Amiodarone Induces Cytochrome c Release and Apoptosis through ...

0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society

Vol. 85, No. 11 Printed in U.S.A.

Amiodarone Induces Cytochrome c Release and Apoptosis through an Iodine-Independent Mechanism* TIZIANA DI MATOLA, FRANCESCA D’ASCOLI, GIANFRANCO FENZI, GUIDO ROSSI, ENIO MARTINO, FAUSTO BOGAZZI, AND MARIO VITALE Dipartimento di Biologia e Patologia Cellulare e Molecolare (M.V., T.D., G.R.) and Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica (F.D., GF.F.), Universita` Federico II, Naples 80131; and Centro di Endocrinologia ed Oncologia Sperimentale G. Salvatore, Consiglio Nazionale delle Ricerche (G.R.), and Dipartimento di Endocrinologia, Universita` di Pisa (E.M., F.B.), Pisa 56100, Italy ABSTRACT Amiodarone (AMD) is one of the most effective antiarrhythmic drugs available. However, its use is often limited by side-effects, mainly hypo- or hyperthyroidism. As AMD displays direct toxic effect on different cell types, we investigated the cytotoxic effect of AMD and its main metabolite, desethylamiodarone (DEA), in thyroid (TAD-2) and nonthyroid (HeLa) cell lines. Both AMD and DEA displayed a dose-dependent toxicity in TAD-2 and HeLa cells, although DEA was more effective. Both TAD-2 and HeLa cells underwent apoptosis, as evidenced by plasma membrane phosphatidylserine exposure and DNA fragmentation. Inhibition of protein synthesis with cycloheximide and inhibition of endogenous peroxidase activity with propylthiouracil did not affect this AMD- and DEA-induced apoptosis in TAD-2 cells. Western blot analysis did not display variations in the

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ODIUM, POTASSIUM, and calcium channels are the primary targets of clinical antiarrhythmic agents (1). Class III antiarrhythmic drugs, the category undergoing the most active development, reduce mainly potassium and calcium ion channel conductance (2). Treatment of cardiac dysrhythmias with the most effective member of the class III antiarrhythmic drugs, the iodinated benzofuran derivative amiodarone (AMD), is limited by its pulmonary toxicity and might be associated with the development of thyroid diseases producing either hypo- or hyperthyroidism (3– 6). AMD-induced thyroid dysfunction occurs in 16 – 49% of patients soon after the initiation of treatment or even long after drug withdrawal (7, 8). AMD treatment can cause both hypothyroidism and thyrotoxicosis due to excess iodine or to a destructive process (6, 9, 10). AMD-induced thyrotoxicosis may occur in apparently normal thyroid glands or in patients with underlying thyroid pathology, such as nodular goiter or Graves’ disease (11). During chronic treatment, AMD and its metabolites reach high concentrations in several tissues, including the thyroid gland (12, 13). The massive increases in iodine and high intrathyroidal concentration of AMD and its metabolites Received May 24, 2000. Revision received July 31, 2000. Accepted August 9, 2000. Address all correspondence and requests for reprints to: Dr. Mario Vitale, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Via S. Pansini 5, Naples 80131, Italy. E-mail: [email protected]. * This work was supported in part by Ministero dell’Universita` e della Ricerca Scientifica (to G.F.F. and E.M.), Consiglio Nazionale delle Ricerche (to M.V.), and the University of Pisa (fondi ateneo).

expression of p53, Bcl-2, Bcl-XL, and Bax proteins during the treatment with AMD and DEA. Generation of reactive oxygen species, investigated by flow cytometry with dichlorofluorescein diacetate, did not show the production of free radicals during drug treatment. Furthermore, Western blot analysis of cytosolic and mitochondrial fractions prepared from AMD-treated cells demonstrated that AMD induces the release of cytochrome c into the cytosol from the mitochondria. These data indicate that AMD induces cytochrome c release from mitochondria, triggering apoptosis through an iodine-independent mechanism, and that this process is not mediated by modulation of p53, Bcl-2, Bcl-XL, or Bax protein expression and does not involve the generation of free radicals. (J Clin Endocrinol Metab 85: 4323– 4330, 2000)

