Towards an improved micronucleus test Studies on 3 model agents,mitomycin C,cyclophosphamide and dimethylbenzanthracene

Although the micronucleus assay has been widely used to detect chromosomal breakage in vivo, none of the protocols that have been used have been fully justified either experimentally or theoretically. Accordingly, we have studied the production of micronuclei by 3 compounds in detail, hoping that an optimal protocol could be defined. 2 factors were investigated, the time course of micronucleus production and the influence of multiple injections. The time course of micronucleus production in polychromatic erythrocytes (PCE) proved to be different for each of the chemicals studied, cyclophosphamide, mitomycin C and dimethylbenzanthracene. After a single intraperitoneal injection, for example, the maximum frequency of micronuclei occurred at ∼36 h for mitomycin C, and at ∼72 h for dimethylbenzanthracene. In no case could an increase in micronuclei be detected earlier than 10 h after injection, as would be predicted on theoretical grounds. From this we conclude, firstly, that multiple times are more valuable than multiple doses in detecting chromosomal breakage in vivo, and, secondly, that any treatment within ∼10 h of sampling is ineffective.The effect of multiple injections was also investigated directly. The conclusion that injections within 10 h of sampling are ineffective was confirmed. In general, the effect of 2 injections 24 h apart was close to the sum of the effects that the individual injections produce at the corresponding times. Obviously if this additivity is the usual situation, and more chemical can be given in multiple injections than in a single one, the sensitivity of the assay will be improved by the use of an appropriate multi-injection protocol. Multiple injections, however, were found to have subtractive effects if carried too far. Although we do not believe that we can define the most effective protocol yet, our results suggest that the following protocol should be more successful than those used previously: (1) inject intraperitoneally at 0 and 24 h, sample at 48, 72 and 96 h, (2) if a negative result is obtained, inject intraperitoneally at 0 h and sample at 30, 48 and 72 h. In each case, a positive result can be confirmed by either a repeat experiment or a dose—response study at the time of the maximum response.