Oikawa Shinji, Kobayashi Hatasu, Tada-Oikawa Saeko, Isono Yoshiaki, Kawanishi Shosuke
Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507, Japan.
Mutat Res. 2009 Feb 10;661(1-2):93-100. doi: 10.1016/j.mrfmmm.2008.11.016. Epub 2008 Dec 3.
Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H(2)O(2))-resistant clone HP100 cells, suggesting the involvement of H(2)O(2). In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using (32)P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H(2)O(2) with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.
阿司匹林已被提议作为一种可能的化学预防剂。另一方面，最近的一项队列研究表明，阿司匹林可能会增加患胰腺癌的风险。为了阐明阿司匹林是否具有潜在致癌性，我们研究了在经阿司匹林代谢物2,3 -二羟基苯甲酸（2,3 - DHBA）处理的培养细胞中，与癌症发生率相关的8 - 氧代 - 7,8 - 二氢 - 2'-脱氧鸟苷（8 - 氧代 - dG）的形成情况。2,3 - DHBA在人胰腺癌细胞系PANC - 1中诱导了8 - 氧代 - dG的形成。使用PANC - 1细胞的彗星试验也揭示了2,3 - DHBA诱导的DNA单链断裂。流式细胞术分析表明，2,3 - DHBA增加了PANC - 1细胞内活性氧（ROS）的水平。在HL - 60白血病细胞系中也观察到了8 - 氧代 - dG的形成和ROS的产生，但在过氧化氢（H₂O₂）抗性克隆HP100细胞中未观察到，这表明H₂O₂参与其中。此外，次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（hprt）突变试验支持了2,3 - DHBA的致突变性。我们使用人肿瘤抑制基因的³²P标记DNA片段研究了2,3 - DHBA诱导DNA损伤的机制。2,3 - DHBA在Cu(II)和烟酰胺腺嘌呤二核苷酸（NADH）存在的情况下诱导DNA损伤。添加组蛋白肽 - 6 [AKRHRK]可增强2,3 - DHBA诱导的DNA损伤。有趣的是，2,3 - DHBA和组蛋白肽 - 6在p53基因的热点序列5'-ACG - 3'和5'-CCG - 3'中引起碱基损伤。铜试剂（一种Cu(I)螯合剂）和过氧化氢酶抑制了DNA损伤。典型的羟基自由基清除剂没有抑制DNA损伤。这些结果表明，H₂O₂与Cu(I)反应产生的ROS参与了DNA损伤。总之，2,3 - DHBA诱导氧化性DNA损伤和突变，这可能导致致癌作用。
