Takatsuka Reine, Ito Shunsuke, Iwai Shigenori, Kuraoka Isao
Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka, Japan.
Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka, Japan.
Mutat Res Genet Toxicol Environ Mutagen. 2017 Aug;820:1-7. doi: 10.1016/j.mrgentox.2017.05.009. Epub 2017 May 22.
Biochemical risk assessment studies of chemicals that induce DNA lesions are important, because lesions in genomic DNA frequently result in cancer, neurodegeneration, and aging in humans. Many classes of DNA lesions induced by chemical agents are eliminated via DNA repair mechanisms, such as nucleotide excision repair (NER) and base excision repair (BER), for the maintenance of genomic integrity. Individuals with NER-defective xeroderma pigmentosum (XP), in which bulky DNA lesions are not efficiently removed, are cancer-prone and suffer neurodegeneration. For research into cancer and neurological diseases, therefore, it might be important to identify DNA damage from agents that induce NER-repairable bulky DNA lesions. However, simple and quick assays to detect such damaging agents have not been developed using human cells. Here, we report a simple, non-isotopic assay for determining DNA damaging agents that induce NER-repairable DNA lesions by visualizing gene expression from treated fluorescent protein vectors in a mammalian cell system. This assay is based on a comparison of fluorescent protein expression in NER-proficient and NER-deficient cells. When we tested UV-irradiated fluorescent protein vectors, the fluorescent protein was observed in NER-proficient cells, but not in NER-deficient cells. Similar results were obtained for vectors treated with the anticancer drug, cisplatin. In contrast, when treated with the DNA alkylating agent methyl methanesulfonate, believed to cause BER-repairable damage, no difference in gene expression between NER-proficient and NER-deficient cells was observed. These results suggest that our assay can specifically detect DNA-damaging agents that induce NER-repairable DNA lesions, and could be used to analyze chemicals with the potential to cause cancer and neurological diseases. With further validation, the assay might be also applicable to XP diagnosis.
对诱导DNA损伤的化学物质进行生化风险评估研究很重要，因为基因组DNA损伤常导致人类患癌症、神经退行性疾病和衰老。化学试剂诱导的许多类DNA损伤可通过DNA修复机制消除，如核苷酸切除修复（NER）和碱基切除修复（BER），以维持基因组完整性。患有NER缺陷的着色性干皮病（XP）的个体无法有效去除大块DNA损伤，易患癌症并遭受神经退行性病变。因此，对于癌症和神经疾病的研究而言，识别诱导NER可修复的大块DNA损伤的化学物质所造成的DNA损伤可能很重要。然而，尚未开发出利用人类细胞检测此类损伤性化学物质的简单快速检测方法。在此，我们报告一种简单的非同位素检测方法，通过观察哺乳动物细胞系统中经处理的荧光蛋白载体的基因表达，来确定诱导NER可修复DNA损伤的DNA损伤剂。该检测方法基于比较NER功能正常和NER缺陷细胞中荧光蛋白的表达。当我们检测紫外线照射的荧光蛋白载体时，在NER功能正常的细胞中观察到了荧光蛋白，而在NER缺陷的细胞中未观察到。用抗癌药物顺铂处理的载体也得到了类似结果。相反，当用据信会导致BER可修复损伤的DNA烷化剂甲磺酸甲酯处理时，NER功能正常和NER缺陷细胞之间未观察到基因表达差异。这些结果表明，我们的检测方法可以特异性地检测诱导NER可修复DNA损伤的DNA损伤剂，并可用于分析具有导致癌症和神经疾病潜力的化学物质。经过进一步验证，该检测方法可能也适用于XP诊断。