are responsible for the changes in thyroid function observed in many patients during chronic treatment. The toxicity of AMD and desethylamiodarone (DEA), a metabolite generated in vivo (14), has also been demonstrated in several cell systems. A cytotoxic effect of AMD has been documented in thyroid cells such as FRTL-5 and primary cultures of human thyroid follicles (15), hamster lung cells (16), and human endothelial cells (17). In FRTL-5 cells, AMD displayed direct dose-dependent cytotoxicity, which was only partially blocked by perchlorate and methimazole. Despite a number of studies in both animals and cultured cells, the pathophysiological mechanisms of AMD-induced cytotoxicity remain poorly understood. AMD-induced hypothyroidism in rats is associated with specific ultrastructural features of necrosis and apoptosis of the thyroid gland, and thyrotoxicosis in human is associated with cytokine release from the damaged gland (18, 19). The question of whether AMD-induced cytotoxicity relies on a necrotic or an apoptotic process, however, remains unanswered. Apoptosis or programmed cell death differs from necrosis, because it is an active process of cell selfdestruction requiring the activation of a genetic program that leads to changes in morphology, DNA fragmentation and protein cross-linking (20, 21). The apoptotic pathways are triggered by environmental signals, cytokines, and growth factors and by pathological stimuli such as radiation and anticancer drugs (22–26). Recently, we demonstrated that iodide excess induces apoptosis by the generation of free radicals (27). In the present study we investigated whether AMD induces apoptosis and whether it exerts direct cyto-

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toxicity or works through high iodine loading, demonstrating that AMD induces cytochrome c release from mitochondria, triggering apoptosis through an iodine-independent mechanism. Materials and Methods Cells and chemicals The TAD-2 cell line, obtained by simian virus 40 infection of human fetal thyroid cells, was a gift from Dr. T. F. Davies, Mount Sinai Hospital (New York, NY). TAD-2 and endometrial carcinoma cells (HeLa) were cultured in a 5% CO2 atmosphere at 37 C in DMEM supplemented with 10% FCS. Medium was changed every 3– 4 days. Cells were detached by 0.5 mmol/L ethylenediamine tetraacetate (EDTA) in calcium- and magnesium-free PBS with 0.05% trypsin. Amiodarone was purchased from Sigma (St. Louis, MO), and desethylamiodarone was provided by Sanofi (Montpelier, France). Both drugs were solubilized and stored in 10% dimethylsulfoxide (DMSO) at 10⫺3 mol/L. Complete solubilization of the drugs was checked by visual inspection after extensive high speed centrifugation. Cycloheximide was purchased from Sigma. A 10⫺3 mol/L stock solution of 6-propyl-2-thiouracil (PTU) (Sigma) was prepared at basic pH, buffered at pH 7.5 by HCl.

DNA electrophoresis Cells collected by centrifugation were washed in PBS, lysed in 300 ␮L 0.5% Triton X-100, 5 mmol/L Tris-buffer (pH 7.4), 20 mmol/L EDTA for 20 min at 4 C and centrifuged at 13,000 rpm for 30 min. Centrifugationresistant low molecular weight DNA was extracted with phenol/chloroform, precipitated with ethanol, and incubated with 0.5 ␮g/ml deoxyribonuclease-free ribonuclease A for 30 min at 37 C. DNA with loading buffer was electrophoresed in 1% agarose and 1 ␮g/ml bromide at 50 V in 45 mmol/L Tris-borate and visualized by UV.

Cell death measurements The annexin V assay for determination of the apoptosis/necrosis ratio was performed as follows. Cells were washed twice with cold PBS, resuspended in 10 mmol/L HEPES (pH 7.4), 140 mmol/L NaCl, and 2.5 mmol/L CaCl2, and incubated for 15 min at room temperature with fluorescein-conjugated annexin V (PharMingen, San Diego, CA) and 5 ␮g/ml propidium iodide. Cells were analyzed within 1 h by flow cytometry using a FACScan (Becton Dickinson and Co., Mountain View, CA). Estimation of cell death by flow cytometry was performed as follows. Floating and adherent cells obtained by trypsin/EDTA were collected, washed in cold phosphate-buffered saline (PBS), and fixed in 70% cold ethanol for 30 min. Ethanol was removed by PBS wash, and cells were incubated in PBS, 50 ␮g/ml propidium iodide, and 10 ␮g/ml deoxyribonuclease-free ribonuclease A overnight at 4 C. Cells were then analyzed by flow cytometry. The percentage of dead cells was calculated by dividing the number of cells displaying red fluorescence lower than the G0-G1 diploid peak by the total number of collected cells ⫻ 100.

