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CHAPTER 10 Mechanisms of Organotin-Induced Apoptosis Željko Jakšić* Ruđer Bošković Institute, Center for Marine Research Rovinj, Croatia Abstract: Apoptosis, a specific type of programmed cell death, is characterized by cell shrinkage, nucleus condensation and fragmentation, plasma membrane blebbing and final engulfment by neighboring cells or professional phagocytes. The molecular mechanisms of organotin-induced apoptosis, which involve a series of biochemical regulators and molecular interactions, have been extensively studied in different cell types but the apoptotic pathway mechanisms and signaling still remain unexplained in some detail. Apoptosis may be triggered and modulated by caspase-independent, and more frequently by caspase-dependent pathways. Pro-caspase activation is driven by death receptors, and/or by a mitochondrion-mediated mechanism. Although both pathways were described, the mitochondrial mechanism seems to be the most important one in organotin-induced apoptosis. Organotin compounds trigger cytoskeletal modifications and disruption of mitochondrial functions. Generally, the apoptotic pathway induced by organotins starts with their interactions with cellular components leading to perturbation of intracellular Ca2+ homeostasis, the latter especially triggered by endoplasmic reticulum stress, and intracellular Ca2+ concentration increase, cessation of ATP and reactive oxygen species production and loss of mitochondrial membrane potential. These events are followed by cytochrome c release from mitochondria to cytosol, apoptosome formation and final executioner caspase activation. The increase in intracellular Ca2+ level and the consequent mitochondrial cytochrome c release play critical steps in organotin-induced apoptosis. The process not only depends on cell type and sensitivity but also on organotin chemical characteristics and insult intensity. New and promising research on mechanisms of organotin-induced apoptosis is focused on the characterization of organotin interactions with apoptosis-related proteins and regulation of gene expression.

Keywords: Apoptosis - organotin-caspase modulation - endoplasmic reticulum stress - intracellular (Ca2+)oxidative stress – mitochondria - MAP-kinase. APOPTOSIS: THE PROGRAMMED CELL DEATH PROCESS Overview The term “Apoptosis”, originating from Greek (apo = from, ptosis = fall), describing the falling off of leaves from trees or of petals from flowers, is used to describe a specific type of programmed cell death characterized by peculiar morphological features driven by biochemical and cellular processes. The occurrence of apoptotic events is recorded in normal physiological and pathological processes. Apoptosis function is to provide normal embryonic and tissue development, metamorphosis, immune system clonal selection, sexual development and organism homeostasis i.e. balances cell death and proliferation, elimination of used cells, and deletion of damaged or viral infected cells. The term was coined at the beginning of the 70’s of the 20th century [1], and the process was characterized by cell rounding-up, retraction of pseudopodes, reduction of cellular and nuclear volume, nucleus condensation and fragmentation, possible slight modification of cytoplasmic organelles, plasma membrane blebbing though maintaining its integrity until the ultimate stage of the process, possible formation of membrane-enclosed particles - apoptotic bodies, and final engulfment by neighboring cells or professional phagocytes [2]. The rapid recognition, ingestion and degradation of apoptotic cells prevent the exudative inflammation of surrounding tissue, unlike the consequence of the accidental cell death, termed necrosis. Necrosis is caused by the irreversible swelling of cytoplasm and its organelles, loss of membrane integrity, cell lysis and noxious cell constituents’ release. Moreover, the Nomenclature Committee of Cell Death continuously improves, recommends and unifies the morphological and enzymological criteria, functional aspects and immunological characteristics to define and classify the cell death types [3]. The initiation of cell death mode mechanisms is based on insult type and intensity, but the crucial factors for initiation of those processes are mitochondrial function status and intracellular ATP level. *Address correspondence to Željko Jakšić: Ruđer Bošković Institute, Center for Marine Research G. Paliage 5, 52210 Rovinj, Croatia; E-mail: [email protected] Alessandra Pagliarani, Fabiana Trombetti and Vittoria Ventrella (Eds) All rights reserved - © 2012 Bentham Science Publishers

