BACKGROUND: Graves' disease is the most frequent cause of hyperthyroidism and is characterized by the presence in patients' serum of autoantibodies directed against the thyrotropin receptor (TRAb) that cause overproduction and release of thyroid hormones. Clinical presentation results from both hyperthyroidism and
underlying autoimmunity. The diagnosis is based on characteristic clinical and biochemical abnormalities: subclinical hyperthyroidism (low serum TSH with normal concentrations of T3 and T4) and overt hyperthyroidism (low serum TSH with increased concentrations of T3 and T4). Although its exact etiology remains to be completely understood, Graves' disease is believed to result from a complex interaction between genetic susceptibility and environmental factors. OBJECTIVE: To study the effect of autoimmunity on oxidative balance in peripheral blood mononuclear cells (PBMC) of patients with overt hyperthyroidism, analyzing its implications on lymphocyte functionality. METHODS: PBMC of patients with Graves´ hypertiroidism and healthy controls were purified by Ficoll-Hypaque. Plasma levels of thyroid hormones and TRAb were measured by radioimmunoassay. Reactive oxygen species (ROS) and apoptosis were evaluated by flow cytometry using DCFH-DA and Annexin-V/PI. Nuclear morphology was analyzed by staining with Hoescht 33342. The expression of antioxidant enzymes was analyzed by real-time PCR and western blot. Enzymatic activity was quantified by spectrophotometry. Malondialdehyde (MDA) levels were assessed by TBARs method. Proliferation was evaluated by [3H]-thymidine incorporation. RESULTS: We observed an increase of ROS in PBMC of patients with Graves´ hyperthyroidism that decreased by preincubation of the cells with the antioxidant N-acetylcysteine. Patients who
received metimazole to restore their thyroid status to normal values showed levels of ROS similar controls. Overt hyperthyroid patients showed a higher activity and genomic expression of Catalase and Glutathione Peroxidase. The increase in ROS was correlated with an increase in the lipid peroxidation, however the levels of apoptosis analyzed by flow cytometry were similar to control. Microscopic analysis of these cells did not show nuclear morphology compatible with apoptosis (DNA fragmentation and condensation). In addition, lymphocytes showed a greater proliferative response to mitogenic stimulation than cells from healthy individuals. CONCLUSIONS: The autoimmune phenomenon in the Graves' pathology induces an increased production and secretion of thyroid hormones that leads to an increase in the production of oxidative species. ROS causes oxidative damage in PBMC, but this is not enough to affect the viability and functionality of lymphocytes. Oxidative species induces the expression of antioxidant enzymes as compensation mechanism tending to restore the oxidative balance.