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(TM) 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 wi th either N-nitroso-N-methylurea (MNU) or benzo[a]pyrene (BaP). Three weeks after mutagen treatment splenic T cells were isolated from the a nimals, and samples were either cultured to measure mutation at the na tive hprt locus or used to extract genomic DNA for transgene mutation analysis. Phage rescued from extracted DNA were plated in the presence of -bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) to scor e lad mutations, or plated on a hflAB lawn to score cll and cl mutants . With MNU hprt mutant frequency increased in a dose-related, sublinea r 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 incr ease at this dose, and the cll and cl transgenes did not show any incr ease. With 150 mg/kg BaP a 5.8- and 8.7-fold increase in mutant freque ncy 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 inc rease was related to BaP exposure. No significant increase in cl mutan t frequency was observed. Therefore, the order of mutation assay sensi tivity 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 a ssay, additional evaluation of its sensitivity is warranted. (C) 1998 Elsevier Science B.V. All rights reserved.