150 Biochemical and Biological Effects of Organotins

Željko Jakšić

The apoptotic process involves several morphological events as well as specific though not exclusive and selfsufficient energy-dependent biochemical and cellular changes such as mitochondrial (trans)-membrane potential dissipation (∆Ψm), caspase activation, proteolysis, DNA fragmentation and phosphatidylserine (PS) residues exposure/flipping which characterize the apoptotic event. Morphologically similar but distinct apoptotic subtypes are triggered trough different biochemical pathways. Mechanisms of Apoptosis Apoptotic cell death may be triggered by several specific signaling pathways [2]. Two of those include the caspase family proteases: the death receptor- and the mitochondrion-mediated procaspase activation. The set of specific cysteine-dependent aspartate-specific proteases known as caspases represents the biochemical bases and play central execution role in the apoptotic process [4]. They are a specifically activated set of proteases with a conservative pentapeptide active site, His-Arg-Phe-Asn/His/Cys-Glu, which cleaves cellular proteins after the aspartic acid residues. This cleavage leads to cellular proteins inactivation, making them responsible for the morphological features typical of apoptosis [2, 5]. Actually, they are activating other caspases and autoactivating themselves and consecutively cleave the structural components such as nuclear laminis and cytoskeletal proteins, negative regulators of apoptosis and either inactivate apoptotic inhibitors or produce proapoptotic fragments. Nevertheless, the endoplasmic reticulum (ER) stress and ER disturbance of Ca2+ transport also trigger the pro-caspase activation and mitochondrion-mediated pathway. Apart from the caspase-driven mechanism, other proteases such as granzyme (GrA), cathepsins, proteasome and serine proteases play a role in the caspase independent pathways and trigger apoptosis [6, 7]. The extrinsic, death receptor-mediated procaspase activation pathway is characteristic for procaspase-8 and -10 activations [5]. It is a fundamental pathway in tissue and immune system homeostasis. The induced proximity mechanism of procaspase-8 and its autoactivation to caspase-8 leads to various substrate activations in different cell types. In some cell types caspase-8 directly activates the downstream procaspase, procaspase-3, by proteolytic cleavage while in other cell types it activates the mitochondrion-mediated pathway by acting on Bid proteins which belong to the Bcl-2 protein family [8]. The direct or indirect death signal to mitochondria is followed by intrinsic or mitochondrion-mediated procaspase activation [5]. This mechanism is extensively used in response to different extracellular causes and intracellular insults like DNA damage. The mitochondrion-dependent intrinsic mechanism is mediated by the release of several mitochondrial proteins to the cytosol. Actually, there are two redundant pathways: the former includes release of cytochrome c, activation of procaspase-9 and procaspase-3, subsequent activation of DNase and oligosomal DNA fragmentation, while the latter involves Apoptosis-Inducing Factor (AIF) release, large-scale DNA fragmentation and peripheral chromatin condensation in a caspase-independent manner. The above mentioned caspase-8-mediated cleavage of pro-apoptotic Bid protein leads to truncated Bid (tBid) translocation from cytoplasm to mitochondria and consequent cytochrome c release to the cytoplasm [9]. Cytochrome c is bound to the inner mitochondrial membrane and associated with the phospholipid cardiolipin. Its dissociation by cardiolipin peroxidation represents the critical step for cytochrome c release and mitochondrionmediated apoptosis induction [10]. Furthermore, the intrinsic or mitochondrial pathway may be driven by a caspaseindependent mechanism, which involves release of AIF and endonuclease G (EndoG) from mitochondria and their translocation to the nucleus. However, following the mitochondrion-mediated procaspase-activation pathway the mitochondria released cytochrome c associates with the Apoptotic Protease Activating Factor 1 (Apaf-1) and dATP/ATP (deoxyadenosine triphosphate/adenosine triphosphate) to form a heptametrical rosette structure, called apoptosome, which works as adaptor complex for procaspase-9 activation [11]. Both activated caspase-8 and -9 promote the activation of an effector caspase, procaspase-3, this latter being the merge point of extrinsic and intrinsic pathways. In addition, caspase-9 may activate the effector procaspase-6, the unique cytosolic caspase able to activate procaspase-8 in the cytochrome c driven pathway. Although Bid protein may represent a proapoptotic link between those two mechanisms they mainly operate independently of each other. Contrary to the caspase-dependent case, the caspase-independent perforin/GrA-mediated signaling pathway is characterized by GrA translocation to the cytosol through the Ca2+-dependent perforin-mediated pores. GrA releases

