Desethylamiodarone antagonizes the effect of thyroid hormone at the molecular level

Objective: To evaluate the molecular mechanisms of the inhibitory effects o f amiodarone and its active metabolite, desethylamiodarone (DEA) on thyroid hormone action. Materials and methods: The reporter construct ME-TRE-TK-CAT or TSH beta -TR E-TK-CAT, containing the nucleotide sequence of the thyroid hormone respons e element (TRE) of either malic enzyme (ME) or TSH beta genes, thymidine ki nase (TK) and chloramphenicol acetyltransferase (CAT) was transiently trans fected with RSV-TRP into NIH3T3 cells. Gel mobility shift assay (EMSA) was performed using labelled synthetic oligonucleotides containing the ME-TRE a nd 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. Amioda rone or DEA (1 mu mol/l) increased CAT activity by 1.4-and 3.4-fold respect ively. Combination of DEA with T-4 or T-3 increased CAT activity by 9.4- an d 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 overexpressi on of the transfected TR into the cells. When the amount of RSV-TRP was red uced 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-T RE, although at low level (35% of the down-regulation produced by T-3), whe reas amiodarone was ineffective. Addition of 1 mu mol/l DEA to T-3-containi ng 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 hormo ne. High concentrations of DEA antagonize the action of T-3 at the molecula r level, interacting with TR and reducing its binding to TREs. This effect may contribute to the hypothyroid-like effect observed in peripheral tissue s of patients receiving amiodarone treatment.