Pig-a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil, thiotepa, melphalan, and 1,3-propane sultone.
To evaluate whether blood-based genotoxicity endpoints can provide temporal and dose-response data within the low-dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry-based micronucleus and Pig-a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN-RET) was used to evaluate chromosomal damage, and the frequency of CD59-negative reticulocytes (RET(CD59-) ) and erythrocytes (RBC(CD59-) ) served as phenotypic reporters of mutation at the X-linked Pig-a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3-propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN-RET were determined at Days 4 and 29, and RET(CD59-) and RBC(CD59-) data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose-related increases were observed for each endpoint, and time to maximal effect was consistently: MN-RET < RET(CD59-)  < RBC(CD59-) . For each of the chemicals studied, the genotoxic events occurred long before tumors or preneoplastic lesions would be expected. Furthermore, in the case of Pig-a gene mutation, the responses were observed at or below the TD50 dose for three out of the four chemicals studied. These data illustrate the potential for quantitative blood-based analyses to provide dose-response and temporality information that relates genetic damage to cancer induction.
Litron Laboratories, Rochester, New York.
Stephen D
SD
Dertinger
Souk
S
Phonethepswath
Svetlana L
SL
Avlasevich
Dorothea K
DK
Torous
Jared
J
Mereness
John
J
Cottom
Jeffrey C
JC
Bemis
James T
JT
Macgregor
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D002699:Chlorambucil / Q000494:pharmacology*; D020552:Comet Assay; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D008297:Male; D008558:Melphalan / Q000494:pharmacology*; D008565:Membrane Proteins / Q000235:genetics*; D015162:Micronucleus Tests; D009152:Mutagenicity Tests; D009154:Mutation / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012156:Reticulocytes / Q000187:drug effects* / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology*; D013852:Thiotepa / Q000494:pharmacology*
D018906:Antineoplastic Agents, Alkylating; D008565:Membrane Proteins; D013876:Thiophenes; C080086:phosphatidylinositol glycan-class A protein; D002699:Chlorambucil; D013852:Thiotepa; C003218:1,3-propane sultone; D008558:Melphalan
Pig-a gene mutation; flow cytometry; genotoxicity; micronuclei; peripheral blood