Interstrand DNA cross-linking has been considered to be the primary action mechanism of cyclophosphamide (CP) and its hydroperoxide derivative, 4-hydroperoxycyclophosphamide (4-HC). To clarify the mechanism of anti-tumor effects by 4-HC, we investigated DNA damage in a human leukemia cell line, HL-60, and its H(2)O(2)-resistant clone HP100. Apoptosis DNA ladder formation was detected in HL-60 cells treated with 4-HC, whereas it was not observed in HP100 cells. 4-HC significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, a marker of oxidative DNA damage, in HL-60 cells. On the other hand, CP did not significantly induce 8-oxodG formation and apoptosis in HL-60 cells under the same conditions as did 4-HC. Using (32)P-labeled DNA fragments from the human p53 tumor suppressor gene, 4-HC was found to cause Cu(II)-mediated oxidative DNA damage, but CP did not. Catalase inhibited 4-HC-induced DNA damage, including 8-oxodG formation, suggesting the involvement of H(2)O(2). The generation of H(2)O(2) during 4-HC degradation was ascertained by procedures using scopoletin and potassium iodide. We conclude that, in addition to DNA cross-linking, oxidative DNA damage through H(2)O(2) generation may participate in the anti-tumor effects of 4-HC.

中文翻译：

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环磷酰胺的氢过氧化物衍生物引起的氧化性DNA损伤。

链间DNA交联被认为是环磷酰胺（CP）及其氢过氧化物衍生物4-氢过氧环磷酰胺（4-HC）的主要作用机理。为了阐明4-HC的抗肿瘤作用机理，我们研究了人类白血病细胞系HL-60及其对H（2）O（2）耐药的克隆HP100的DNA损伤。在用4-HC处理的HL-60细胞中检测到凋亡DNA梯形形成，而在HP100细胞中未观察到。4-HC在HL-60细胞中显着增加了8-oxo-7,8-dihydro-2'-deoxyguanosine（8-oxodG）的形成，这是氧化DNA损伤的标志。另一方面，在与4-HC相同的条件下，CP在HL-60细胞中没有显着诱导8-oxodG的形成和凋亡。使用来自人类p53抑癌基因的（32）P标记的DNA片段，发现4-HC会导致Cu（II）介导的氧化DNA损伤，但CP不会。过氧化氢酶抑制4-HC诱导的DNA损伤，包括8-oxodG的形成，表明H（2）O（2）的参与。通过使用碱和碘化钾的程序确定了4-HC降解过程中H（2）O（2）的产生。我们得出的结论是，除了DNA交联之外，通过H（2）O（2）生成的氧化DNA损伤可能参与了4-HC的抗肿瘤作用。