title: Efficient monitoring of in vivo pig-a gene mutation and chromosomal damage: summary of 7 published studies and results from 11 new reference compounds.
abstract: The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and flow cytometric micronucleus analysis. Eight reference genotoxicants and three presumed nongenotoxic compounds were studied: chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea, methyl methanesulfonate, o-anthranilic acid, sulfisoxazole, and sodium chloride. These experiments extend previously published results with seven other chemicals. Male Sprague Dawley rats were treated via gavage for 3 or 28 consecutive days with several dose levels of each chemical up to the maximum tolerated dose. Blood samples were collected at several time points up to day 45 and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on day 4, and day 29 for the 28-day study, were evaluated for MN-RET frequency. The three nongenotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of Pig-a mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, when total dose was greatest, the highest induction of MN-RET was observed in the 3-day studies, when dose per day was greatest. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken with other previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained.
authors.affiliation: Litron Laboratories, Rochester, New York 14623, USA. sdertinger@litronlabs.com
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Dertinger
authors.forename: Souk
authors.initials: S
authors.lastname: Phonethepswath
authors.forename: Svetlana L
authors.initials: SL
authors.lastname: Avlasevich
authors.forename: Dorothea K
authors.initials: DK
authors.lastname: Torous
authors.forename: Jared
authors.initials: J
authors.lastname: Mereness
authors.forename: Steven M
authors.initials: SM
authors.lastname: Bryce
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Bemis
authors.forename: Sara
authors.initials: S
authors.lastname: Bell
authors.forename: Pamela
authors.initials: P
authors.lastname: Weller
authors.forename: James T
authors.initials: JT
authors.lastname: Macgregor
mesh_terms: D000818:Animals; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology; D005434:Flow Cytometry; D018189:Immunomagnetic Separation; D007962:Leukocytes / Q000187:drug effects / Q000473:pathology; D008297:Male; D008565:Membrane Proteins / Q000097:blood / Q000235:genetics*; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced*; D015162:Micronucleus Tests; D016296:Mutagenesis / Q000187:drug effects*; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012156:Reticulocytes / Q000187:drug effects / Q000201:enzymology; D018570:Risk Assessment; D013997:Time Factors
chemical_list: D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein