Bioimmunoassay of Aryl Hydrocarbon (Ah) Receptor Transformation in Vitro by 2,3,7,8- Tetrachlorodibenzo-p-Dioxin(TCDD)

Abstract

An assay is described that detects 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) indirectly by quantitating transformation of guinea pig aryl hydrocarbon (Ah) receptor in vitro. Transformed Ah receptor is isolated on an affinity column and measured by dot-blot immunoassay. Antisera were developed against the highly conserved amino-terminal region of mammalian Ah receptor and affinity-purified. Hepatic cytosol from guinea pigs was treated with TCDD. Treatment resulted in the transformation of the Ah receptor to the DNA binding form. Affinity chromatography of the treated cytosol using dioxin-responsive element (DRE) oligonucleotides isolated a single immunoreactive and TCDD treatment-dependent protein with a molecular mass of 105 kDa. The protein was identified as guinea pig Ah receptor based on TCDD treatment dependence, molecular weight, and immunoreactivity. A bioimmunoassay was designed to detect the transformation of guinea pig Ah receptor by TCDD. Transformation of guinea pig Ah receptor by TCDD was dose-dependent. Maximal transformation was seen at doses of 4 and 8 nM TCDD. Half-maximal response was estimated to be 0.548 nM ≤ 0.002 SE from nonlinear curve fitting of the dose-response data to a hyperbolic function. The limit of quantitation was 0.125 nM. A standard curve of 10 to 320 fmol of antigen on dot blots was generated. The limit of antigen quantitation was 10 fmol. Comparison of densities from the TCDD-dose responses to densities of the antigen standard curve indicated that the recovery of transformed Ah receptor was between 1.8 and 14 fmol/mg cytosol protein in the 0.125 to 8-nm TCDD dose range. Seven additional dioxin and furan congeners of TCDD and three PCBs were tested in the assay. Dose responses to 1,2,3,7,8-PCDD; 1,2,3,4,7,8-HCDD; 2-MCDD; 2,3,7,8-TCDF; 1,2,3,7,8-PCDF; 2,3,4,7,8-PCDF; 2,3,4,6,7,8-HCDF; 3,3′,4,4′-TCB; 3,3′,4,4′,5-PCB; and 2,3,3′,4,4′,5-HCB were generated. The half-maximal response of these compounds was proportional to their respective toxic equivalency factors. These results suggest that this assay could be used for detection of TCDD and related congeners, quantitation of transformed Ah receptor, and estimations of toxic equivalency factors in vitro. The potential uses of this assay are discussed.

Key Words: :

• Aryl hydrocarbon receptor
• Dioxins
• Guinea pig
• Immunoassay
• 2,3,7,8-tetrachlorodibenzo-p-dioxin
• Toxic equivalency factors