Hexachlorobenzene induces thyroid dysfunction and an imbalance in the homeostasis of FRTL-5 thyroid cells growth, involving apoptosis and cycle arrest.

FLORENCIA CHIAPPINI; CAROLINA PONTILLO; LAURA ALVAREZ; ANDREA RANDI.; DIANA KLEIMAN DE PISAREV

Lisboa

Congreso; International Congress on Environmental Health; 2012

Hexachlorobenzene (HCB) is a widespread and persistent environmental pollutant with deleterious effect on human health. It is a weak ligand of the aryl hydrocarbon receptor (AhR). Chronic administration of HCB to laboratory animals elicits a number of effects including, thyroid and reproductive dysfunctions and immunopathology. We have previously demonstrated that HCB induced TGF-â1 expression and apoptosis in thyroid gland and thyroid follicular cell line FRTL-5. It is known that TGF-â1 inhibits cell proliferation, upregulating the expression of cell cycle regulatory proteins such as p21, p27, p15 and p16, resulting in cell growth arrest or apoptosis in normal epithelial cells. Moreover, TGF-â1 inhibits a number of functional parameters. The aim of the present study was to assess the effect of HCB on the functional parameter, thyroglobulin (TG), the oxidative status, and cell cycle progression in the rat normal thyroid cell line FRTL-5. We also determined the role of TGF-â1, AhR, and ROS in HCB induced apoptosis. Cells were exposed to HCB (0.005, 0.05, 0.5, and 5 ìM) for different periods of time. TG, p27 and cyclin D1 protein levels were assessed by Western blot. mRNA levels of antioxidant enzymes catalase and superoxide dismutase-1 (SOD1), was determined by RT-PCR. Intracellular ROS generation was measured using an oxidation sensitive fluorescent probe, DCFH-DA. Cell cycle progression was assayed by flow cytometry with propidium iodine staining. Cell viability, was determined using the MTT assay, in the absence or presence of specific inhibitors. FRTL-5 cells exposed to HCB (5 ìM, for 24 h) decreased TG protein levels (50 % p<0.05). HCB (5 ìM) induced ROS generation (4h) (455% p<0.01) and decreased the expression of SOD1 (6 h) (50 % p<0.05). However the expression of catalase was significantly increased (106, 120, 146 and 150%) after 8 h of treatment with HCB (0.005, 0.05, 0.5 and 5 ìM), respectively. Cell viability loss, induced by HCB, was restored to control levels, when assayed in the presence of the ROS scavenger, TROLOX. p27 levels significantly increased (165, 114 and 96%) in the nuclear fraction at 2, 4 and 6 h after HCB (5 ìM) exposure. HCB (5 ìM) decreased nuclear cyclin D1 protein levels (40, 50, 60 and 80%, p<0.05), at 4, 6, 24 and 30 h, respectively. The cell cycle progression was arrested in G1 (72 h) and G2/M (24 h) phases with HCB 5 ìM. Assays in the presence of TGF-â receptor type-I inhibitor, SB431542, or an AhR antagonist, 4,7-orthophenantroline, showed that neither TGF-â1 or AhR were involved in HCB induced apoptosis. In conclusion, our results show for the first time, that HCB induces thyroid dysfunction and an imbalance in the homeostasis of FRTL-5 thyroid cells growth, involving apoptosis and cycle arrest.