Antibodies and Western blot analysis Mouse monoclonal antibody to p53 was purchased from Transduction Laboratories (Lexington, KY); mouse monoclonal antibody to Bcl-2 and rabbit polyclonal antibodies to Bcl-X and Bax were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody to cytochrome c was purchased from Alexis (Laufelfingen, Switzerland). Cells were washed in cold PBS and lysed for 10 min at 4 C with 1 mL lysis buffer [50 mmol/L Tris (pH 7.4), 0.5% Nonidet P-40, and 0.01% SDS] containing protease inhibitors. Lysates from adherent cells collected by scraping and from floating cells were centrifuged at 12,000 ⫻ g for 15 min at 4 C. The protein concentration in cell lysates was determined by protein assay (BioRad Laboratories, Inc., Richmond, CA), and 50 ␮g total protein from each sample were boiled for 5 min in Laemmli sample buffer [125 mmol/L Tris (pH 6.8), 5% glycerol, 2% SDS, 1% ␤-mercaptoethanol, and 0.006% bromophenol blue]. Proteins were separated by SDS-

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PAGE and transferred onto nitrocellulose membrane (Hybond-ECL nitrocellulose, Amersham Pharmacia Biotech, Rainham, UK). The acrylamide concentration was 12% for p53 and Bcl-XL, 15% for Bcl-2 and Bax. Membranes were blocked by 5% nonfat dry milk, 1% ovalbumin, 5% FCS, and 7.5% glycine. After three washes, the membranes were incubated for 1 h at 4 C with 0.5 ␮g/ml mouse monoclonal or rabbit polyclonal primary antibodies in PBS. After three additional washes, filters were incubated for 1 h at 4 C with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies (Bio-Rad Laboratories, Inc.) diluted 1:2000 in PBS and Tween-20. After a final wash, protein bands were detected by an enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Fluorescent measurement of intracellular reactive oxygen species (ROS) TAD-2 cells were collected by mild trypsinization; washed in PBS; resuspended in PBS, 10 ␮mol/L 5,6-carboxy-2⬘,7⬘-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Inc., Eugene, OR), and 5 ␮g/ml propidium iodide at 37 C; and kept in DCFH-DA thereafter. DCFH-DA is a compound taken up by the cells and trapped in a nonfluorescent deacylated form (DCFH). DCFH is oxidized by ROS to a fluorescent form. After 1-h incubation, cells were analyzed by FACScan with excitation at 495 nm and emission at 525 nm wavelength. Damaged cells leaking DCFH because no longer intact were stained by the nonmembrane-permeable dye propidium iodide and excluded.

Preparation of cytosolic and mitochondrial fractions Untreated and AMD-treated TAD-2 cells were resuspended in 20 mmol/L HEPES (pH 7.5), 10 mmol/L EDTA, 1 mmol/L dithiothreitol, 300 mmol/L sucrose, and protease inhibitors. After several passages through a fine needle, intact cells and nuclei were removed by centrifugation at 1,000 ⫻ g for 10 min, and the supernatant was subjected to centrifugation at 10,000⫻ g for 30 min. The pellet fraction, containing mitochondria, and the supernatant, containing cytosol, were analyzed by Western blot.

