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MC-LR can interfere with thyroid function in fish, but the underlying mechanism is still unclear. Current study focuses to study the intergenerational inheritance of MC-LR-induced thyroid toxicity in zebrafish and in rat thyroid cells. In vivo experiments, adult female zebrafish (F0) were exposed to MC-LR (0, 5, and 25 µg/L) for 90 days and mated with male zebrafish without MC-LR exposure to generate F1 generation. F1 embryos were allowed to develop normally to 7 days post-fertilization (dpf) in clear water. In the F0 generation, MC-LR induced disturbance of the hypothalamic-pituitary-thyroid (HPT) axis, leading to a decrease in the production of thyroid hormones. Maternal MC-LR exposure also induced growth inhibition by altering thyroid hormones (THs) homeostasis and interfering with thyroid metabolism and development in F1 offspring. Mechanistically, MC-LR caused excessive accumulation of ROS and induced ER stress that further lead to activation of UPR in the F0 and F1 offspring of zebrafish. Interestingly, our findings suggested that MC-LR exposure hampered thyroglobulin turnover by triggering IRE1 and PERK pathway in zebrafish and FRTL-5 thyroid cells, thus disturbing the thyroid endocrine system and contributing to the thyroid toxicity from maternal to its F1 offspring of zebrafish. Particularly, inhibition of the IRE1 pathway by siRNA could alleviate thyroid development injury induced by MC-LR in FRTL-5 cells. In addition, MC-LR induced thyroid cell apoptosis by triggering ER stress. Taken together, our results demonstrated that maternal MC-LR exposure causes thyroid endocrine disruption by ER stress contributing to transgenerational effects in zebrafish offspring.

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Glyphosate is a pesticide, which contaminates the environment and exposes workers and general population to its residues present in foods and waters. In soil, Glyphosate is degraded in metabolites, amino-methyl-phosphonic acid (AMPA) being the main one. Glyphosate is considered a potential cancerogenic and endocrine-disruptor agent, however its adverse effects on the thyroid were evaluated only in animal models and in vitro data are still lacking. Aim of this study was to investigate whether exposure to Glyphosate could exert adverse effects on thyroid cells in vitro. Two models (adherent-2D and spheroid-3D) derived from the same cell strain Fisher-rat-thyroid-cell line-5 (FRTL-5) were employed. After exposure to Glyphosate at increasing concentrations (0.0, 0.1-0.25- 0.5-1.0-2.0-10.0 mM) we evaluated cell viability by WST-1 (adherent and spheroids), results being confirmed by propidium-iodide staining (only for spheroids). Proliferation of adherent cells was assessed by crystal violet and trypan-blue assays, the increasing volume of spheroids was taken as a measure of proliferation. We also evaluated the ability of cells to form spheroids after Glyphosate exposure. We assessed changes of reactive-oxygen-species (ROS) by the cell-permeant H2DCFDA. Glyphosate-induced changes of mRNAs encoding for thyroid-related genes (TSHR, TPO, TG, NIS, TTF-1 and PAX8) were evaluated by RT-PCR. Glyphosate reduced cell viability and proliferation in both models, even if at different concentrations. Glyphosate at the highest concentration reduced the ability of FRTL-5 to form spheroids. An increased ROS production was found in both models after exposure to Glyphosate. Finally, Glyphosate increased the mRNA levels of some thyroid related genes (TSHR, TPO, TG and TTF-1) in both models, while it increased the mRNAs of PAX8 and NIS only in the adherent model. The present study supports an adverse effect of Glyphosate on cultured thyroid cells. Glyphosate reduced cell viability and proliferation and increased ROS production in thyroid cells.

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Sulfonation is an important step in the metabolism of dopamine, estrogens, dehydroepiandrosterone, as well as thyroid hormones. However, the regulation of cytosolic sulfotransferases in the thyroid is not well understood. In a DNA microarray analysis of rat thyroid FRTL-5 cells, we found that the mRNA expression of 10 of 48 sulfotransferases was significantly altered by thyroid stimulating hormone (TSH), with that of sulfotransferase family 1A member 1 (SULT1A1) being the most significantly affected. Real-time PCR and Western blot analyses revealed that TSH, forskolin and dibutyryl cyclic AMP significantly suppressed SULT1A1 mRNA and protein levels in a time- and concentration-dependent manner. Moreover, immunofluorescence staining of FRTL-5 cells showed that SULT1A1 is localized in the perinuclear area in the absence of TSH but is spread throughout the cytoplasm with reduced fluorescence intensity in the presence of TSH. Sulfotransferase activity in FRTL-5 cells, measured using 3'-phosphoadenosine-5'-phosphosulfate as a donner and p-nitrophenol as an acceptor substrate, was significantly reduced by TSH. These findings suggest that the expression and activity of SULT1A1 are modulated by TSH in thyrocytes.

