Repair replication of DNA in the intact animal following treatment with dimethylnitrosamine and with methyl methanesulphonate, studied by fractionation of nuclei in a zonal centrifuge

Abstract

To relate repair of lesions in DNA with carcinogenesis, it is necessary to study repair in the intact animal under conditions which will and will not induce cancer. The methods used to study the replications stage of repair in isolated cells are not suitable for use in the intact animal. A method is presented which depends on the fact that during cell replication the nuclei enlarge, and may be separated from nuclei of non-replicating cells by rate zonal centrifugation on a sucrose gradient in a zonal rotor. Thus incorporation of [3H]thymidine as a result of de novo synthesis of DNA in replicating nuclei can be separated from incorporation due to repair synthesis in non-replicating nuclei. Treatment of animals with dimethylnitrosamine or with methyl methanesulphonate produced a "repair-type" profile, which contrasted with that given by liver nuclei from untreated animals, or from animals in which DNA synthesis had been reduced by treatment with cycloheximide, a compound which is not known to cause direct damage to DNA. Evidence is presented which suggests that the method is a rapid sensitive test for the occurrence of repair replication of some kind in the liver of the intact animal.