Results AMD and DEA are cytotoxic to TAD-2 cells

The immortalized fetal thyroid cell line TAD-2 was treated with varying concentrations of AMD and DEA for 24 h. Both drugs induced a dramatic change in the morphology of the cells, which appeared small, rounded, and floating in the medium (not shown). As degradation and loss of DNA occur in death cells, the number of death cells with hypodiploid DNA content was determined by flow cytometric analysis of both floating and adherent cells (Fig. 1A). The cytotoxic effect of AMD and DEA was dose dependent and reached 50% at 25 and 15 ␮mol/L, respectively, whereas the diluent DMSO alone failed to induce any effect. Time-course experiments using 40 ␮mol/L AMD and 20 ␮mol/L DEA indicated a loss of DNA content by the cells initiated after 12 h of stimulation (Fig. 1B). Cytotoxicity by AMD and DEM is an apoptotic process

Apoptosis and necrosis can be induced by the same toxin at different concentrations, and both are characterized by DNA loss of cellular content. Thus, to determine whether cytotoxicity by AMD and DEA is an apoptotic process, TAD-2 cells were treated with the drugs, then DNA fragmentation was analyzed by agarose gel electrophoresis, and plasma membrane phosphatidylserine exposure was analyzed by annexin binding. DNA analysis by agarose gel electrophoresis after 24-h

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FIG. 1. Dose-response and time course of AMD- and DEA-induced cytotoxicity measured by flow cytometry. TAD-2 cells were cultured for 24 h, then treated with varying concentrations of AMD and DEA for 24 h (A) or with 40 ␮mol/L AMD or 20 ␮mol/L DEA for different times (B). Floating and adherent cells were collected, stained with propidium iodide, and analyzed by flow cytometry. The percentage of death cells was determined by gating the cells with hypodiploid DNA content. F, AMD; E, DEA. A, Open rhombuses represent the diluent DMSO alone at the same concentrations as in the presence of the drugs. Results are reported as the percentage of hypodiploid cells and are from three separate experiments.

cells bound annexin V, and the majority of annexin V-stained cells still retained plasma membrane integrity, remaining impermeable to propidium iodide, thus demonstrating that the cells died with apoptotic modalities. Induction of apoptosis by AMD and DEA is not restricted to thyroid cells

Toxicity of AMD was previously demonstrated in lung and liver cells both in vivo and in culture. To determine whether apoptosis by AMD and DEA is a phenomenon restricted to thyroid cells, we replicated the experiments in HeLa cells. Analysis of hypodiploid DNA content by flow cytometry (Fig. 4A) demonstrated that the toxicity of AMD and DEA occurred in HeLa cells at concentrations similar to those acting on TAD-2 cells (Fig. 4B), whereas DNA analysis by agarose gel electrophoresis showed the characteristic DNA fragmentation pattern of apoptosis (not shown). FIG. 2. Analysis of low molecular weight DNA by gel electrophoresis. TAD-2 cells were cultured for 24 h in the presence of 40 ␮mol/L AMD or DEA. Centrifugation-resistant low molecular weight DNA was extracted from the cells, electrophoresed in 1% agarose and 1 ␮g/ml propidium bromide in Tris-borate buffer, and visualized by UV. Low molecular weight DNA with characteristic apoptotic internucleosomal fragmentation was evident in the presence of the drugs. CTRL, DNA of untreated cells.

exposure to AMD and DEA, showed the characteristic DNA fragmentation pattern of apoptosis (Fig. 2). Loss of plasma membrane asymmetry before loss of membrane integrity was searched by simultaneous staining of the cells with annexin V and propidium iodide (Fig. 3). After 12 h of treatment with 40 ␮mol/L AMD and 20 ␮mol/L DEA, 40 – 45% of the

Inhibition of protein synthesis and thyroperoxidases (TPOs) are ineffective on AMD- and DEA-induced apoptosis

The involvement of protein synthesis in this type of drug-induced apoptosis was determined by treating TAD-2 cells for 24 h with 40 ␮mol/L AMD or 20 ␮mol/L DEA in the presence of varying concentrations of cycloheximide. This inhibitor of protein synthesis was used at a concentration of 1 ␮mol/L or lower, which was nontoxic for TAD-2 cells while inhibiting macromolecular synthesis (25, 27). Cycloheximide was completely ineffective on the apoptosis induced by the two drugs, as determined by flow cytometry (Fig. 5A). We previously demonstrated that molecular iodine, gen-

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FIG. 3. Annexin V assay for determination of the apoptosis/necrosis ratio. TAD-2 cells were untreated (CTRL) or treated with AMD or DEA for 12 h. Then, the cells were incubated with fluorescein-conjugated annexin V (abscissa) and propidium iodide (ordinate) and analyzed by flow cytometry. Intact cells are located in the lower left quadrant, necrotic cells permeable to propidium iodide are in the upper right and left quadrants, the apoptotic cells stained by annexin V and unstained by propidium iodide are in the lower right quadrant.

