Abstract
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
Highlights
Phytochemicals from vegetable diet and pharmaceutical products are usually considered safe
We performed analyses of transcriptomic data using Ingenuity Pathway Analysis software (IPA) tool for comparing different datasets represented as different concentrations
We found an increase in the expression of genes coding for vanin 1 (VNN1), matrix metallopeptidase 7 (MMP7), cathepsin E (CTSE), laminin subunit α3 (LAMA3), cytoglobin (CYGB), sulfatase 2 (SULF2), and keratin 23 (KRT23) as well as a downregulation of the gene coding for H2B clustered histone 14 (HIST1H2BM)
Summary
Phytochemicals from vegetable diet and pharmaceutical products are usually considered safe. Nutritional products and herbal medicines can be contaminated with highly toxic compounds, including pyrrolizidine alkaloids (PAs). They are hepatotoxic and carcinogenic even at very low doses, and their intake has to be minimized (Bode and Dong 2015; Schrenk et al 2020). This is a large class of more than 600 natural compounds, occurring in estimated 6000 plant species, representing 2% of all flowering plants. The basic structure consists of a necine base coupled with one or two necine acids by ester linkages They are classified based on the necine saturation either as fully saturated or 1,2-unsaturated PAs (Schrenk et al 2020). PAs play a major role as plant defense mechanisms against fungal or bacterial infections and against attacks by mammalian herbivores and insects (Hartmann, 1994; Hartmann 1999)
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