs are unknown, which makes it difficult to determine a universal threshold of toxic dose of individual PAs for safe regulation of PA-containing natural products. The aim of the present study is to develop a simple and convenient in vitro model to assess the hepatotoxicity of different PAs. MATERIAL AND METHODS: Six common cytotoxicity assays were used to evaluate the hepatotoxicity of different PAs in human hepatocellular carcinoma HepG2 cells. RESULTS: The combination of MTT and bromodeoxyuridine incorporation (BrdU) assays demonstrated to be a suitable method to evaluate the toxic potencies of various PAs in HepG2 cells, and the results indicated that otonecine-type PA (clivorine: IC₂₀=0.013±0.004mM (MTT), 0.066±0.031mM (BrdU)) exhibited significantly higher cytotoxic and anti-proliferative effects than retronecine-type PA (retrorsine: IC₂₀=0.27±0.07mM (MTT), 0.19±0.03mM (BrdU)). While as expected, the known less toxic platyphylline-type PA (platyphylline: IC₂₀=0.85±0.11mM (MTT), 1.01±0.40mM (BrdU)) exhibited significantly less toxicity. The different cytotoxic and anti-proliferative potencies of various PAs in the same retronecine-type could also be discriminated by using the combined MTT and BrdU assays. In addition, the developed assays were further utilized to test alkaloid extract of Gynura segetum, a senecionine and seneciphylline-containing herb, the overall cytotoxicity of two PAs in the extract was comparable to that of these two PAs tested individually. CONCLUSION: Using the developed in vitro model, the cytotoxicity of different PAs and the extract of a PA-containing herb were investigated in parallel in one system, and their different hepatotoxic potencies were determined and directly compared for the first time. The results suggested that the developed model has a great potential to be applied for the quick screening of the toxicity of PAs and PA-containing natural products.