FIG. 4. Flow cytometry and dose responses of AMD- and DEA-induced cytotoxicity in HeLa cells. A, HeLa cells were treated with 40 ␮mol/L AMD for 24 h, then floating and adherent cells were collected, stained with propidium iodide, and analyzed for DNA content by flow cytometry. B, HeLa cells were cultured for 24 h, then treated with varying concentrations of AMD (F) or DEA (E) for 24 h. The percentage of death cells was determined by gating the cells with hypodiploid DNA content, as shown in A.

erated by oxidation of iodide by endogenous TPO, induces apoptosis in thyroid cells (27). To determine whether the iodide released by AMD and DEA was involved in this drug-induced apoptosis, TPO activity was inhibited by PTU in TAD-2 cells, and drug toxicity was determined. PTU inhibits in a dose-dependent manner the TPO activity in TAD-2

cells at nontoxic concentrations (⬍600 ␮mol/L) (27). Although iodine-induced apoptosis was completely blocked by 300 ␮mol/L PTU, neither AMD- nor DEA-induced apoptosis was sensitive to PTU at any concentration, demonstrating that AMD and DEA do not induce apoptosis through an iodine-mediated mechanism (Fig. 5B).


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FIG. 6. Western blot analysis of p53, Bcl-2, Bcl-XL, and Bax expression in TAD-2 cells treated with 40 ␮mol/L AMD or 20 ␮mol/L DEA for 0, 12, and 24 h. Fifty micrograms of each sample were loaded in the gel. The acrylamide concentration was 12% for p53 and Bcl-XL and 15% for Bcl-2 and Bax.

FIG. 5. Effects of cycloheximide and PTU on AMD- and DEA-induced apoptosis. TAD-2 cells were treated for 24 h with 40 ␮mol/L AMD (F) or 20 ␮mol/L DEA (E) alone or with different concentrations of cycloheximide (A) or PTU (B). Apoptosis induced by 48-h treatment with 50 ␮mol/L KI is also shown (䡺). The percentage of apoptotic cells was determined by flow cytometry. Results are reported as the percentage of dead cells and are from three separate experiments.

Expression of p53, Bcl-2, Bax, and Bcl-XL is unchanged in AMD- and DEA-induced apoptosis

The expression of pro- and antiapoptotic proteins was investigated by Western blot analysis. Thyroid cells were cultured in the presence of 40 ␮mol/L AMD or 20 ␮mol/L DEA for 0, 12, and 24 h, and total protein extracts were analyzed with specific antibodies (Fig. 6). The expression of the proapoptotic protein p53 did not change upon drug stimulation. Also, the proteins belonging to the Bcl-2 family (Bcl-2, Bcl-XL, and Bax), did not show any quantitative variation during drug treatment. These results agree with cycloheximide results and demonstrate that apoptosis induced by AMD and DEA is not associated with a variation in the ratios between the death agonist Bax and antagonists Bcl-2 and Bcl-XL. ROS are not generated during AMD- and DEA-induced apoptosis

To asses whether ROS were generated during apoptosis, we used the oxidation-sensitive fluorescent probe DCFH-DA and propidium iodide in cells treated with varying AMD or DEA concentrations or with a constant drug concentration at different times. DCFH-DA is a compound readily taken up by the cells and trapped in a nonfluorescent deacylated form (DCFH). DCFH is oxidized by ROS to a fluorescent form that can be measured

by FACS. Cells stained by propidium iodide were excluded because DCFH leaks out of late apoptotic cells whose membrane is no longer intact. Treatment with both AMD and DEA for 24 h did not induce variations in ROS cell content at any concentration (Fig. 7A). Time-course measurement of the cellular content of ROS during constant drug treatment did not reveal generation of free radicals (Fig. 7B). These results, also supported by the observation that the antioxidant N-acetyl-l-cysteine (Sigma) did not inhibit apoptosis (not shown), demonstrate that ROS production is not involved in this type of druginduced apoptosis. AMD and DEA induce cytochrome c release into the cytosol

