A comparative study of in vivo mutation assays:: analysis of hprt, lacI, and cII/cI as mutational targets for N-nitroso-N-methylurea and benzo[a]pyrene in Big Blue™ mice

We have compared the response of the native hprt gene and the lad, cll, and cl transgenes in Big Blue(TM) B6C3F1 mice following treatment with either N-nitroso-N-methylurea (MNU) or benzo[a]pyrene (BaP). Three weeks after mutagen treatment splenic T cells were isolated from the animals, and samples were either cultured to measure mutation at the native hprt locus or used to extract genomic DNA for transgene mutation analysis. Phage rescued from extracted DNA were plated in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) to score lad mutations, or plated on a hflAB lawn to score cll and cl mutants. With MNU hprt mutant frequency increased in a dose-related, sublinear manner up to 78-fold above background at the highest dose tested (20 mg/kg). In comparison, the lad transgene yielded only a 3.1-fold increase at this dose, and the cll and cl transgenes did not show any increase. With 150 mg/kg BaP a 5.8- and 8.7-fold increase in mutant frequency was observed at hprt and loci, respectively, while only a 1.3-fold increase was observed at cll. DNA sequencing revealed an increase in GC --> TA transversions among the cll mutants, suggesting that the increase was related to BaP exposure. No significant increase in cl mutant frequency was observed. Therefore, the order of mutation assay sensitivity was hprt > loci > cII/cI with MNU, and hprt approximate to lad > cII/cI with BaP. While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lad assay, additional evaluation of its sensitivity is warranted. (C) 1998 Elsevier Science B.V. All rights reserved.