Siegel D, Gibson N W, Preusch P C, Ross D
Molecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado, Boulder 80309.
Cancer Res. 1990 Dec 1;50(23):7483-9.
The role of DT-diaphorase in bioreductive activation of mitomycin C was examined using HT-29 and BE human carcinoma cells which have high and low levels of DT-diaphorase activity, respectively. HT-29 cells were more sensitive to mitomycin C-induced cytotoxicity than the DT-diaphorase-deficient BE cell line. Mitomycin C induced DNA interstrand cross-linking in HT-29 cells but not in BE cells. Both mitomycin C-induced cytotoxicity and induction of DNA interstrand cross-links could be inhibited by pretreatment of HT-29 cells with dicoumarol. Metabolism of mitomycin C by HT-29 cell cytosol was pH dependent and increased as the pH was lowered to 5.8, the lowest pH tested. Metabolism of mitomycin C by HT-29 cytosol was inhibited by prior boiling of cytosol or by the inclusion of dicoumarol. Little metabolism was detected in BE cytosols. When purified rat hepatic DT-diaphorase was used, metabolism of mitomycin C increased as the pH was decreased and could be detected at pH 5.8, 6.4, 7.0, 7.4, but not at 7.8. Metabolism of mitomycin C was NADH dependent and inhibited by dicoumarol or by prior boiling of enzyme. An approximate 1:1 stoichiometry between NADH and mitomycin C removal was demonstrated and no oxygen consumption could be detected. Metabolism of mitomycin C by purified HT-29 DT-diaphorase was also dicoumarol inhibitable and pH dependent. The major metabolite formed during metabolism of mitomycin C by HT-29 cytosol, purified HT-29, and rat hepatic DT-diaphorase was characterized as 2,7-diaminomitosene. These data suggest that two-electron reduction of mitomycin C by DT-diaphorase may be an important determinant of mitomycin C-induced genotoxicity and cytotoxicity.
利用分别具有高和低水平 DT-黄递酶活性的 HT-29 和 BE 人癌细胞，研究了 DT-黄递酶在丝裂霉素 C 生物还原激活中的作用。HT-29 细胞比缺乏 DT-黄递酶的 BE 细胞系对丝裂霉素 C 诱导的细胞毒性更敏感。丝裂霉素 C 在 HT-29 细胞中诱导 DNA 链间交联，但在 BE 细胞中不诱导。用双香豆素预处理 HT-29 细胞可抑制丝裂霉素 C 诱导的细胞毒性和 DNA 链间交联的诱导。HT-29 细胞胞质溶胶对丝裂霉素 C 的代谢呈 pH 依赖性，随着 pH 降至 5.8（测试的最低 pH）而增加。HT-29 胞质溶胶对丝裂霉素 C 的代谢可被胞质溶胶预先煮沸或加入双香豆素所抑制。在 BE 胞质溶胶中几乎检测不到代谢。当使用纯化的大鼠肝 DT-黄递酶时，丝裂霉素 C 的代谢随着 pH 降低而增加，在 pH 5.8、6.4、7.0、7.4 时可检测到，但在 7.8 时未检测到。丝裂霉素 C 的代谢依赖于 NADH，并被双香豆素或酶预先煮沸所抑制。证明了 NADH 与丝裂霉素 C 去除之间的近似 1:1 化学计量关系，且未检测到氧消耗。纯化的 HT-29 DT-黄递酶对丝裂霉素 C 的代谢也可被双香豆素抑制且呈 pH 依赖性。HT-29 胞质溶胶、纯化的 HT-29 和大鼠肝 DT-黄递酶在丝裂霉素 C 代谢过程中形成的主要代谢产物被鉴定为 2,7-二氨基丝裂霉素。这些数据表明，DT-黄递酶对丝裂霉素 C 的双电子还原可能是丝裂霉素 C 诱导的遗传毒性和细胞毒性的重要决定因素。