Mitochondria play a pivotal role in several apoptotic processes. Cytochrome c can be released from the mitochondria into the cytosol, where it binds Apaf-1, which then activates caspase-9 that, in turn, activates caspase-3. To determine whether this pathway is activated by AMD and DEA, we examined whether these drugs induce cytochrome c release into the cytosol. Drug-treated TAD-2 cells were collected and fractionated into cytosolic and mitochondrial fractions. The presence of cytochrome c in these fractions was detected by Western blot analysis using an anticytochrome c monoclonal antibody. Cytochrome c was detected in the cytosol after 24 h of treatment with AMD (Fig. 8) as well as DEA (not shown), with a concomitant decrease in cytochrome c in the mitochondrial fraction. Discussion

Although AMD-induced thyroid dysfunction can produce either hyperthyroidism or hypothyroidism, several lines of evidences support the concept that a common central event is follicular cell disruption. Degeneration of individual thyrocytes up to total follicular disruption have been described in the glands of some patients by thyroidectomy and by needle aspiration biopsy (28, 29). In animal models, AMD

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FIG. 7. Estimation of intracellular ROS levels. Cells were treated with varying concentrations of AMD and DEA for 24 h (A) or with 40 ␮mol/L AMD or 20 ␮mol/L DEA for different times (B). Floating and adherent cells were collected, incubated with a DCFH-DA probe for 1 h, and analyzed by FACS. Contemporaneous staining with propidium iodide was used to exclude cells whose membrane was no longer intact. F, AMD; E, DEA. Results are reported as the percentage of dead cells and are from three separate experiments.

FIG. 8. Cytochrome c release from mitochondria in AMD-treated cells. TAD-2 cells were untreated (CTRL) or treated with 40 ␮mol/L AMD for 24 h. Cells were harvested, and cytosolic and mitochondrial fractions were prepared. The levels of cytochrome c were determined by Western blot analysis with anticytochrome c monoclonal antibody.

induces ultrastructural changes, including marked distortion of thyroid architecture and cell degeneration (18). In vitro studies of AMD also demonstrated a cytotoxic effect of this drug in a number of cell types (15–17). The present study demonstrates that AMD induces programmed cell death in thyroid cells as well as in a cell line of nonthyroid origin. These findings confirm at a molecular level the observation that the thyroid of AMD-treated rats displays a histological pattern of apoptosis (18). This type of cell death is an active process that can require the regulation of expression of proand antiapoptotic proteins. Apoptosis is differentially affected by protein synthesis inhibition, depending on the cell system and the triggering factor (30). Some types of apoptosis do not require protein synthesis, and entry into the apoptosis pathway does not involve transcriptional activity. This type of cell death involves interaction of protein death domains and activation of proteases of the caspase cascade already present in the cell. Many membrane receptors, such as Fas/ APO-1/CD95 and tumor necrosis factor receptor-1, induce

cell death with these modalities, and inhibition of protein synthesis increases susceptibility to apoptosis through the blockage of survey mechanisms (31). In AMD-induced apoptosis, as in iodine excess-induced apoptosis in the thyroid cell, cycloheximide is completely unable to modulate the self-destruction process (27). The lack of modulation of apoptotic proteins was also confirmed by Western blot, which demonstrated a constant expression of the major components of the Bcl-2 family. The p53 tumor suppressor gene has been demonstrated to play a crucial role in some forms of apoptosis such as that induced by irradiation or after treatment with some chemotherapeutic compounds (32, 33), whereas other types of apoptosis are independent of this transcription factor (25). As in iodine excess (27), AMD treatment is not associated with modulation of p53 expression. These results indicate that AMD acts by interfering with biochemical pathways or disrupting subcellular organelle functions that activate the apoptotic pathway. In patients developing thyrotoxicosis, a mixture of apoptosis and necrosis might occur, and when AMD and iodine reach a very high intrathyroid concentration, necrosis might be dominant, as shown by elevated serum interleukin-6 levels (10). Our results in immortalized cell lines demonstrate that cytotoxicity is caused by a direct AMD effect on the cell, although lymphocytes and cytokines may also contribute, as a secondary mechanism, to the in vivo cytotoxic process. The role of iodine vs. a direct drug toxicity of AMD has been a long-standing debate. AMD contains 37.2% iodine by weight, and it is stored in adipose tissue, with the consequence that a large amount of iodide is continuously released up to several months after drug withdrawn (14, 34). Although AMD and iodide excess induce different patterns of ultrastructural changes in rat thyroid, iodide released from