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Recently, ER stress induced by tunicamycin (TM) was reported to inhibit the expression of key genes involved in thyroid hormone synthesis, such as sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), and their regulators such as thyrotropin receptor (TSHR), thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2) and paired box gene 8 (PAX-8), in FRTL-5 thyrocytes. The present study tested the hypothesis that resveratrol (RSV) alleviates this effect of TM in FRTL-5 cells. While treatment of FRTL-5 cells with TM alone (0.1 µg/mL) for 48 h strongly induced the ER stress-sensitive genes heat shock protein family A member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3) and repressed NIS, TPO, TG, TSHR, TTF-1, TTF-2 and PAX-8, combined treatment with TM (0.1 µg/mL) and RSV (10 µM) for 48 h attenuated this effect of TM. In conclusion, RSV alleviates TM-induced ER stress and attenuates the strong impairment of expression of genes involved in thyroid hormone synthesis and their regulators in FRTL-5 thyrocytes exposed to TM-induced ER stress. Thus, RSV may be useful for the treatment of specific thyroid disorders, provided that strategies with improved oral bioavailability of RSV are applied.

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PURPOSE: Per- and poly-fluoroalkyl-substances (PFASs) are synthetic compounds that raised concern due to their potential adverse effects on human health. Long-chain PFAS were banned by government rules in many states, and thus, new emerging PFAS were recently introduced as substitutes. Among these, Perfluoro{acetic acid, 2-[(5-methoxy-1,3-dioxolan-4-yl)oxy]}, ammonium salt (C6O4) was recently introduced to produce a range of food contact articles and literature data about this compound are scanty. The aim of this study was to evaluate the in vitro effects of exposure to C6O4, compared with PFOA and PFOS on thyroid cells. METHODS: FRTL5 rat-thyroid cell lines and normal human thyroid cells (NHT) were incubated with increasing concentrations of C6O4 for 24, 48, 72, and 144 h to assess cell viability by WST-1. Cell viability was confirmed by AnnexinV/PI staining. Long-chain PFAS (PFOA and PFOS) were used at same concentrations as positive controls. The proliferation of cells exposed to C6O4, PFOA, and PFOS was measured by staining with crystal violet and evaluation of optical density after incubation with SDS. Changes in ROS production by FRTL5 and NHT after exposure to C6O4 at short (10, 20, and 30 min) and long-time points (24 h) were evaluated by cytofluorimetry. RESULTS: C6O4 exposure did not modify FRTL5 and NHT cell viability at any concentration and/or time points with no induction of necrosis/apoptosis. At difference, PFOS exposure reduced cell viability of FRTL5 while and NHT, while PFOA only in FRTL5. FRTL5 and NHT cell proliferation was reduced by incubation with by PFOA and PFOS, but not with C6O4. ROS production by NHT and FRTL5 cells was not modified after C6O4 exposure, at any time/concentration tested. CONCLUSIONS: The present in vitro study constitutes the first evaluation of the potential adverse effects of the new emerging PFAS C6O4 in cultured rat and human thyroid cells, suggesting its safety for thyroid cells in vitro.

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PURPOSE: Perfluorinated chemicals are widespread pollutants persistent in the environment with links to some major health issues. The two main compounds, perfluoro-octanoic acid (PFOA) and perfluoro-alkyl sulphonate (PFOS), were recently classified as carcinogenetic and thus their use has been restricted. Short-chain PFCs were recently developed as an alternative, but no data regarding the possible endocrine toxicities of these compounds are available. Aim of this study was to investigate whether short-chain PFCs could jeopardize thyroid cell viability and/or interfere with the functional effect TSH. METHODS: Fisher rat thyroid line-5 (FRTL-5) was treated with increasing concentrations of PFOA, PFOS, perfluorobutanesulfonic acid (PFBS), perfluorobutanoic acid (PFBA), pentafluoropropionic anhydride (PFPA), perfluoropentanoic acid (PFPeA) to evaluate modifications in cell viability and TSH-stimulated cAMP production. RESULTS: Neither long nor short-chain PFCs affected cell viability (apart from PFOS 100 µM), or interfered with cAMP production. CONCLUSIONS: The results of the present study demonstrate for the first time that short-chain PFCs have no acute cytotoxic effect on thyroid cells in vitro and that cAMP production is not modulated by any of the tested PFCs.

