Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs.

Graphical abstract

中文翻译：

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通过细胞色素 P450 3A4 过表达 HepG2 克隆 9 细胞的转录组学分析，吡咯里西啶生物碱导致细胞周期和 DNA 损伤修复缺陷

吡咯里西啶生物碱 (PAs) 是一大类剧毒化合物，在许多食品（例如蜂蜜）、膳食补充剂、凉茶和药用草药中被发现为交叉污染物。PA 污染是造成严重肝毒性和肝癌发生的原因。卫生当局必须设定法定限值，以保证安全食用植物性营养和医疗产品而不会损害健康。毒理学和化学分析方法通常用于确定法律允许的 PA 限值。在本研究中，我们应用了一种高度敏感的转录组学方法来研究低浓度的五种 PA（拉西奥卡品、瑞德林、番茄红素、棘脒、和野百合碱）对人细胞色素 P450 3A4 过表达 HepG2 克隆 9 肝细胞的影响。失调基因表达的转录组学分析表明，PAs 破坏了转染肝细胞中与细胞周期调节和 DNA 损伤修复相关的重要信号通路，这可能解释了 PA 的致癌作用。由于 PA 影响参与细胞周期调节的基因的表达，我们应用流式细胞术细胞周期分析来验证转录组数据。有趣的是，PA 处理导致细胞周期的 S 期停滞，与毒性较低的野百合碱相比，毒性更大的 PA（即拉西奥卡品和瑞德林）更明显。使用免疫荧光，在 PA 处理后检测到高比例的细胞存在染色体聚集缺陷，表明有丝分裂失败。总之，测试的 PA 揭示了阈值浓度，高于该浓度，关键的信号通路被解除管制，导致细胞损伤和致癌。细胞周期停滞和 DNA 损伤修复指向 PA 的致突变性。染色体聚集的紊乱是Pas的一种新机制，它也可能有助于PA介导的致癌作用。转录组学、细胞周期和免疫荧光分析应补充毒理学的标准技术，以揭示肝细胞中 PA 暴露作为 PA 代谢过程中的主要目标的生物学效应。细胞周期停滞和 DNA 损伤修复指向 PA 的致突变性。染色体聚集的紊乱是Pas的一种新机制，它也可能有助于PA介导的致癌作用。转录组学、细胞周期和免疫荧光分析应补充毒理学的标准技术，以揭示肝细胞中 PA 暴露作为 PA 代谢过程中的主要目标的生物学效应。细胞周期停滞和 DNA 损伤修复指向 PA 的致突变性。染色体聚集的紊乱是Pas的一种新机制，它也可能有助于PA介导的致癌作用。转录组学、细胞周期和免疫荧光分析应补充毒理学的标准技术，以揭示肝细胞中 PA 暴露作为 PA 代谢过程中的主要目标的生物学效应。

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