Byrnes R W, Petering D H
Department of Chemistry, University of Wisconsin-Milwaukee 53201.
Biochem Pharmacol. 1994 Aug 3;48(3):575-82. doi: 10.1016/0006-2952(94)90288-7.
The sources of iron (Fe) and reductant required for DNA strand breakage by the antitumor drug bleomycin (Blm), H2O2 and ascorbate were investigated using nuclei instead of whole cells in order to study a simpler, related system that was subject to better control and easier chemical manipulation. Ehrlich ascites tumor cells were isolated and treated directly on filters, and analysed for DNA damage by alkaline and nondenaturing elution. Extraction and treatment buffers were depleted of trace Fe by passage through Mg(OH)2 gel. Nuclei were treated for 1 hr at 37 degrees. High levels of single- and double-strand breakage were obtained using Fe(III)Blm in the range 0.01 to 0.08 microM. In contrast, Blm was effective only at two orders of magnitude greater concentration. Cu(II)Blm was totally ineffective in causing damage. Depletion of nuclear protein thiols with N-ethylmaleimide reduced double-strand breakage at the upper end of the FeBlm concentration-response curve. A 1 mM concentration of NADPH or NADH greatly increased the extent of double-strand breakage by 0.01 microM FeBlm, suggesting roles for cytochrome P450 or cytochrome b5 reductase in strand breakage. Fe(III)ATP (1:20 metal to ligand and 50 microM in Fe) and Fe(III)EDTA (1:2 metal to ligand and 50 microM in Fe) did not cause single-strand breaks. In the absence of added Fe, H2O2 or ascorbic acid (50 microM) caused less than one Gy-equivalent single-strand breakage. Addition of ascorbate plus Fe(III)ATP or Fe(III)EDTA produced breakage beyond the capacity of alkaline elution to analyse (5-6 Gy). Overall, the results indicate that Fe, which may contribute to DNA damage by Blm and forms of activated oxygen within cells, is not strongly bound in the nucleus and that nuclear thiols other than glutathione contribute reducing equivalents to Fe(III)Blm for the DNA damaging chemistry.
为了研究一个更简单、更易控制且便于化学操作的相关系统，使用细胞核而非完整细胞来研究抗肿瘤药物博来霉素（Blm）、过氧化氢（H₂O₂）和抗坏血酸导致DNA链断裂所需的铁（Fe）和还原剂来源。分离出艾氏腹水瘤细胞并直接在滤膜上进行处理，通过碱性和非变性洗脱分析DNA损伤情况。提取和处理缓冲液通过Mg(OH)₂凝胶过滤以去除痕量铁。细胞核在37℃处理1小时。使用浓度范围为0.01至0.08微摩尔的Fe(III)Blm可获得高水平的单链和双链断裂。相比之下，Blm仅在浓度高两个数量级时才有效。Cu(II)Blm在造成损伤方面完全无效。用N-乙基马来酰亚胺耗尽核蛋白硫醇会降低FeBlm浓度-反应曲线上限处的双链断裂。1毫摩尔浓度的NADPH或NADH极大地增加了0.01微摩尔FeBlm导致的双链断裂程度，表明细胞色素P450或细胞色素b5还原酶在链断裂中起作用。Fe(III)ATP（金属与配体比例为1:20且铁浓度为50微摩尔）和Fe(III)EDTA（金属与配体比例为1:2且铁浓度为50微摩尔）不会导致单链断裂。在不添加铁的情况下，H₂O₂或抗坏血酸（50微摩尔）导致的单链断裂小于1戈瑞当量。添加抗坏血酸加Fe(III)ATP或Fe(III)EDTA产生的断裂超出了碱性洗脱分析能力（5 - 6戈瑞）。总体而言，结果表明，可能导致Blm和细胞内活性氧形式造成DNA损伤的铁，在细胞核中结合不紧密，并且除谷胱甘肽外的核硫醇为Fe(III)Blm的DNA损伤化学过程提供还原当量。
