No abstract

Differential CYPlAl inducibility, reflecting variations in aromatic hydrocarbon receptor (AHR) affinity among inbred mouse strains, is an important determinant of environmental toxicity. We took advantage of the Air polymorphism in C57BL16 and DBA/2 mice to develop an oligonudeotide-hybridization screening approach for the rapid identification of DNA sequence differences between Air alleles. Oligonudeotides containing single-base changes at polymorphic sites were immobilized on a solid support and hybridized with C57BL/6 or DBA/2 AHR cDNA radiolabeled probes. The observed hybridization patterns demonstrate that this approach can be used to detect nudeotide differences in the Air coding region with very high accuracy. In parallel experiments, we used a yeast two-hybrid system to assess phenotypic differences in AHR function. AHR activation, as measured by 13-galactosidase reporter activity in Saceharomyces cerevisiae strain SFY526, was determined following treatment with varying doses of the AHR ligand P-naphthoflavone (BNF (9). Ahr nucleotide differences, and corresponding AHR amino-acid changes between the C57BL/6 (B6) and DBA/2 (D2) mouse strains that carry the AhA'-1 and Ahkd alleles, respectively, have been studied extensively (9-15). An A375V change has been reported to confer most of the observed phenotypic differences between B6 and D2 (15). With hepatic cytosol from B6 and D2 mice, ligand-affinity differences were found to range between 4-and 10-fold for the B6 and D2 AHRs (1618, whereas ligand-affinity differences range between 2-and 6-fold for the B6 and D2 AHRs when cDNA-expressed AHRs are studied (9,15 (15,22,

Differential CYP1A1 inducibility, reflecting variations in aromatic hydrocarbon receptor (AHR) affinity among inbred mouse strains, is an important determinant of environmental toxicity. We took advantage of the Ahr polymorphism in C57BL/6 and DBA/2 mice to develop an oligonucleotide-hybridization screening approach for the rapid identification of DNA sequence differences between Ahr alleles. Oligonucleotides containing single-base changes at polymorphic sites were immobilized on a solid support and hybridized with C57BL/6 or DBA/2 AHR cDNA radiolabeled probes. The observed hybridization patterns demonstrate that this approach can be used to detect nucleotide differences in the Ahr coding region with very high accuracy. In parallel experiments, we used a yeast two-hybrid system to assess phenotypic differences in AHR function. AHR activation, as measured by beta-galactosidase reporter activity in Saccharomyces cerevisiae strain SFY526, was determined following treatment with varying doses of the AHR ligand beta-naphthoflavone (BNF). We found that the C57BL/6 AHR has about a 15-fold higher affinity for BNF than the DBA/2 AHR, in much better agreement with results reported for whole-animal studies than the values observed by in vitro ligand-binding assays. Using C57BL/6 and DBA/2 AHR chimeric proteins, we also confirmed the previously reported observation that an A375V change is principally responsible for the high- to low-affinity AHR phenotype. There has been no straightforward method to reliably and reproducibly phenotype large numbers of humans for CYP1A1 inducibility or AHR affinity. Screening human AHR cDNAs by oligonucleotide-hybridization and yeast two-hybrid methodologies will be invaluable for the rapid and unequivocal determination of changes in DNA sequence and receptor-ligand affinities associated with human AHR polymorphisms.