Zhitkovich A, Song Y, Quievryn G, Voitkun V
Brown University, Department of Pathology and Laboratory Medicine, Providence, Rhode Island 02912, USA.
Biochemistry. 2001 Jan 16;40(2):549-60. doi: 10.1021/bi0015459.
Intracellular reduction of carcinogenic Cr(VI) generates Cr-DNA adducts formed through the coordination of Cr(III) to DNA phosphates (phosphotriester-type adduct). Here, we examined the role of Cr(III)-DNA adducts in mutagenesis induced by metabolism of Cr(VI) with cysteine. Reduction of Cr(VI) caused a strong oxidation of 2', 7'-dichlorofluoroscin (DCFH) and extensive Cr-DNA binding but no DNA breakage. Cr-DNA adducts induced unwinding of supercoiled plasmids and structural distortions in the DNA helix as detected by decreased ethidium bromide binding. Propagation of Cr-treated pSP189 plasmids in human fibroblasts led to a dose-dependent formation of the supF mutants and inhibition of replication. Blocking of Cr(III)-DNA binding by occupation of DNA phosphates with Mg(2+) or by sequestration of Cr(III) by inorganic phosphate or EDTA eliminated mutagenic responses and restored a normal yield of replicated plasmids. Dissociation of Cr(III) from DNA by a phosphate-based reversal procedure returned mutation frequency to background levels. The mutagenic responses at the different phases of the reduction reaction were unrelated to the amount of reduced Cr(VI) but reflected the number and the spectrum of Cr(III)-DNA adducts that were formed. Ternary cysteine-Cr(III)-DNA adducts were approximately 4-5 times more mutagenic than binary Cr(III)-DNA adducts. Although intermediate reaction products (CrV/IV, thiyl radicals) were capable of oxidizing DCFH, they were insufficiently reactive to damage DNA. Single-base substitutions at G/C pairs were the predominant type of Cr-induced mutations. The majority of mutations occurred at the sites where G had adjacent purine in the 3' or 5' position. Overall, our results present the first evidence that Cr(III)-DNA adducts play the dominant role in the mutagenicity caused by the metabolism of Cr(VI) by a biological reducing agent.
致癌性六价铬（Cr(VI)）在细胞内的还原会生成通过三价铬（Cr(III)）与DNA磷酸酯配位形成的铬-DNA加合物（磷酸三酯型加合物）。在此，我们研究了Cr(III)-DNA加合物在六价铬与半胱氨酸代谢诱导的诱变中的作用。六价铬的还原导致2',7'-二氯荧光素（DCFH）强烈氧化以及广泛的铬-DNA结合，但未导致DNA断裂。如通过溴化乙锭结合减少所检测到的，铬-DNA加合物诱导超螺旋质粒解旋以及DNA螺旋结构扭曲。经铬处理的pSP189质粒在人成纤维细胞中的复制导致剂量依赖性的supF突变体形成并抑制复制。通过用镁离子（Mg(2+)）占据DNA磷酸酯或通过无机磷酸或乙二胺四乙酸（EDTA）螯合三价铬来阻断Cr(III)-DNA结合，消除了诱变反应并恢复了复制质粒的正常产量。通过基于磷酸盐的逆转程序使三价铬从DNA上解离，使突变频率恢复到背景水平。还原反应不同阶段的诱变反应与还原的六价铬量无关，而是反映了所形成的Cr(III)-DNA加合物的数量和谱型。三元半胱氨酸-Cr(III)-DNA加合物的诱变性比二元Cr(III)-DNA加合物大约高4-5倍。尽管中间反应产物（CrV/IV、硫自由基）能够氧化DCFH，但它们的反应性不足以损伤DNA。鸟嘌呤/胞嘧啶（G/C）碱基对处的单碱基替换是铬诱导突变的主要类型。大多数突变发生在鸟嘌呤在3'或5'位置有相邻嘌呤的位点。总体而言，我们的结果首次证明Cr(III)-DNA加合物在生物还原剂对六价铬代谢所致的诱变性中起主导作用。
