吡咯烷基生物碱（Pyrrolizidine alkaloids, PAs）作为污染物存在于基于植物的食物和草药中。在被细胞色素P450（cytochrome P450, CYP）酶代谢激活后，PAs能引起DNA损伤、肝毒性并在啮齿动物中导致肝癌。有充足证据表明PAs的化学结构决定了其毒性。但是，还需要更多的数量基因毒性数据，尤其是在原代人类肝细胞（primary human hepatocytes, PHH）中。本实验利用体外测试进行了针对十一种不同结构的PAs在CYP3A4过表达的人类HepG2细胞和PHH中的基因毒性研究。此外，还对数据进行基准剂量（benchmark dose, BMD）建模以推导单个PAs的基因毒性效力。首先在HepG2-CYP3A4细胞中测定了细胞毒性，揭示了PAs的结构毒性关系。重要的是，在PHH中的实验验证了被测试的PAs的结构依赖性毒性和细胞毒性效力排序。γH2AX和p53基因毒性标记以及碱性Comet实验一致地证明了HepG2-CYP3A4细胞中PAs的结构依赖性基因毒性，与它们的细胞毒性效力相关。BMD建模得出了大多数环内和开放双酯的BMD值在0.1-10µM的范围内，其次是单酯。虽然Retrorsine（返巢烷）表现出最高的基因毒性效力，但是Monocrotaline（毒地黄碱）和Lycopsamine（狼刺碱）显示了最低的基因毒性。最后，在PHH中的实验证实了基因毒性效力排序，并揭示了即使在没有可检测的细胞毒性的情况下也存在基因毒性效应。总之，我们的发现强烈支持将PAs分为效力类，有助于为风险评估的相对效力因子的广泛接受铺平道路。 © 2023作者。

Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure-toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1-10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment.© 2023. The Author(s).