AMD may participate in thyroid toxicity (18). Although in TAD-2 cells, apoptosis by iodide excess and AMD have in common some molecular features (independence from protein synthesis, no modulation of p53 and Bcl-2 family proteins), the present study demonstrates that induction of apoptosis by AMD in cells in culture is due to a direct effect of the drug, and iodide does not contribute to its toxicity, although higher concentrations of AMD might release sufficient iodide to participate in cell damage through a TPOdependent mechanism (15). This conclusion is supported by the following evidences: 1) PTU does not inhibit AMD and DEA toxicity, but it completely blocks iodide-induced apoptosis; 2) AMD and DEA also induce apoptosis in nonthyroid cells, whereas iodide does not; and 3) ROS are generated by iodide excess, whereas AMD and DEA treatment is not associated with free radical production. Apoptosis mediated by death receptors, such as FAS or tumor necrosis factor receptors, is regulated and executed by a group of cysteine proteases, known as caspases, that become activated by proteolytic processing (35–39). In a different apoptotic pathway, UV irradiation, chemotherapeutic drugs, and growth factor withdrawal generate death signals that change the conductance properties of the outer mitochondrial membrane, culminating in loss of outer mitochondrial membrane integrity that, in turn, provokes the translocation of cytochrome c from the mitochondria to the cytosol (40 – 44). Cytochrome c release from the mitochondria initiates a cascade that leads to the activation of caspase-3 through caspase-9 and Apaf-1 association (45, 46). However, the apoptotic mitochondrial pathway can be activated by cytosolic factors generated by a mitochondria-independent pathway, thus amplifying the caspase cascade and ensuring rapid and massive cell death (47). Our results support the idea that the mitochondrial pathway is involved in AMDinduced apoptosis. Further work may clarify whether cytochrome c is released by a direct AMD effect on mitochondrial potassium or calcium ion channel conductance or by mitochondrial-independent cytosolic factors. In conclusion, these data indicate that AMD and its metabolite DEA induce apoptosis in thyroid and nonthyroid cells. In the range of concentrations used in this study, iodine does not contribute to the toxicity of these iodine-rich drugs. Apoptosis induced by AMD and DEA is not mediated by modulation of p53, Bcl-2, Bcl-XL, or Bax protein expression and does not involve the generation of free radicals, whereas it induces the release of mitochondrial cytochrome c into the cytosol. References 1. Nattel S. 1999 The molecular and ionic specificity of antiarrhythmic drug actions. J Cardiovasc Electrophysiol. 10:272–289. 2. Varro A, Virag L, Papp JG. 1996 Comparison of the chronic and acute effects of amiodarone on the calcium and potassium currents in rabbit isolated cardiac myocytes. Br J Pharmacol. 117:1181–1186. 3. Martin WJ, Howard DM, Chest. 1985 Amiodarone-induced lung toxicity. In vitro evidence for the direct toxicity of the drug. Am J Pathol. 120:344 –350. 4. Wilson BD, Clarkson CE, Lippmann ML. 1991 Amiodarone-induced pulmonary inflammation. Correlation with drug dose and lung levels of drug, metabolite, and phospholipid. Am Rev Respir Dis. 143:1110 –1114. 5. Harjai KJ, Licata AA. 1997 Effects of amiodarone on thyroid function. Ann Intern Med. 126:63–73. 6. Martino E, Aghini-Lombardi F, Bartalena L, et al. 1994 Enhanced suscepti-

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