title: Pig-a mutation: kinetics in rat erythrocytes following exposure to five prototypical mutagens.
abstract: 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.
authors: Litron Laboratories, Rochester, New York 14623, USA.
authors: Souk
authors: S
authors: Phonethepswath
authors: Dean
authors: D
authors: Franklin
authors: Dorothea K
authors: DK
authors: Torous
authors: Steven M
authors: SM
authors: Bryce
authors: Jeffrey C
authors: JC
authors: Bemis
authors: Sarojini
authors: S
authors: Raja
authors: Svetlana
authors: S
authors: Avlasevich
authors: Pamela
authors: P
authors: Weller
authors: Ollivier
authors: O
authors: Hyrien
authors: James
authors: J
authors: Palis
authors: James T
authors: JT
authors: Macgregor
authors: Stephen D
authors: SD
authors: Dertinger
mesh_terms: D015112:4-Nitroquinoline-1-oxide / Q000633:toxicity; D015127:9,10-Dimethyl-1,2-benzanthracene / Q000633:toxicity; D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005038:Ethylnitrosourea / Q000633:toxicity; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics*; D008770:Methylnitrosourea / Q000633:toxicity; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar
chemical_list: D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein; D001564:Benzo(a)pyrene; D015112:4-Nitroquinoline-1-oxide; D015127:9,10-Dimethyl-1,2-benzanthracene; D008770:Methylnitrosourea; D005038:Ethylnitrosourea