Detection of micronuclei after exposure to mitomycin C, cyclophosphamide and diethylnitrosamine by the in vivo micronucleus test in mouse splenocytes.
A micronucleus detection test using mouse splenocytes has been adapted from a method previously carried out using human lymphocytes. An ex vivo protocol was chosen: male C57B16 mice were treated with various compounds. Splenocytes were then isolated and placed in culture for 48 h and stimulated with concanavalin A and conditioned medium. The cytokinesis-block method reported by Fenech and Morley was used to detect and score micronuclei in the proliferating lymphocytes (3 micrograms/ml of cytochalasin B for 16 h). Three mutagenic clastogens, mitomycin C (MMC), a direct alkylating agent (0.4, 0.8 and 1.6 mg/kg), cyclophosphamide (CP), an indirect alkylating agent (25, 50 and 100 mg/kg) and diethylnitrosamine (DEN), an indirect alkylating agent with labile metabolites (25, 50 and 100 mg/kg), were tested at four sampling times (2, 4, 8 and 15 days). All three compounds were detected from 48 h after treatment. This method was indeed able to detect clastogenic compounds normally detected by the mouse bone marrow micronucleus test (MMC, CP) as well as a compound with labile metabolites which is not usually detected by this test (DEN). Maximum micronucleus induction was observed after 4 days for MMC, 2 days for CP and 15 days for DEN. This method thus appears to offer a potentially useful toxicological test for assessing in vivo clastogenicity.
Benning
V
V
Rhône-Poulenc Rorer, Centre de Recherche de Vitry-Alfortville, Département Sécurité du Médicament, Vitry sur Seine, France.
Depasse
F
F
Melcion
C
C
Cordier
A
A
D000818:Animals; D002478:Cells, Cultured; D003520:Cyclophosphamide / Q000633:toxicity*; D004052:Diethylnitrosamine / Q000633:toxicity*; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015162:Micronucleus Tests; D016685:Mitomycin / Q000633:toxicity*; D013154:Spleen / Q000166:cytology / Q000187:drug effects
D004052:Diethylnitrosamine; D016685:Mitomycin; D003520:Cyclophosphamide