Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany.
Molecular Cell Biology, Brandenburg University of Technology, Senftenberg, Germany.
Toxicology. 2020 Nov;444:152566. doi: 10.1016/j.tox.2020.152566. Epub 2020 Aug 25.
Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N-(isoestragole-3'-yl)-2'-desoxyguanosine (E3'NdG) and N-(isoestragole-3'-yl)-desoxyadenosine (E3'NdA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3'NdA at 10 μM, E3'NdG at 1μM estragole). E3'NdG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 μM after 24 h and < 0.5 μM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 μM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 μM estragole led to a 3.2 fold and 300 μM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a 'practical threshold' dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.
黄樟素是草药和香料中的天然成分，也是精油或草药茶等产品的成分。在细胞色素 P450 催化的羟化和随后的磺化作用后，黄樟素作为一种遗传毒性肝致癌物，在啮齿动物肝脏中形成 DNA 加合物。由于遗传毒性作用模式和在食品和植物药中的广泛存在，需要对黄樟素进行精细的风险评估。我们分析了 DNA 加合物 N-(异黄樟素-3'-基)-2'-脱氧鸟苷（E3'NdG）和 N-(异黄樟素-3'-基）-脱氧腺苷（E3'NdA）在原代培养的大鼠肝细胞（pRH）中的时间和浓度依赖性水平，这些加合物据报道是大鼠肝脏中形成的主要加合物。通过使用稳定同位素稀释分析的 UHPLC-ESI-MS/MS 测量 DNA 加合物水平。两种加合物均在 pRH 中形成，在孵育 1 小时后即可定量（E3'NdA 在 10 μM，E3'NdG 在 1μM 黄樟素）。E3'NdG 是所有孵育时间和浓度下的主要加合物，在 24 小时后可以在低于 0.1 μM 的黄樟素浓度下检测到，在 48 小时后可以在低于 0.5 μM 的黄樟素浓度下检测到。加合物水平在 6 小时时最高，随后在稍后的时间点呈下降趋势，可能是由于 DNA 修复和/或细胞凋亡。虽然加合物形成的浓度反应特征在整个浓度范围内明显呈线性，但当仅基于 <1 μM 的浓度进行建模时，明显存在显著的次线性。在 HepG2 细胞的微核试验中，未发现黄樟素具有致突变性，而在 HepG2-CYP1A2 细胞中，1 μM 黄樟素导致微核数增加 3.2 倍，300 μM 黄樟素导致微核数增加 7.1 倍。我们的研究结果表明，作为黄樟素致癌性的起始关键事件，DNA 加合物形成存在一个“实用阈值”剂量，这表明浓度-反应线性的默认假设是值得怀疑的，至少对于我们在这里研究的两种主要加合物是如此。
