DNA碱基加合物M1G的体内和体外代谢。,Chemical Research in Toxicology

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DNA碱基加合物M1G的体内和体外代谢。
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2007 Mar , DOI: 10.1021/tx600334x
Charles G. Knutson ₁ , Dapo Akingbade ₁ , Brenda C. Crews ₁ , Markus Voehler ₁ , Donald F. Stec ₁ , Lawrence J. Marnett ₁

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氧化损伤被认为是导致包括癌症在内的遗传疾病的主要因素。我们的实验室正在评估内源性形成的DNA加合物作为氧化损伤的基因组生物标记。最近的努力集中在研究加合物在体外和体内的代谢稳定性。在这里，我们证明了碱性加合物M1G在体外在大鼠肝细胞溶质（RLC，Km = 105 microM和vmax / Km = 0.005 min-1 mg-1）中以及在静脉内给予雄性Sprague Dawley时经历氧化代谢大鼠。LC-MS分析揭示了两种代谢物，其中包含连续添加的16 amu。一维和二维NMR实验表明，氧化首先在嘧啶环的6位发生，形成6-oxo-M1G，然后在咪唑环的2位发生，生成2,6-dioxo-M1G 。化学合成了真实的6-oxo-M1G，并在RLC中观察到代谢为2,6-dioxo-M1G（Km = 210 microM，vmax / Km = 0.005 min-1 mg-1）。别嘌醇在RLC中部分抑制M1G代谢（75％），并完全抑制6-oxo-M1G代谢。这些抑制研究表明，黄嘌呤氧化酶是作用于RLC中M1G的主要酶，并且是唯一将6-oxo-M1G转化为2,6-dioxo-M1G的酶。M1G和6-oxo-M1G在RLC中都是比脱氧核苷M1dG更好的氧化代谢底物（5倍）。M1dG的替代修复途径或生物加工过程使感兴趣的M1G的命运成为体内氧化损伤的潜在标志。别嘌醇在RLC中部分抑制M1G代谢（75％），并完全抑制6-oxo-M1G代谢。这些抑制研究表明，黄嘌呤氧化酶是作用于RLC中M1G的主要酶，并且是唯一将6-oxo-M1G转化为2,6-dioxo-M1G的酶。M1G和6-oxo-M1G在RLC中都是比脱氧核苷M1dG更好的氧化代谢底物（5倍）。M1dG的替代修复途径或生物加工过程使感兴趣的M1G的命运成为体内氧化损伤的潜在标志。别嘌醇在RLC中部分抑制M1G代谢（75％），并完全抑制6-oxo-M1G代谢。这些抑制研究表明，黄嘌呤氧化酶是作用于RLC中M1G的主要酶，并且是唯一将6-oxo-M1G转化为2,6-dioxo-M1G的酶。M1G和6-oxo-M1G在RLC中都是比脱氧核苷M1dG更好的氧化代谢底物（5倍）。M1dG的替代修复途径或生物加工过程使感兴趣的M1G的命运成为体内氧化损伤的潜在标志。M1G和6-oxo-M1G在RLC中比脱氧核苷M1dG更好的是氧化代谢的底物（5倍）。M1dG的替代修复途径或生物加工过程使感兴趣的M1G的命运成为体内氧化损伤的潜在标志。M1G和6-oxo-M1G在RLC中都是比脱氧核苷M1dG更好的氧化代谢底物（5倍）。M1dG的替代修复途径或生物加工过程使感兴趣的M1G的命运成为体内氧化损伤的潜在标志。

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Metabolism in vitro and in vivo of the DNA base adduct, M1G.

Oxidative damage is considered a major contributing factor to genetic diseases including cancer. Our laboratory is evaluating endogenously formed DNA adducts as genomic biomarkers of oxidative injury. Recent efforts have focused on investigating the metabolic stability of adducts in vitro and in vivo. Here, we demonstrate that the base adduct, M1G, undergoes oxidative metabolism in vitro in rat liver cytosol (RLC, Km = 105 microM and vmax/Km = 0.005 min-1 mg-1) and in vivo when administered intravenously to male Sprague Dawley rats. LC-MS analysis revealed two metabolites containing successive additions of 16 amu. One- and two-dimensional NMR experiments showed that oxidation occurred first at the 6-position of the pyrimido ring, forming 6-oxo-M1G, and then at the 2-position of the imidazole ring, yielding 2,6-dioxo-M1G. Authentic 6-oxo-M1G was chemically synthesized and observed to undergo metabolism to 2,6-dioxo-M1G in RLC (Km = 210 microM and vmax/Km = 0.005 min-1 mg-1). Allopurinol partially inhibited M1G metabolism (75%) and completely inhibited 6-oxo-M1G metabolism in RLC. These inhibition studies suggest that xanthine oxidase is the principal enzyme acting on M1G in RLC and the only enzyme that converts 6-oxo-M1G to 2,6-dioxo-M1G. Both M1G and 6-oxo-M1G are better substrates (5-fold) for oxidative metabolism in RLC than the deoxynucleoside, M1dG. Alternative repair pathways or biological processing of M1dG makes the fate of M1G of interest as a potential marker of oxidative damage in vivo.

更新日期：2017-01-31

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