Byrnes R W, Antholine W E, Petering D H
Department of Chemistry, University of Wisconsin, Milwaukee 53201.
Free Radic Biol Med. 1992 Nov;13(5):469-78. doi: 10.1016/0891-5849(92)90141-3.
The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.
研究了2,9-二甲基-1,10-菲咯啉（新铜试剂或NC）及其铜配合物与艾氏腹水瘤细胞的相互作用。在通过菲咯啉铜进行体外DNA降解的研究中，NC经常用作阴性对照，并且还被发现可作为生物系统中氧化应激损伤的潜在抑制剂。在单层培养中，经过48小时处理，0.05 nmol/10⁵个细胞的NC可抑制艾氏细胞生长50%。添加5至100倍比例的CuCl₂与NC（0.035 nmol NC/10⁵个细胞）会产生逐渐增强的生长抑制作用。在0.08 - 0.2 nmol/10⁵个细胞/mL的NC浓度范围内，添加1:0.5比例的NC与CuCl₂，经1小时处理后，通过DNA碱性洗脱法测定，会导致DNA单链断裂。同时添加过氧化氢酶或二甲基亚砜（DMSO）可抑制DNA链断裂，而超氧化物歧化酶则增强断裂。过氧化氢酶和DMSO也可抑制NC铜配合物诱导的膜通透性。这些细胞效应显然是由细胞内H₂O₂产生的羟基自由基引起的。NC促进了铜进入细胞，尽管它最初是以类似铜 - 组氨酸的配合物形式结合的。内化的铜被还原为Cu(I)，主要以(NC)₂Cu(I)的形式结合。为了解释(NC)₂Cu依赖的羟基自由基生成，推测谷胱甘肽成功竞争Cu(I)，将其转化为可催化分子氧还原为·OH的氧化还原活性形式。模型研究支持这一观点。自由基清除剂不能逆转由NC或NC + CuCl₂产生的生长抑制作用。
