A Truncation in the Aryl Hydrocarbon Receptor of the CRL:WI(Han) Rat Does Not Affect the Developmental Toxicity of TCDD

Tao Jiang,Brian G Miller,David R Bell,Martin Rose,Shaun White,Ming Qi Fan,Lang Tran,George Loizou,Alan Macnicoll,Paul M D Foster,Sally Clode,Alwyn Fernandes

doi:10.1093/toxsci/kfn252

Abstract

The aryl hydrocarbon receptor (AhR) is required for the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and so the AhR of CRL:WI and CRL:WI(Han) rats was characterized. Western blot showed AhR proteins of approximately 110 and approximately 97 kDa in individual rats from both strains. The AhR cDNA from a CRL:WI(Han) rat with the approximately 110-kDa protein revealed a sequence that was identical to that of the CRL:WI and SD rat. However, cloning of the AhR from a rat with the approximately 97-kDa protein revealed a point mutation, and five variants encoding two C-terminally truncated variants of the AhR protein, arising from a point mutation in the intron/exon junction and consequent differential splicing. These C-terminally truncated variants were expressed and shown to give rise to a protein of approximately 97 kDa; the recombinant AhR bound TCDD with an affinity that was not statistically different from the full-length protein. A single-nucleotide polymorphism assay was developed, and showed that both alleles were represented in a Hardy-Weinberg equilibrium in samples of CRL:WI and CRL:WI(Han) populations; both alleles are abundant. Rats from two studies of TCDD developmental toxicity were genotyped, and the association with toxicity investigated using statistical analysis. There was no plausible evidence that the AhR allele had a significant effect on the toxic endpoints examined. These data show that the two AhR alleles are common in two strains of Wistar rat, and that the AhR alleles had no effect on TCDD-induced developmental toxicity in two independent studies.

Highlights

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous toxin, and a prototypical representative of a series of chemicals which effect toxicity through a common mechanism, binding to the Aryl Hydrocarbon Receptor (AhR) (Poland et al, 1976a) (Poland and Knutson, 1982)
Given that AhR polymorphisms are known to be determinative of TCDD-induced toxicity in the mouse, we sought to investigate the AhR of the CRL:WI(Han) rat, whether there were any genetic polymorphisms in AhR, and characterising what those polymorphisms were
Any polymorphisms might have affected the toxicity of TCDD in our studies with this rat strain (Bell et al, 2007a) (Bell et al, 2007b) (Bell et al, 2007c), and so we investigated whether polymorphic AhR variants have altered affinity for TCDD, and whether these polymorphisms in the AhR of the CRL:WI(Han) rat are associated with susceptibility to the developmental toxicity of TCDD in two separate studies

Summary

Introduction

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous toxin, and a prototypical representative of a series of chemicals which effect toxicity through a common mechanism, binding to the Aryl Hydrocarbon Receptor (AhR) (Poland et al, 1976a) (Poland and Knutson, 1982). Maternal dosing with low doses of TCDD during pregnancy is reported to cause adverse effects in the reproductive system of male offspring, notably decreased sperm count in the cauda epididymis of Holtzmann (Mably et al, 1992) or Wistar (Faqi et al, 1998) rats. These effects were used to set a Tolerable Daily Intake of 2pg kg-1 day-1 for TCDD and related compounds by the UK Committee on Toxicity (Committee on Toxicity, 2001). Any polymorphisms might have affected the toxicity of TCDD in our studies with this rat strain (Bell et al, 2007a) (Bell et al, 2007b) (Bell et al, 2007c), and so we investigated whether polymorphic AhR variants have altered affinity for TCDD, and whether these polymorphisms in the AhR of the CRL:WI(Han) rat are associated with susceptibility to the developmental toxicity of TCDD in two separate studies

Methods

Results

Conclusion