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Previous studies have shown that quercetin inhibits thyroid function both in vitro and in vivo. An attempt to evaluate the effect of quercetin at the promoter level of the thyroid-specific genes led to the observation that this compound induces the basal activity of the reporter vector. Therefore, the action of quercetin has been evaluated on the basal activity of several reporter vectors: The PGL3 basic, promoter and control vectors from Promega, and a pSV-based chloramphenicol acetyltransferase (CAT) reporter vector. In the Fisher Rat Thyroid cell Line FRTL-5 thyroid cells transiently transfected, quercetin 10 µM increased the basal activity of all the reporter vectors evaluated, although the degree of the effect was significantly different among them. The analysis of the difference among the regulatory regions of these vectors identified the activator protein 1 (AP-1) binding site as one of the potential sites involved in the quercetin effect. Electromobility shift assay experiments showed that the treatment with quercetin induced the binding of a protein complex to an oligonucleotide containing the AP-1 consensus binding site. This is the first study showing an effect of quercetin on AP-1 activity in thyroid cells. Further studies are in progress to understand the role of AP-1 activation in the effects of quercetin on thyroid function.

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PATZ1 is a transcriptional factor downregulated in thyroid cancer whose re-expression in thyroid cancer cells leads to a partial reversion of the malignant phenotype, including the capacity to proliferate, migrate, and undergo epithelial-to-mesenchymal transition. We have recently shown that PATZ1 is specifically downregulated downstream of the Ras oncogenic signaling through miR-29b, and that restoration of PATZ1 in Ha-Ras transformed FRTL5 rat thyroid cells is able to inhibit their capacities to proliferate and migrate in vitro. Here, we analyzed the impact of PATZ1 expression on the in vivo tumorigenesis of these cells. Surprisingly, FRTL5-Ras-PATZ1 cells showed enhanced tumor initiation when engrafted in nude mice, even if their tumor growth rate was reduced compared to that of FRTL5-Ras control cells. To further investigate the cause of the enhanced tumor engraftment of FRTL5-Ras-PATZ1 cells, we analyzed the stem-like potential of these cells through their capacity to grow as thyrospheres. The results showed that restoration of PATZ1 expression in these cells increases stem cell markers' expression and self-renewal ability of the thyrospheres while limiting their growth capacity. Therefore, we suggest that PATZ1 may play a role in enhancing the stem cell potential of thyroid cancer cells, but, at the same time, it impairs the proliferation of non-stem cells.

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Thyroid hormones are involved in many important physiological activities including regulation of energy metabolism, development of nervous system, maintenance of cerebral functions, and so on. Endocrine-disrupting chemicals (EDCs) that interfere with thyroid functions raise serious concerns due to their frequent misuse in areas where regulations are poorly implemented. In addition, chemicals that are originally regarded safe may now be considered as toxic with the development of life sciences. Malathion is an organophosphate insecticide that is widely applied and distributed in agricultural and residential settings. Due to the low acute toxicity and rapid degradation, malathion is not listed as a primary thyroid disrupting chemical. However, emerging evidences reported that malathion affected thyroperoxidase catalyzed iodide oxidation which in turn influenced thyroid hormone transportation, and enhanced parathyroid hyperplasia prevalence. Nevertheless, direct effect of malathion on thyroid hormone biosynthesis remains to be elucidated. This study investigated the effects of thyroid disruption of malathion in Fischer rat thyroid follicular cell line, FRTL-5. Transcriptional and translational analyses on thyroglobulin demonstrated that both mRNA and protein expression levels were significantly inhibited by malathion. Cellular cAMP level and TSH receptor expression were distinctly reduced by malathion (6.0 µg/ml). These results suggested that malathion directly disrupted the biosynthesis of thyroid hormone and the mechanism involved down-regulation of TSH receptor and cellular cAMP. This subsequently led to the suppression of TSH dependent signal transduction, TG transcription inhibition, and obstruction of thyroid hormone biosynthesis.

