Flow Cytometric Detection ofPig-AMutant Red Blood Cells Using an Erythroid-Specific Antibody: Application of the Method for Evaluating the In Vivo Genotoxicity of Methylphenidate in Adolescent Rats

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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 × /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/μl, 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. Environ. Mol. Mutagen. 51:138–145, 2010. Published 2009 by Wiley-Liss, Inc.

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