University of Kaiserslautern, Food Chemistry and Toxicology, Erwin-Schroedinger-Strasse 52, 67663, Kaiserslautern, Germany.
University of Kaiserslautern, Food Chemistry and Toxicology, Erwin-Schroedinger-Strasse 52, 67663, Kaiserslautern, Germany.
Food Chem Toxicol. 2018 Jun;116(Pt B):138-146. doi: 10.1016/j.fct.2018.04.025. Epub 2018 Apr 11.
While alpha-asarone (aA) and beta-asarone (bA) are genotoxic and were shown to be carcinogenic the mechanisms underlying these effects are not understood. Major metabolites of both compounds are epoxides which are mutagenic in the Ames test. We investigated their reactivity towards nucleosides and identified epoxide-derived DNA adducts with 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) using UPLC-UV/VIS, LC-MS/MS and NMR spectroscopy. The adducts were characterized as N-1'-hydroxy-dihydro-asarone-dA and N-1'-hydroxy-dihydro-asarone-dG. Chemical synthesis of these adducts, isotope labeled standards and development of a sensitive and specific isotope dilution mass spectrometric method allowed the quantification of DNA adducts formed in primary rat hepatocytes incubated with aA or bA over up to 48 h. We observed a concentration-dependent, nearly linear formation of DNA adducts, which was higher for bA than for aA. In time course experiments, the amount of DNA adducts reached a maximum within the first 6 h. Over the next 42 h, the amount of DNA adducts decreased, however DNA adducts were still detectable even at the lowest substrate concentration of 10 μM. These results clearly show that aA and bA are able to form epoxide-derived DNA adducts in mammalian cells which may be responsible for their genotoxic, mutagenic and carcinogenic mode of action.
虽然α-细辛脑（aA）和β-细辛脑（bA）具有遗传毒性，并已被证明具有致癌性，但这些影响的机制尚不清楚。这两种化合物的主要代谢物是环氧乙烷，在Ames 试验中具有致突变性。我们研究了它们与核苷的反应性，并使用 UPLC-UV/VIS、LC-MS/MS 和 NMR 光谱鉴定了与 2'-脱氧腺苷（dA）和 2'-脱氧鸟苷（dG）形成的环氧衍生 DNA 加合物。这些加合物被鉴定为 N-1'-羟基二氢细辛脑-dA 和 N-1'-羟基二氢细辛脑-dG。这些加合物的化学合成、同位素标记标准品的开发以及灵敏和特异的同位素稀释质谱方法的建立，允许定量分析在原代大鼠肝细胞中孵育 aA 或 bA 长达 48 小时形成的 DNA 加合物。我们观察到 DNA 加合物的形成呈浓度依赖性，几乎呈线性，bA 比 aA 更高。在时间过程实验中，DNA 加合物的量在最初的 6 小时内达到最大值。在接下来的 42 小时内，DNA 加合物的量减少，但即使在最低的 10 μM 底物浓度下，仍能检测到 DNA 加合物。这些结果清楚地表明，aA 和 bA 能够在哺乳动物细胞中形成环氧衍生的 DNA 加合物，这可能是它们遗传毒性、致突变性和致癌作用的机制。