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The rotary cell culture system(RCCS)was used to simulate the microgravity environment, and FRTL-5 cells were divided into simulated microgravity group(SMG)and normal gravity group(NG). FRTL-5 cells were harvested after treatment for 6, 12, 24, and 36 h, the cell viability was measured by MTT assay, and the cells cycles were detected by flow cytometry. The ultrastructure of FRTL-5 cells was observed under laser confocal microscope with FITC-labeled technique. The MTT assay showed that the proliferation of FRTL-5 cells was significantly inhibited after RCCS treatment for 6, 12, 24, and 36h compared with NG(P<0.05), in which the most obvious effect was observed at 24h. The flow cytometry showed that the proportion of FRTL-5 cells at G1 stage in RCCS group was increased significantly after 6, 12, 24, and 36h compared with NG(P<0.05), while the proportion of FRTL-5 cells at S stage was decreased significantly(P<0.05)except that cultured with RCCS for 6 h. The proportion of FRTL-5 cells at G2/M stage was decreased in early phase(6-12 hours)of RCCS culture, with the lowest at 12h and transient increase at 24h of RCCS culture. The laser confocal microscope revealed that there were local microfilament depolymerization, tension fibers decrease, structure disorder, cellular pseudopodia reduction, and irregular shape among FITC-labeled FRTL-5 cells cultured with RCCS for 36h. (Chin J Endocrinol Metab, 2018, 34: 598-601)

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Many secretory cells increase the synthesis and secretion of cargo proteins in response to specific stimuli. How cells couple increased cargo load with a coordinate rise in secretory capacity to ensure efficient transport is not well understood. We used thyroid cells stimulated with thyrotropin (TSH) to demonstrate a coordinate increase in the production of thyroid-specific cargo proteins and ER-Golgi transport factors, and a parallel expansion of the Golgi complex. TSH also increased expression of the CREB3L1 transcription factor, which alone caused amplified transport factor levels and Golgi enlargement. Furthermore, CREB3L1 potentiated the TSH-induced increase in Golgi volume. A dominant-negative CREB3L1 construct hampered the ability of TSH to induce Golgi expansion, implying that this transcription factor contributes to Golgi expansion. Our findings support a model in which CREB3L1 acts as a downstream effector of TSH to regulate the expression of cargo proteins, and simultaneously increases the synthesis of transport factors and the expansion of the Golgi to synchronize the rise in cargo load with the amplified capacity of the secretory pathway.

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Resveratrol is a natural polyphenol with antioxidant, anti-inflammatory, and antiproliferative properties. We have shown previously that resveratrol decreases sodium/iodide symporter expression and iodide uptake in thyrocytes, both in vitro and in vivo. In the present study, we further investigated the effects of resveratrol, with evaluation of the expression of additional thyroid-specific genes in the FRTL-5 rat thyroid cell line: thyroglobulin, thyroid peroxidase, TSH receptor, Nkx2-1, Foxe1 and Pax8. We observed decreased expression of these genes in FRTL-5 cells treated with 10 µM resveratrol. The effects of resveratrol was further evaluated in vivo using Sprague-Dawley rats treated with resveratrol 25 mg/kg body weight intraperitoneally, for 60 days. No clinical signs of hypothyroidism were seen, although the treated rats showed significant increase in thyroid size. Serum TSH and thyroid hormone levels were in the normal range, with significantly higher TSH seen in resveratrol-treated rats, compared with control rats. Histological and immunohistochemical analyses confirmed increased proliferative activity in the thyroid from resveratrol-treated rats. These data suggest that resveratrol acts as a thyroid disruptor and a goitrogen, which indicates the need for caution as a supplement and for therapeutic uses.

