Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential.
Glycosylphosphatidylinositol (GPI) anchors attach specific proteins to the cell surface of hematopoietic cells. Of the genes required to form GPI anchors, only Pig-a is located on the X-chromosome. Prior work with rats suggests that the GPI anchor deficient phenotype is a reliable indicator of Pig-a mutation [Bryce et al., Environ. Mol. Mutagen., 49 (2008) 256-264]. The current report extends this line of investigation by describing simplified blood handling procedures, and by testing the assay principle in a second species, Mus musculus. With this method, erythrocytes are isolated, incubated with anti-CD24-PE, and stained with SYTO 13. Flow cytometric analyses quantify GPI anchor-deficient erythrocytes and reticulocytes. After reconstruction experiments with mutant-mimicking cells demonstrated that the analytical performance of the method is high, CD-1 mice were treated on three occasions with 7,12-dimethyl-1,2-benz[a]anthracene (DMBA, 75 mg/kg/day) or ethyl-N-nitrosourea (ENU, 40 mg/kg/day). Two weeks after the final treatment, DMBA-treated mice were found to exhibit markedly elevated frequencies of GPI anchor deficient erythrocytes and reticulocytes. For the ENU experiment, blood specimens were collected at weekly intervals over a 5-week period. Whereas the frequencies of mutant reticulocytes were significantly elevated 1 week after the last administration, the erythrocyte population was unchanged until the second week. Thereafter, both populations exhibited persistently elevated frequencies for the duration of the experiment (mean frequency at termination=310x10(-6) and 523x10(-6) for erythrocyte and reticulocyte populations, respectively). These data provide evidence that Pig-a mutation does not convey an appreciable positive or negative cell survival advantage to affected erythroid progenitors, although they do suggest that affected erythrocytes have a reduced lifespan in circulation. Collectively, accumulated data support the hypothesis that flow cytometric enumeration of GPI anchor deficient erythrocytes and/or reticulocytes represents an effective in vivo mutation assay that is applicable across species of toxicological interest.
Phonethepswath
Souk
S
Litron Laboratories, 200 Canal View Boulevard, Rochester, NY, United States.
Bryce
Steven M
SM
Bemis
Jeffrey C
JC
Dertinger
Stephen D
SD
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000633:toxicity; D000818:Animals; D004906:Erythrocyte Count; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D009152:Mutagenicity Tests; D009154:Mutation
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D015127:9,10-Dimethyl-1,2-benzanthracene
Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7479-83
10377440
Environ Mol Mutagen. 2008 Oct;49(8):622-30
18626996
Environ Mol Mutagen. 2008 May;49(4):256-64
18288722
Blood Cells Mol Dis. 1998 Sep;24(3):370-84
10087994
Mutagenesis. 1995 Nov;10(6):467-70
8596464
Blood. 1995 Nov 1;86(9):3277-86
7579428
Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5209-14
10220445
Genomics. 1994 Oct;23(3):566-74
7851884
Environ Mol Mutagen. 2008 Oct;49(8):614-21
18626999
Ann N Y Acad Sci. 2002 Jun;963:290-6
12095954
Cytometry B Clin Cytom. 2007 Jan 15;72(1):34-42
17051524
Am J Hematol. 1999 Nov;62(3):175-82
10539884
Environ Mol Mutagen. 1996;28(4):313-6
8991058