Desethylamiodarone antagonizes the effect of thyroid hormone at the molecular level

Objective: To evaluate the molecular mechanisms of the inhibitory effects of amiodarone and its active metabolite, desethylamiodarone (DEA) on thyroid hormone action. Materials and methods: The reporter construct ME-TRE-TK-CAT or TSH beta -TRE-TK-CAT, containing the nucleotide sequence of the thyroid hormone response element (TRE) of either malic enzyme (ME) or TSH beta genes, thymidine kinase (TK) and chloramphenicol acetyltransferase (CAT) was transiently transfected with RSV-TRP into NIH3T3 cells. Gel mobility shift assay (EMSA) was performed using labelled synthetic oligonucleotides containing the ME-TRE and in vitro translated thyroid hormone receptor (TR)beta. Results: Addition of 1 mu mol/l T-4 or T-3 to the culture medium increased the basal level of ME-TRE-TK-CAT by 4.5- and 12.5-fold respectively. Amiodarone or DEA (1 mu mol/l) increased CAT activity by 1.4-and 3.4-fold respectively. Combination of DEA with T-4 or T-3 increased CAT activity by 9.4- and 18.9-fold respectively, These data suggested that DEA, but not amiodarone, had a synergistic effect with thyroid hormone on ME-TRE, rather than the postulated inhibitory action; we supposed that this was due to overexpression of the transfected TR into the cells. When the amount of RSV-TRP was reduced until it was present in a limited amount, allowing competition between thyroid hormone and the drug, addition of 1 mu mol/l DEA decreased the T-3-dependent expression of the reporter gene by 50%. The inhibitory effect of DEA was partially due to a reduced binding of TR to ME-TRE, as assessed by EMSA. DEA activated the TR-dependent down-regulation by the negative TSH-TRE, although at low level (35% of the down-regulation produced by T-3), whereas amiodarone was ineffective. Addition of 1 mu mol/l DEA to T-3-containing medium reduced the T-3-TR-mediated down-regulation of TSH-TRE to 55%. Conclusions: Our results demonstrate that DEA, but not amiodarone, exerts a direct, although weak, effect on genes that are regulated by thyroid hormone. High concentrations of DEA antagonize the action of T-3 at the molecular level, interacting with TR and reducing its binding to TREs. This effect may contribute to the hypothyroid-like effect observed in peripheral tissues of patients receiving amiodarone treatment.