Mechanisms of Organotin-Induced Apoptosis

Biochemical and Biological Effects of Organotins 151

the active form of the GrA-activated DNase (GAAD) by cleaving GAAD inhibitors in the cytosol and triggering single strand DNA break formation [12]. About 30 years ago Ca2+ was noted as mediator of specific endonuclease activity and inducer of apoptotic DNA fragmentation which may be electrophoretically revealed by agarose gels [13]. The ER serves as Ca2+ storage in cells, and its pool depletion or overload can lead to changes in newly synthesized protein load thus resulting in ER stress [14]. With the presence of incorrectly folded proteins and/or accumulated protein signals, removal or remodeling of such proteins in the ER is provided by induction of specific mechanisms which in response to severe stress can contribute to apoptotic cell death. During prolonged ER stress and depletion or alteration in the Ca2+ transport system, the Ca2+-activated cysteine protease, calpain [15], cleaves and activates the procaspase-12 localized in the ER membrane. Caspase-12 continues the caspase cascade-activation without cytochrome c involvement and induces apoptosis [16]. Furthermore, several ER membrane proteins like Bax inhibitor 1, BAP31 and Spike participate in the apoptotic process by procaspase-8 activation, release of Ca2+ from the ER to the cytosol, and Ca2+ electrogenic uptake by mitochondria through the Ca2+ uniport. Both events, the pore formation by the Bcl-2 family proteins and Ca2+ regulation of inner-membrane permeability transition and pore opening, lead to the outer mitochondrial membrane permeabilization and to the release of proapoptotic proteins as mentioned above. Therefore, the mitochondrial (caspase-dependent) pathway is characterized by the release of cytochrome c from mitochondria to cytosol and subsequent activation of initiator caspase-9. The death receptor pathway involves Fas Associated Death Domain (FADD) translocation from cytosol to the cell membrane and consequent activation of caspase-8. Both activated apical caspases promote the activation of executioner caspase-3. Activated caspase-3 cleaves several substrates such as the inhibitor of caspase-activated DNase (ICAD) or DNA fragmentation factor-45 (DFF45), and releases caspase-activated DNase (CAD) which degrades DNA. The cleavage of another caspase-3 substrate, poly-(ADP-ribose) polymerase (PARP), disables the catalytic transfer of ADP-ribose polymers in response to DNA strand breaks. In some cell types the activated caspase-8 may activate cross-linkage between two pathways, by pro-apoptotic Bid protein cleavage, tBid translocation to the mitochondrial membrane and further enhancement of the mitochondrial pathway. ORGANOTIN-MEDIATED APOPTOSIS Mitochondria – the Initial Target of Organotins The ability of organotin compounds to induce apoptosis has been known for a long time [17, 18]. In particular, tributyltin chloride (TBTC) and triphenyltin chloride (TPhTC) are able to trigger cytoskeletal modifications and disruption of mitochondrial function by cessation of ATP synthesis, leading to incomplete reduction of O2 and subsequent production of reactive oxygen species (ROS) and ∆Ψm loss. However, while the very latest research indicates the involvement and possible mediation role of the Fas receptor in organotin-induced apoptosis [19], general knowledge of the basic mode of organotin action in the activation of the apoptotic process includes a large increase in intracellular (cytosolic) free Ca2+ concentrations ([Ca2+]i), subsequent mitochondrial permeability transition (MPT), release of cytochrome c from mitochondria, protein phosphorylation, caspase activation and programmed cell death promotion [20]. Based on differences in mitochondrial depolarization kinetics, cell type and organotin chemical reactivity and concentration, different scenarios of the cell death process are promoted. Tributyltin (TBT)-induced stress release of Ca2+ from the ER and/or mitochondria to cytosol, and perturbation of [Ca2+]i homeostasis due to massive Ca2+ release from intracellular stores, increased influx and/or extrusion inhibition, modulates protease, phospholipase and nuclease activities thus leading to various structural and functional disturbances of cell organelles [21]. Therefore the influence of Ca2+ on the apoptotic process is provided by stimulating cytochrome c release from mitochondria and/or participating in executioner caspase activation. The mechanisms of cytochrome c release from mitochondria to cytoplasm differ depending on the agents which disrupt mitochondrial functions. Cytochrome c export from mitochondria plays a central and critical step in organotin-induced caspase activation and apoptosis. Known and hypothesized organotin interactions with cell components and apoptotic signaling mechanisms are schematically drawn in Fig. (1).