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Since the discovery 60 years ago of the "long-acting thyroid stimulator" by Adams and Purves, great progress has been made in the detection of thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) in Graves' disease. Today, commercial assays are available that can detect TRAbs with high accuracy and provide diagnostic and prognostic evaluation of patients with Graves' disease. The present review focuses on the development of TRAbs bioassays, and particularly on the role that Leonard D. Kohn had in this. Indeed, 30 years ago, the Kohn group developed a bioassay based on the use of FRTL-5 cells that was characterized by high reproducibility, feasibility, and diagnostic accuracy. Using this FRTL-5 bioassay, Kohn and his colleagues were the first to develop monoclonal antibodies (moAbs) against the TSHR. Furthermore, they demonstrated the multifaceted functional nature of TRAbs in patients with Graves' disease, with the identification of stimulating and blocking TRAbs, and even antibodies that activated pathways other than cAMP. After the cloning of the TSHR, the Kohn laboratory constructed human TSHR-rat luteinizing hormone/chorionic gonadotropin receptor chimeras. This paved the way to a new bioassay based on the use of non-thyroid cells transfected with the Mc4 chimera. The new Mc4 bioassay is characterized by high diagnostic and prognostic accuracy, greater than for other assays. The availability of a commercial kit based on the Mc4 chimera is spreading the use of this assay worldwide, indicating its benefits for these patients with Graves' disease. This review also describes the main contributions made by other researchers in TSHR molecular biology and TRAbs assay, especially with the development of highly potent moAbs. A comparison of the diagnostic accuracies of the main TRAbs assays, as both immunoassays and bioassays, is also provided.

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The tissues of the body are routinely subjected to various forms of mechanical vibration, the frequency, amplitude, and duration of which can contribute both positively and negatively to human health. The vocal cords, which are in close proximity to the thyroid, may also supply the thyroid with important mechanical signals that modulate hormone production via mechanical vibrations from phonation. In order to explore the possibility that vibrational stimulation from vocalization can enhance thyroid epithelial cell function, FRTL-5 rat thyroid cells were subjected to either chemical stimulation with thyroid stimulating hormone (TSH), mechanical stimulation with physiological vibrations, or a combination of the two, all in a well-characterized, torsional rheometer-bioreactor. The FRTL-5 cells responded to mechanical stimulation with significantly (p<0.05) increased metabolic activity, significantly (p<0.05) increased ROS production, and increased gene expression of thyroglobulin and sodium-iodide symporter compared to un-stimulated controls, and showed an equivalent or greater response than TSH only stimulated cells. Furthermore, the combination of TSH and oscillatory motion produced a greater response than mechanical or chemical stimulation alone. Taken together, these results suggest that mechanical vibrations could provide stimulatory cues that help maintain thyroid function.

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BACKGROUND: Thyroid-derived cell models are commonly used to investigate the characteristics of thyroid cancers. It is noteworthy that each in vitro single cell model system imitates only a few characteristics of thyroid cancer depending on e.g. source of cells or oncogene used to transform the cells. METHODS: In the current work we utilized rat thyroid cancer cell models to determine their clinical relevance in redox gene studies by comparing in vitro expression data to thyroid Oncomine microarray database. To survey the cell lines we analyzed mRNA expression of genes that produce superoxide anion (nox family), genes that catalyze destruction of superoxide anion to hydrogen peroxide (sod family), and genes that remove hydrogen peroxide from cellular environment (catalase, gpx family and prdx family). RESULTS: Based on the current results, rat thyroid PC Cl3, PC PTC1, PC E1A, or FRLT5 cell models can be used to study NOX2, NOX4, SOD2, SOD3, CATALASE, GPX1, GPX2, GPX5, PRDX2, and PRDX3 gene expression and function. CONCLUSIONS: Redox gene expression in rat originated single cell model systems used to study human thyroid carcinogenesis corresponds only partly with human redox gene expression, which may be caused by differences in redox gene activation stimulus. The data suggest careful estimation of the data observed in rat thyroid in vitro models.

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AIM: We hypothesized that radiation-induced fibrosis was, in part, a result of altered signal transduction that directly modulates integrin expression and may indirectly affect the extracellular matrix (ECM). Major focus was given on protein kinase C (pKC). MATERIALS AND METHODS: Rat FRTL-5 and primary thyroid cells were exposed to proton radiation (5 and 10 Gy). Hours to days after exposure, a series of assays were performed. In addition, the neck region of Lewis rats was proton-irradiated to 40 Gy (5 Gy/day or 10 Gy/day). At 11 weeks after exposure, thyroid tissue was evaluated. RESULTS: Accumulation of ECM in irradiated FRTL-5 and primary thyroid cells was coincident with loss of tissue organization and follicularization at one or more doses and time points. Several pKC isoforms increased post-irradiation, which coincided with modulated integrin expression; fibronectin, laminin and collagen were also altered (p<0.05 vs. 0 Gy). Modulation of thyroid cells in culture with 12-O-tetradecanoylphorbol-13-acetate (TPA)±calphostin C supported a direct role of pKC in these altered properties. Thyroid tissue from irradiated rats had significantly more fibrotic lesions and increases in several pKC isoforms, integrins and fibronectin compared to 0-Gy (p<0.05). CONCLUSION: pKC is a likely contributor to alteration of key players associated with radiation-induced fibrosis.

