Horibata Katsuyoshi, Ukai Akiko, Ishikawa Shigeo, Sugano Ayako, Honma Masamitsu
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Mutat Res Genet Toxicol Environ Mutagen. 2016 Sep 15;808:20-6. doi: 10.1016/j.mrgentox.2016.08.002. Epub 2016 Aug 10.
The recently introduced Pig-a in vivo gene mutation assay measures endogeneous mutations of Pig-a (human, PIG-A), an X-linked gene that is conserved across species from rodents to humans. Flow cytometric analysis enables the enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient erythrocytes, resulting from a mutation in Pig-a/PIG-A, in only a few microliters of peripheral blood. Pig-a/PIG-A mutations appear to function in a neutral manner, allowing evaluation of the accumulated genotoxic effects of repeated exposures. To date, most Pig-a studies have been conducted in rodents; only a few reports regarding human applications of the PIG-A assay have been published. We have conducted a PIG-A assay in the context of human genotoxicity monitoring. Peripheral blood was collected from healthy human donors and chemotherapy-treated cancer patients at Yamagata University Hospital. To investigate the PIG-A mutant frequency (MF) induced by chemotherapy, red blood cells were analyzed via flow cytometry following staining with allophycocyanin-conjugated anti-CD235ab (erythrocyte specific) and fluorescein isothiocyanate-conjugated anti-CD59 antibodies (GPI-anchored protein specific). Reticulocyte frequencies (%RET) were also analyzed using a phycoerythrin-conjugated anti-CD71 antibody to monitor bone marrow suppression and reticulocytosis. Two of 27 patients exhibited a significantly elevated frequency of PIG-A mutants. Although we observed either a reduced or an increased %RET in all patients, no association was observed between this factor and the PIG-A MF. Unfortunately, we could not analyze blood samples collected before treatment during therapeutic processes. Additionally, the sampling time point for some patients was too short to express the PIG-A mutant phenotypes. Therefore, the possibility of natively high PIG-A MFs prior to treatment must be considered. The human PIG-A assay shows promise as a human genotoxicity monitoring method.
最近引入的体内Pig-a基因突变试验可检测Pig-a（人类为PIG-A）的内源性突变，Pig-a是一种X连锁基因，在从啮齿动物到人类的物种中都保守。流式细胞术分析能够在仅几微升外周血中计数因Pig-a/PIG-A突变而产生的糖基磷脂酰肌醇（GPI）锚定缺陷红细胞。Pig-a/PIG-A突变似乎以中性方式起作用，从而能够评估重复暴露积累的遗传毒性效应。迄今为止，大多数Pig-a研究是在啮齿动物中进行的；关于PIG-A试验在人类中的应用仅有少数报道发表。我们在人类遗传毒性监测背景下开展了PIG-A试验。从山形大学医院的健康人类供体和接受化疗的癌症患者采集外周血。为了研究化疗诱导的PIG-A突变频率（MF），在用别藻蓝蛋白偶联的抗CD235ab（红细胞特异性）和异硫氰酸荧光素偶联的抗CD59抗体（GPI锚定蛋白特异性）染色后，通过流式细胞术分析红细胞。还使用藻红蛋白偶联的抗CD71抗体分析网织红细胞频率（%RET），以监测骨髓抑制和网织红细胞增多。27例患者中有2例显示PIG-A突变体频率显著升高。尽管我们在所有患者中都观察到%RET降低或升高，但未观察到该因素与PIG-A MF之间存在关联。遗憾的是，我们无法在治疗过程中分析治疗前采集的血样。此外，一些患者的采样时间点过短，无法表达PIG-A突变表型。因此，必须考虑治疗前PIG-A MF天然就高的可能性。人类PIG-A试验有望成为一种人类遗传毒性监测方法。