EXTRACELULAR SPACE

Željko Jakšić

CELL MEMBRANE

organotin compounds

CYTOPLASM -Sn proteosome

Fas ligand

MPT ∆Ψm

Fas / CD95 DISC

caspase-8

TMT DMT

MITOCHONDRIA O2

FADD

Bcl2

Snn

Bid / p22

O2-•

ROS

H2O2

tBid / p15

procaspase-8 cytochrome c

apoptosome caspase-6

Ca2+

GrA procaspase-9

Apaf 1

PUMA

calpain GAAD

procaspase-6

TMT

EndoG caspase-12

procaspase-3

Smac/Diablo

DMT

AIF

caspase-9 IAP

CAD DNA f ragmentation

ICAD/DFF45 NUCLEUS caspase-3

PARP Cell shrinking

DNA damage

ENDOPLASMATIC RETICULUM p53

APOPTOSIS

Membrane blebbing p21 FADD

- Fas associated death domain

DISC MPT ∆Ψm ROS Snn

- death-inducing signal complex - membrane permeability transition - mitohondrial (trans)membrane potential - reactive oxygen species - stannin

AIF EndoG IAP Smac/Diablo

- apoptosis inducing f actor - endonuclease G - inhibitor of apoptosis protein - second mitochondrial activator of caspase /direct IAP binding protein with low pH protein

CAD ICAD/DFF45 PARP GrA GAAD

- caspase-activated DNase - inhibitor CAD / DNA f ragmentation f actor-45 - poly-(ADP-ribose) poymerase - granzyme - GrA activated DNase

DNA repair

activation inhibition translocation organotin action

Figure 1: The mechanisms of organotin-induced apoptosis. A proposed mechanistic model of interactions and related signaling mechanisms in organotin-induced apoptosis.

TBT is one of the best known mitochondrial toxins. It triggers apoptosis via the mitochondrial pathway and blocks mitochondrial ATP production. At low in vitro concentrations, usually up to 3 μM TBT, gradual ∆Ψm loss and membrane swelling is induced, whiles higher concentrations induce rapid MPT and necrosis. This apoptotic



