Mechanistic studies of the interactive toxicology of dioxin-like compounds
Dioxin-like compounds (DLCs) elicit a variety of biological responses that are mediated by the aryl hydrocarbon (Ah) receptor. Mixtures of compounds display interactive effects that modify observed endpoints both 'in vivo' and 'in vitro', suggesting a divergence of mechanistic pathways when the AhR is bound by different compounds. An AhR agonist, antagonists and binary mixtures were used to probe three stages in the mechanism of action of the AhR (transformation, activation, and CYP 1A1 induction) to determine where mechanistic pathways diverge for the AhR agonist TCDD (2,3,7,8-tetrachlorodibenzo-'p'-dioxin), and AhR antagonists (various PCBs), and to elucidate the biochemical basis of the interactive effects. Transformation of the AhR was examined by monitoring a shift in the conformation of the receptor in which the ligand becomes bound with increased affinity. An AhR agonist and antagonist induced transformation equally well, thus no divergence was found at this stage. An attempt was also made to synthesize and study a [125I]-labeled PCB. Activation of the AhR to the form possessing a high affinity for binding to specific DNA sequences, was examined using the gel mobility shift assay. None of the DLCs examined activated the AhR to the same extent as TCDD. When the AhR was partially occupied by TCDD, combination with a DLC produced additive responses through occupation of unliganded AhR molecules. When the AhR was saturated with TCDD, combination with a DLC resulted in displacement of TCDD. This decreased amount of activated AhR produced antagonistic responses relative to those observed with TCDD alone. The findings were further examined in a metabolically functional living system, by examining the induction of cytochrome P450 1A1 in primary rat hepatocyte cultures. The interactive effects were reproducible in this system, confirming the results obtained with the model systems. In addition, it was discovered that the inducing compounds acted as competitive inhibitors of ethoxyresorufin-' O'-deethylase, an enzyme that is commonly used to quantitate DLCs in environmental samples. Kinetic models were successfully developed to simulate the behaviour of DLCs as EROD inducers and inhibitors. The interactive effects were concluded to arise from incomplete activation of the AhR by DLCs compared with TCDD.