Pharmaceutical Development Research Laboratories, Teijin Institute for Bio-medical Research, Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo 191-8512, Japan.
Mutat Res. 2013 Aug 15;755(2):126-34. doi: 10.1016/j.mrgentox.2013.06.006. Epub 2013 Jun 20.
The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.
外周血 Pig-a 检测法已显示出作为评估体内致突变性的工具的潜力。在这项研究中，五个实验室参与了一项合作试验，评估了使用 HIS49 抗体与红细胞和红细胞祖细胞上的抗原反应的大鼠 Pig-a 检测法的可转移性和重现性。在初步工作中，建立了流式细胞术方法，使所有实验室都能够检测到对照大鼠中 <10×10(-6) 的 CD59 阴性红细胞频率（Pig-a 突变频率）。然后，四个实验室（活体实验室）用单次口服 N-亚硝基-N-乙基脲、7,12-二甲基苯并[a]蒽（DMBA）或 4-硝基喹啉-1-氧化物（4NQO）处理雄性大鼠。在处理后最多 4 周采集血液样本，并通过流式细胞术分析总红细胞（RBC；RBC Pig-a 检测法）中 CD59 阴性细胞的频率。RBC Pig-a 检测法在四个活体实验室中进行，外加一个从其他实验室接收血液样本的第五个实验室。此外，五个实验室中的三个进行了网织红细胞（RETs；PIGRET 检测法）上的 Pig-a 检测法，使用来自用 DMBA 和 4NQO 处理的大鼠的血液。四个活体实验室检测到所有三种测试物质的 RBC Pig-a 突变频率（MF）的一致、时间和剂量相关增加。此外，从其他实验室接收血液样本的第五个实验室获得了可比的结果。进行 PIGRET 检测法的三个实验室也检测到 Pig-a MF 的一致、时间和剂量相关增加，与 RBC MF 相比，RET MF 随时间的增加更快。这些结果表明，使用 HIS49 抗体的大鼠 Pig-a 检测法在实验室之间可转移，并且检测法产生的数据是可重现的。研究结果还表明，与 RBC Pig-a 检测法相比，PIGRET 检测法可能更早检测到测试化合物的体内致突变性。