pathway depends on mitochondrial outer membrane permeabilization status which is in turn regulated by the Bcl-2 family proteins, [Ca2+]i level, and ROS production. ROS play a critical role in both caspase-dependent and caspaseindependent pathways, by changing the mitochondrial membrane potential or activating PARP. The release of specific proteins (cytochrome c, AIF, EndoG etc.) from the mitochondrial intermembrane space into the cytosol enables either caspase cascade activation or caspase-independent orchestration of cell death [22]. Since the first records of TBT-induced apoptosis in rat thymocytes [17, 18] and of a signed mechanism independent of protein synthesis under conditions of depleted nuclear ATP [23], several cell types and organotin compounds have been used in research on organotin-induced programmed cell death. Disturbance of Intracellular Ca2+ Homeostasis In vitro treatment of human leukemia T cell line Jurkat T lymphocytes with 2 M TBT resulted into rapid ∆Ψm dissipation, leading to maximal cytochrome c release within 5-10 min and consequent caspase activation within 1 hour of exposure. Previous findings of direct mitochondrial ATP synthase inhibition and oxidative phosphorylation block by organotin compounds, and their known ability to act as a Cl-/OH- exchangers, provided a possible explanation for the rapid ∆Ψm loss. Although the chronology between cytochrome c release and ∆Ψm dissipation could not be assessed, concomitant cytochrome c export as a consequence of MPT induction and ∆Ψm dissipation was suggested [24]. In order to address this question Ca2+-loaded liver mitochondria, isolated by differential centrifugation, were exposed to 0.5 and 5.0 M TBT. Only Ca2+ release induced by the lowest 0.5 M TBT dose was inhibited by 1 M cyclosporine A, specific MPT inhibitory agent [25]. Nevertheless, mitochondria exposed to 0.5 M TBT showed cytochrome c release both in the presence and absence of increased [Ca2+]i. This finding suggested that at least two distinct mechanisms promoted cytochrome c release in mammalian liver mitochondria. The critical one was largely Ca2+-independent and involved a slow loss of ∆Ψm due to inhibition of respiration and uncoupling of oxidative phosphorylation. This process started soon after TBT exposure and lasted until TBT access in intact cells was restricted. ∆Ψm loss was then followed by mitochondrial swelling, osmotic imbalance across the inner mitochondrial membrane, rupture of the outer membrane and subsequent cytochrome c release from the inner mitochondrial membrane without MPT involvement. This mechanism was specific and independent of high [Ca2+]i concentrations. Conversely a second mechanism was strongly Ca2+-dependent and involved MPT. Undoubtedly, organotins act as effectors of intracellular calcium homeostasis by increasing plasma membrane permeability to calcium ions. At the same time, mitochondria actively sequester [Ca2+]i and possess high Ca2+ uptake efficiency which makes them act as intracellular buffer devices [26]. Employing the Fluorescence Activated Cell Sorting methodology and double staining strategy, it was possible to precisely detect responses and to distinguish among the molecular events induced by organotins in cells [27]. Immediately 30 s after addition of 2 M TBT to Jurkat T cells, mitochondrial hyperpolarization was evident and reached a maximum value in about 1 min. Mitochondrial permeability returned to the control value by 5 min and was completely lost after 15 min. The observed ∆Ψm loss could be prevented by Ca2+ chelating agents. A [Ca2+]i rise was recorded in the same cells in the first minute after TBT addition. Further experiments on Jurkat T cells cultivated in glucose-free media (to avoid glycolytic ATP production) upon 2 M TBT addition, showed an almost immediate increase in [Ca2+]i which attained a maximum value within 3 min, and maintained ∆Ψm up to 80 min. Jurkat T cells incubated in Ca2+-free medium prior to TBT exposure resulted in a slightly [Ca2+]i increase and rapid ∆Ψm loss. The overall of results suggested that [Ca2+]i elevation and ∆Ψm loss were uncoupled events in Jurkat T cells upon TBT exposure. The rapid rise in [Ca2+]i could result from direct interaction of TBT with mitochondrial component(s) controlling pore transition. Induction of caspase proteolytic activity and consequent apoptotic morphology were not observed in Jurkat T cells incubated in glucose- or Ca2+-free media but only in cells cultivated in RPMI 1640 media (cell culture media developed at Roswell Park Memorial Institute, Buffalo, NY, USA, frequently used for the culture of human normal and neoplastic leukocytes) thus suggesting that TBT induced apoptosis in Jurkat T cells was [Ca2+]i-dependent [27]. Several proapoptotic events were simultaneously analyzed by flow cytometry in Jurkat T cells exposed to 0.3 - 10 M TBT [28]. Although the cells showed a transient [Ca2+]i increase, the subpopulation with increased and steady-state [Ca2+]i correlated with cell membrane permeability loss and volume decrease in a dose-dependent manner. It was shown that DNA leader formation and the increase in [Ca2+]i levels in TBT- and TPhT- induced apoptosis follow a time- and dose-dependent manner not only in Jurkat T cells but also in rat testicular Leydig cells. Since the chelating agent 1,1-bis(2-aminophenoxy)ethane-N,N,N,N´tetraacetic acid (BAPTA) efficiently suppressed [Ca2+]i increase and apoptosis in TBT exposed Leydig cells, apoptosis was ascribed to a [Ca2+]i-mediated mechanism [29].