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Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the environment. In this study we have demonstrated that HCB induced loss of cell viability and alterations in cell cycle regulation in FRTL-5 rat thyroid cells. Analysis of cell cycle distribution by flow cytometric analysis demonstrated that HCB induced cell cycle arrest at G2/M and at G0/G1 phase, inhibiting cell cycle progression at the G1/S phase, after 24 h and 72 h of treatment. HCB-treatment resulted in an increase in transforming growth factor-beta (TGF-ß1) mRNA levels, a negative regulator of cell growth in thyroid epithelial cells. Time-dependent studies showed that both cytosolic and nuclear p27 protein levels were increased by 5 µM HCB. After 24 h of treatment, total p27 in whole cells lysate was increased. Dose-dependent studies, demonstrated that HCB (0.005, 0.05, 0.5 and 5 µM) increased p27, both in the cytosol and nucleus. HCB (5 µM) induced a concomitant decrease in nuclear cyclin D1 protein levels, in a time-dependent manner. We have also demonstrated that TGF-ß1 Smad signaling is involved in HCB-induced alterations of p27 and cyclin D1 protein levels. On the other hand, ERK1/2 activation is not involved in the alteration of cell cycle regulatory proteins.

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Quercetin is the most abundant flavonoid present in a broad range of fruit and vegetables. Furthermore, quercetin is available as dietary supplements that are based on its antioxidant, antiproliferative and anti-inflammatory properties. However, concerns have been raised about the potential toxic effects of excessive intake of quercetin, and several studies have demonstrated that flavonoids, included quercetin, can interfere with thyroid function. In a previous report, we showed that quercetin inhibits thyroid-cell growth and iodide uptake. The latter effect was associated with down-regulation of sodium/iodide symporter gene expression. In the present study, we have evaluated the effects of quercetin on the expression of other thyroid-restricted genes, and we show that quercetin decreases the expression of the thyrotropin receptor, thyroid peroxidase and thyroglobulin genes. We further investigated the inhibitory effects of quercetin on thyroid function in vivo through evaluation of radioiodine uptake in the Sprague-Dawley rat, which was significantly decreased after 14 days of quercetin treatment. These data confirm that quercetin can act as a thyroid disruptor, and they suggest that caution is needed in its supplemental and therapeutic use.

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Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the environment. We have previously shown that chronic HCB exposure triggers apoptosis in rat thyroid follicular cells. This study was carried out to investigate the molecular mechanism by which the pesticide causes apoptosis in FRTL-5 rat thyroid cells exposed to HCB (0.005, 0.05, 0.5, and 5µM) for 2, 6, 8, 24, and 48h. HCB treatment lowered cell viability and induced apoptotic cell death in a dose- and time-dependent manner, as demonstrated by morphological nuclear changes and the increase of DNA fragmentation. The pesticide increased activation of caspases-3, -8, and full-length caspase-10 processing. HCB induced mitochondrial membrane depolarization, release of cytochrome c and apoptosis-inducing factor (AIF), from the mitochondria to the cytosol, and AIF nuclear translocation. Cell death was accompanied by an increase in reactive oxygen species (ROS) generation. Blocking of ROS production, with a radical scavenger (Trolox), resulted in inhibition of AIF nuclear translocation and returned cells survival to control levels, demonstrating that ROS are critical mediators of HCB-induced apoptosis. The pesticide increased ERK1/2, JNK, and p38 phosphorylation in a time- and dose-dependent manner. However, when FRTL-5 cells were treated with specific MAPK inhibitors, only blockade of MEK1/2 with PD98059 prevented cell loss of viability, as well as caspase-3 activation. In addition, we demonstrated that HCB-induced production of ROS has a critical role in ERK1/2 activation. These results demonstrate for the first time that HCB induces apoptosis in FRTL-5 cells, by ROS-mediated ERK1/2 activation, through caspase-dependent and -independent pathways.

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