A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and 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 lacI, cII, and cI transgenes in Big Blue 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 lacI mutations, or plated on a hflAB lawn to score cII and cI 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 lacI transgene yielded only a 3.1-fold increase at this dose, and the cII and cI 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 lacI, respectively, while only a 1.3-fold increase was observed at cII. DNA sequencing revealed an increase in GC-->TA transversions among the cII mutants, suggesting that the increase was related to BaP exposure. No significant increase in cI mutant frequency was observed. Therefore, the order of mutation assay sensitivity was hprt>lacI>cII/cI with MNU, and hprt approximately lacI> cII/cI with BaP. While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lacI assay, additional evaluation of its sensitivity is warranted.
Monroe
J J
JJ
Department of Genetic and Cellular Toxicology, Merck Research Laboratories, WP45-333, West Point, PA 19486, USA. james_monroe@merck.com
Kort
K L
KL
Miller
J E
JE
Marino
D R
DR
Skopek
T R
TR
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda; D001564:Benzo(a)pyrene / Q000633:toxicity*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D029968:Escherichia coli Proteins; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D056968:Lac Repressors; D008770:Methylnitrosourea / Q000633:toxicity*; D051379:Mice; D008822:Mice, Transgenic; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013154:Spleen; D013601:T-Lymphocytes; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D009153:Mutagens; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C039000:cII protein, bacteriophage lambda; C016649:phage repressor proteins; D001564:Benzo(a)pyrene; D008770:Methylnitrosourea; D007041:Hypoxanthine Phosphoribosyltransferase