Željko Jakšić

The organotin compounds, TBTC and dibutyltin dichloride (DBTC), were used to induce an apoptotic pathway in rat thymocytes. In TBTC-exposed cells the increased [Ca2+]i was a consequence of stimulation of Ca2+ entry and reduced Ca2+ efflux from the cell, but also due to mobilization from the intracellular Ca2+ pool(s) [30]. The organotin-induced plasma membrane permeability allowed extracellular Ca2+ influx through the plasma membrane to provide the main contribution to significant [Ca2+]i increase. The time- and dose-dependent instant increase of [Ca2+]i and ROS production in rat thymocytes treated by DBT or TBT were followed by cytochrome c release and caspase activity. The pre-treatment of those cells with the Ca2+-chelating agent BAPTA, the mitochondrial electron transport chain-inhibitor rotenone and ruthenium red an inhibitor of mitochondrial Ca2+ channels, significantly reduced DBT- and TBT-induced ROS production in all cases [31]. These findings not only confirmed mitochondria as a primary source of ROS, but stressed the importance and leading role of [Ca2+]i increase in promoting ROS generation. In addition, the release of the cytochrome c from the inner mitochondrial membrane, caspase-activity and characteristic DNA fragmentation were negatively modulated by BAPTA and rotenone, indicating the involvement of [Ca2+]i and ROS in these processes. In the same way, the dose-related [Ca2+]i increase upon organotin treatment was the earliest observed intracellular molecular event in murine keratinocyte cells, and was followed by a consecutive increase of cellular or more precisely mitochondrial oxidative activity [32]. Moreover, a dose-dependent proapoptotic effect of TBTC was detected in rainbow trout leukocytes and nucleated erythrocytes. Mitochondrial swelling, observed by transmission electron microscopy, and ∆Ψm decrease were much more pronounced in erythrocytes than in leukocytes. The cytosolic cytochrome c content quickly increased in lymphocytes, while in erythrocytes a gradual elevation of cytochrome c and caspase-3 activity was observed [33]. The key event in TBT-driven apoptosis of trout hepatocytes was a significant increase in [Ca2+]i by mobilization from internal pools. Increased [Ca2+]i levels also activated a Ca2+-dependent cysteine protease, calpain, which degrades the protein kinase C  (PKC) thus modulating phosphorylation of Bcl-2 proteins in trout hepatocytes [34]. Similarly, extracellular Ca2+ influx, ROS generation and DNA fragmentation were observed on human breast adenocarcinoma epithelial cells, MCF-7, and human erythromyeloblastoid leukemia cells, K562, exposed to tributyltin benzoate (TBTB) and its halogenated derivates. The highest initial extracellular Ca2+ influx and prior DNA fragmentation were observed in TBTB-treated K562 cells. Apoptotic MCF-7 cells were significantly characterized by ROS production. The effects of organotin compounds were ascribed to the extent of extracellular Ca2+ influx [35]. The dose or impulse-strength dependence is not only typical of programmed cell death promoted by toxicants, but even the mechanism itself of the apoptotic pathway may depend on the impulse intensity. Pheochromocytoma cells exposed to different concentrations of TBTC showed an increased [Ca2+]i level, nuclear fragmentation and activation of caspase-3, but differences in the apoptotic pathways were detected depending on the TBTC dose. A moderate [Ca2+]i increase induced by 0.5 M TBT was mediated through the voltage-dependent calcium channel (VDCC). Under these conditions the apoptotic mechanism involved c-Jun N-terminal kinase (JNK) phosphorylation and activation. Conversely, 2 M TBTC promoted a large Ca2+ release from inositol 1,4,5-trisphosphate, involved ryanodine receptors, and was followed by ROS production [36]. In addition, the dose dependent effects of TBT on [Ca2+]i and viability of the rat pheochromocytoma cell line (PC12) were prevented by BAPTA or by the L-type VDCC blocker nicardipine [37]. Regarding the failed inhibition effect of calphostin C on PKC, which is known to act through phosphorylation of ionic channels including VDCCs [38], the proposed action of TBT on L-type VDCC was a direct impact with the lipophilic domain within the channels that is responsible for channel gating [37]. Caspase Modulation As caspase proteolytic cleavage inactivates the cell proteins, from a biochemical point of view, the apoptosis may be considered as the cell’s self-induced process of programmed proteolytic activity. The mechanism and kinetics of organotin-induced caspase activity has been studied in various cell types. The dependence of cell death scenario on intensity of cell death stimuli or toxicant concentration was investigated by studying the effect of TBTC and TPhTC [39] on Jurkat T cell line exposed to 0 - 2 M TBTC in vitro for 3 h. A clear dose-dependent increase in caspase activity accompanied by evident membrane blebbing and nuclear condensation was shown. The maximum of a 30fold increase in caspase activity at 2 μM TBTC was reached within 1 hour, while the decline in caspase activity and



