DETECTION OF MICRONUCLEI AFTER EXPOSURE TO MITOMYCIN-C, CYCLOPHOSPHAMIDE AND DIETHYLNITROSAMINE BY THE INVIVO MICRONUCLEUS TEST IN MOUSE SPLENOCYTES

A micronucleus detetion 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-mu-g/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.