背景:马兜铃酸(AAs)是天然存在的硝基菲羧酸,主要存在于马兜铃科植物中。马兜铃酸D(AAD)是中草药西新(细辛的根和根茎)的根和根茎中的主要成分,这是准备一套上市中药的关键材料。在结构上,AAD与肾毒性马兜铃酸I(AAI)几乎相同,在C-6位点有一个额外的酚基团。尽管AAI的肾毒性和代谢途径已经得到了很好的研究,AAD在人类中的代谢途径以及AAD代谢对其肾毒性的影响尚未被研究。
目的:为了鉴定人组织中AAD的主要代谢产物,并表征不同酶源的AADO-葡糖醛酸化动力学,以及探讨AADO-葡糖醛酸化对其肾毒性的影响。
方法:生物合成了AAD的O-葡糖苷酸,其化学结构通过1H-NMR和13C-NMR充分表征。反应表型分析,化学抑制试验,进行了酶动力学分析,以评估参与人类AADO-葡糖醛酸化的关键酶。进行对接模拟以模拟人UDP-葡糖醛酸基转移酶(UGT)中AAD的催化构象,同时测量了每个测试的人UGT同工酶中AAD的去质子化C-6酚基与UDPGA的葡糖醛酸基部分之间的预测结合能和距离。用AAI处理的HK-2细胞中的线粒体膜电位(MMP)和活性氧(ROS)水平,或AAD,或AADO-葡糖苷酸进行了测试,阐明O-葡糖醛酸化对AAD肾毒性的影响。
结果:AAD可以在人的肝脏和肠道微粒体中快速代谢(HLM和HIM,分别)形成单葡糖苷酸,将其纯化并通过NMR完全表征为AAD-6-O-β-D-葡糖苷酸(AADG)。UGT1A1是负责AAD-6-O-葡糖醛酸化的主要酶,而UGT1A9的贡献程度较小。HLM中的AAD-6-O-葡糖醛酸化,他,UGT1A1和UGT1A9遵循米氏-门顿动力学,Km值为4.27μM,9.05μM,3.87μM,和7.00μM,分别。对接模拟表明AAD可进入UGT1A1或UGT1A9的催化腔并形成催化构象。进一步的研究表明,AAI和AAD都可以触发细胞内ROS水平升高并诱导线粒体功能障碍,在HK-2细胞中,但AADG很难引发ROS积累和线粒体功能障碍。
结论:总的来说,UGT1A催化的AAD6-O-葡糖醛酸化代表了这种天然存在的AAI类似物在人类中的关键解毒途径,这与AAI非常不同。
BACKGROUND: Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet.
OBJECTIVE: To identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity.
METHODS: The O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD.
RESULTS: AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-β-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 μM, 9.05 μM, 3.87 μM, and 7.00 μM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction.
CONCLUSIONS: Collectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.