Aromatic hydrocarbon receptor polymorphism: development of new methods to correlate genotype with phenotype.

Abstract

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.

Metabolomics Reveals Aryl Hydrocarbon Receptor Activation Induces Liver and Mammary Gland Metabolic Dysfunction in Lactating Mice

“…BNF shows higher affinity for the Ahr b allele. 20 Doses were chosen based on previous reports; 21 , 22 however, no studies to date have directly assessed the impact of BNF on lactation. Females were maintained on the diet treatment from conception until birth of their pups.…”

Induction of Oxidative Stress Responses by Dioxin and other Ligands of the Aryl Hydrocarbon Receptor

“…Most of the amino acid changes distinguishing these strains occur within the transactivation domain and have little or no known functional consequence (Chang et al, 1993). However, a point mutation at position 375 of the DBA/2 AHR results in an ALA to VAL substitution in the second PAS domain of the C57BL/6 strain that is responsible for the difference in ligand binding affinity and transactivation (Poland et al, 1994;Maier et al, 1998). These findings have been further supported in mice with homozygous loss of functional AHR (Fernandez-Salguero et al, 1995;Schmidt et al, 1996).…”

Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down

“…Metabolism of TCDD is known to be low [54] (a factor that contributes to the persistence of chloracne [7] ) but inducible by AhR activation [10,55] , while the metabolism of β-NF is known to be more rapid, through cytochromes P450 1A1 and 1A2 [56,57] . Xenobiotic metabolising cytochrome P450 enzymes in keratinocytes and in human epidermal equivalents are of low activity [46] and with the multiple dosing regimen used in these studies the concentrations of ligands would have been maintained.…”