Pig-a Mutation: Kinetics in Rat Erythrocytes Following Exposure to Five Prototypical Mutagens
Pig-a Mutation: Kinetics in Rat Erythrocytes Following Exposure to Five Prototypical Mutagens
复制标题
DOI：
10.1093/toxsci/kfp289
复制
发表时间：
2010-03-01
影响因子：
3.8
通讯作者：
Dertinger, Stephen D.
中科院分区：
文献类型：
--
作者：
Phonethepswath, Souk;Franklin, Dean;Dertinger, Stephen D.
基于糖基磷脂酰肌醇（GPI）锚定缺陷大鼠红细胞的流式细胞计数，开发了体内突变试验。使用该方法，将血液与抗CD 59-PE和SYTO 13染料一起孵育，并使用流式细胞术对CD 59阴性红细胞的频率进行评分。本文所述的实验被设计为定义突变红细胞从外周血出现和消失的动力学，以支持用于测定的适当处理和取样设计。Wistar Han大鼠用五种典型诱变剂之一处理：N-乙基-N-亚硝基脲（ENU）、7，1,2-二甲基-1，2-苯并[a]蒽（DMBA）、4-硝基喹啉-1-氧化物、苯并[a]芘和N-甲基-N-亚硝基脲。还在Sprague道利大鼠中评价了ENU和DMBA。通过经口灌胃连续三天（第1-3天）对动物进行处理，并在第-1、4、15、30、45和90天（ENU为第180天）采集血液样本。在第4天测量遗传毒性的第二个终点，外周血微核网织红细胞的频率。每种化学品均诱导微核和GPI锚缺失表型。在第15天，突变体细胞频率明显增加。突变网织红细胞频率保持相对稳定的一些化学品，但其他人达到峰值，然后显着下降。观察到的动力学差异可能与造血干细胞相对于自我更新能力有限的更定型细胞发生突变的程度有关。总的来说，结果表明，计数GPI锚缺陷红细胞是一种有效的手段，评价体内的化学品的致突变潜力。动力学和对该血液终点评分的容易性表明，将其纳入常规毒理学研究是可行的。
An in vivo mutation assay has been developed based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient rat erythrocytes. With this method, blood is incubated with anti-CD59-PE and SYTO 13 dye, and flow cytometry is used to score the frequency of CD59-negative erythrocytes. The experiments described herein were designed to define the kinetics of mutant erythrocyte appearance and disappearance from peripheral blood to support appropriate treatment and sampling designs for the assay. Wistar Han rats were treated with one of five prototypical mutagens: N-ethyl-N-nitrosourea (ENU); 7,12-dimethyl-1,2-benz[a]anthracene (DMBA); 4-nitroquinoline-1-oxide; benzo[a]pyrene; and N-methyl-N-nitrosourea. ENU and DMBA were also evaluated in Sprague Dawley rats. Animals were treated on three consecutive days (days 1-3) via oral gavage, and blood specimens were obtained on days -1, 4, 15, 30, 45, and 90 (and day 180 for ENU). A second endpoint of genotoxicity, the frequency of peripheral blood micronucleated reticulocytes, was measured on day 4. Each chemical induced micronuclei and the GPI anchor-deficient phenotype. Increased mutant cell frequencies were evident at day 15. Mutant reticulocyte frequencies remained relatively stable for some chemicals, but others peaked and then dropped significantly. The differences in kinetics observed are presumably related to the degree to which mutation occurs in hematopoietic stem cells versus more committed cells with limited self-renewal capacity. Collectively, the results suggest that enumerating GPI anchor-deficient erythrocytes is an efficient means of evaluating the in vivo mutagenic potential of chemicals. The kinetics and ease of scoring this blood-based endpoint suggest that integration into routine toxicology studies will be feasible.