Flow cytometric detection of Pig-A mutant red blood cells using an erythroid-specific antibody: application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats.
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.
Dobrovolsky
Vasily N
VN
Division of Genetic and Reproductive Toxicology; National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA. Vasily.Dobrovolsky@fda.hhs.gov
Boctor
Sherin Y
SY
Twaddle
Nathan C
NC
Doerge
Daniel R
DR
Bishop
Michelle E
ME
Manjanatha
Mugimane G
MG
Kimoto
Takafumi
T
Miura
Daishiro
D
Heflich
Robert H
RH
Ferguson
Sherry A
SA
D000477:Alkylating Agents / Q000008:administration & dosage / Q000009:adverse effects; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001289:Attention Deficit Disorder with Hyperactivity / Q000188:drug therapy; D000697:Central Nervous System Stimulants / Q000008:administration & dosage / Q000009:adverse effects* / Q000097:blood; D004249:DNA Damage; D004912:Erythrocytes / Q000187:drug effects* / Q000276:immunology; D005038:Ethylnitrosourea / Q000008:administration & dosage / Q000009:adverse effects; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000172:deficiency*; D008774:Methylphenidate / Q000008:administration & dosage / Q000009:adverse effects* / Q000097:blood; D015162:Micronucleus Tests; D009152:Mutagenicity Tests / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D000697:Central Nervous System Stimulants; D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D008774:Methylphenidate; D005038:Ethylnitrosourea