Division of Genetic and Reproductive Toxicology; National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA.
Environ Mol Mutagen. 2010 Mar;51(2):138-45. doi: 10.1002/em.20519.
A modified flow cytometry assay for Pig-A mutant rat red blood cells (RBCs) was developed using an antibody that positively identifies rat RBCs (monoclonal antibody HIS49). The assay was used in conjunction with a flow cytometric micronucleus (MN) assay to evaluate gene mutation and clastogenicity/aneugenicity in adolescent male and female rats treated with methylphenidate hydrochloride (MPH). Sprague-Dawley rats were treated orally with 3 mg/kg MPH (70/sex) or water (40/sex) 3 x /day on postnatal days (PNDs) 29-50. Eight additional rats (4/sex) were injected i.p. with N-ethyl-N-nitrosourea (ENU) on PND 28. Blood was collected on PNDs 29, 50, and 90, and used for determining serum MPH levels and/or conducting genotoxicity assays. On the first and last days of MPH treatment (PNDs 29 and 50), serum MPH levels averaged 21 pg/microl, well within the clinical treatment range. Relative to our previously published method (Miura et al. [2008]; Environ Mol Mutagen 49: 614-629), the HIS49 Pig-A mutation assay significantly reduced the background RBC mutant frequency; in the experiments with ENU-treated rats, the modification increased the overall sensitivity of the assay 2-3 fold. Even with the increased assay sensitivity, the 21 consecutive days of MPH treatment produced no evidence of Pig-A mutation induction (measured at PND 90); in addition, MPH treatment did not increase MN frequency (measured at PND 50). These results support the consensus view that the genotoxicity of MPH in pediatric patients reported earlier (El-Zein et al. [2005]: Cancer Lett 230: 284-291) cannot be reproduced in animal models, suggesting that MPH at clinically relevant levels may be nongenotoxic in humans.
建立了一种改良的流式细胞术检测 Pig-A 突变大鼠红细胞（RBC）的方法，该方法使用一种能够特异性识别大鼠 RBC 的抗体（单克隆抗体 HIS49）。该方法与流式细胞术微核（MN）检测相结合，用于评估接受盐酸哌甲酯（MPH）治疗的青春期雄性和雌性大鼠的基因突变和断裂/变导原性。Sprague-Dawley 大鼠于出生后第 29-50 天（PND）每天经口给予 3mg/kg MPH（每组 70 只）或水（每组 40 只），3 次/天。另外 8 只大鼠（每组 4 只）于 PND 28 时腹腔注射 N-乙基-N-亚硝基脲（ENU）。在 PND 29、50 和 90 时收集血液，用于测定血清 MPH 水平和/或进行遗传毒性检测。在 MPH 治疗的第一天和最后一天（PND 29 和 50），血清 MPH 水平平均为 21pg/µl，远低于临床治疗范围。与我们之前发表的方法（Miura 等人，[2008]；Environ Mol Mutagen 49：614-629）相比，HIS49 Pig-A 突变检测法显著降低了背景 RBC 突变频率；在接受 ENU 处理的大鼠实验中，该改良法使检测的总体敏感性提高了 2-3 倍。即使增加了检测的敏感性，21 天的 MPH 治疗也没有产生 Pig-A 突变诱导的证据（在 PND 90 时测量）；此外，MPH 治疗并未增加 MN 频率（在 PND 50 时测量）。这些结果支持了之前报告的关于儿童患者 MPH 的遗传毒性（El-Zein 等人，[2005]：Cancer Lett 230：284-291）在动物模型中无法重现的共识观点，这表明在临床相关水平下，MPH 可能对人类没有遗传毒性。
