ROS production induced by 3,5-diiodothyronine : a new extranuclear effect for thyroid hormone?

Extranuclear effects of thyroid hormones L-T3 and L-T4 are now widely recognized, but in the last few years evidence for a similar activity of a new thyroid hormone, 3,5-diiodo-L-thyronine (3,5-T2), has been reported. The 3,5-T2 is a deiodination metabolite of L-T3 that is able to mimic some important rapid nongenomical effects of L-T3. High levels of 3,5-T2 have been found in connection with some types of brain tumors and other nonthyroidal illnesses, such as liver cirrhosis, and it appears likely that 3,5-T2 may have a specific physiological role. Previous results from our laboratory demonstrated that 3,5-T2 in the physiological range (around 0.1 nM) mimics L-T3 activation of the Na/H exchanger, causing an increase in the intracellular pH of L-6 myoblasts (Incerpi et al., Endocrinology 140:693-689, 1999) and chick embryo hepatocytes (Incerpi et al., Endocrinology, 143:1660-1668, 2002). We here show that 3,5-T2 increases the fluorescence of diclorofluorescein in L-6 myoblasts, probably due to the production of reactive oxygen species (ROS). The increase is partially prevented by extracellular superoxide dismutase (SOD) or by treatment of cells with an inhibitor of the plasma membrane NADPH oxidase, that is believed to be an important source of superoxide formation both inside and outside cells. Experiments with Electron Paramagnetic Resonance spectroscopy, using Cu,Zn SOD as a probe, show that 3,5-T2 over a wide concentration range induces superoxide formation, whereas other analogs of thyroid hormones do not give the same effect. The results also indicate that superoxide is produced on the outside of the cell membrane, probably due to activation of the plasma membrane NADPH oxidase. Experiments are in progress in order to establish whether ROS production induced by 3,5-T2 represents a physiological signal, possibly as part of a response to a pathological condition.