plasma membrane breakdown occurred within 3 hours of exposure to higher TBTC concentrations (5 and 10 M TBTC). The human T cell lymphoma cell line, Hut-78 cells, showed the same pattern of dose-dependent increase in caspase activity, with a peak at 5 M TBTC, followed by activity decrease at higher TBTC concentrations. The general apoptotic/necrotic effect promoted by trialkylated organotin compounds was confirmed by exposure of Hut78 cells to TPhTC. In spite of a long lasting activation, Hut-78 cells exposed to low TPhTC concentrations (10 - 100 nM) showed a significant increase in caspase activity, while higher doses (0.5 and 1 mM) of TPhTC resulted into caspase activity inhibition and induction of necrosis. The concentration dependent activation of caspases is a crucial factor which governs cell death mechanisms. On considering the structural similarity between organotin compounds (three alkyl/phenyl groups coordinated with a central Sn) and a known caspase inhibitor, phenylarsine oxide (phenyl and oxygen coordinated with As), the formation of organotin complexes with vicinal thiol groups was hypothesed. Actually, caspase inactivation by the TBTC and TPhTC, by the thiol-dependent manner, was reversed the thiol reducing agent dithiothreitol, and provide evidence for a possible direct interaction of organotins with vicinal thiols but not monothiols, and confirmed the proposed inhibitory mechanism. In spite of these findings and the requirement of a reduced state of thiol groups for the proteolytic function, the caspase-3 crystal structure did not show a second cysteine in the vicinity of the caspase-3 cysteine active site, or cysteine pair in another region of the caspase-3 protein, to fully explain the observed effect [39]. A dose-dependent caspase activation accompanied by consequent apoptotic morphology was observed in TBT-exposed human peripheral blood lymphocytes. The early ∆Ψm loss and cytochrome c release in both cases suggested the involvement of the mitochondrial apoptotic pathway. On the other hand in peripheral blood lymphocytes the time-dependent caspase activation without loss of ∆Ψm, cytochrome c release and apoptotic morphology suggested a response to immunotoxic agents [40]. Contrary to human leukemia T cell lines and peripheral T lymphocytes, human granulocytes showed a different mechanism of apoptotic induction. The study of preapoptotic events in TBT-treated human eosynophils and neutrophils confirmed cell type specificity towards the induction of apoptosis. In vitro exposure of eosynophils to 2 M TBT resulted in a 14-fold increase of caspase-3 activity in 10 min and a slower increase in the next 2 h. In human neutrophils maximal 4-5-fold increase of caspase-3 activity was reached 30 min after incubation with 2 M TBT. Moreover, nuclear condensation and PS-exposure in TBT-exposed cells were observed after 30 min in eosynophils and after 120 min in neutrophils. The observed lower sensitivity and delay in TBT-induced apoptosis in neutrophils may be associated with a reduced level of pro-apoptotic Bax protein. However, rapid induction of 3caspase activity in human granulocytes was not accompanied by prior mitochondrial changes such as induction of MPT, significant ∆Ψm loss and cytochrome c release. The observed late dissipation of ∆Ψm and subsequent cytochrome c release raises the possibility of a mitochondrial-independent caspase-3 induction scenario in granulocytes [41]. The recent research of the novel organotin supramolecular coordination polymers 3∞[Me3SnCu(CN)2•(EN)2] and 3∞[Ph3SnCu(CN)2•(3-mpy)2] as a possible anticancer drugs showed their cell viability and growth inhibition specificity towards human breast cancer cell line ZR-75-1 in a dose-dependent manner. The flow-cytometric cell cycle analysis of treated ZR-75-1 cells showed specific apoptotic peak and G1 arrest compared to the control nontumorigenic breast tissue cells MCF10A, whereas the induced caspase-3 activity in treated ZR-75-1 cells confirmed its general mediator role in stress induced programmed cell death [42]. The in vitro studies of T-lymphocytes, isolated from nave rats’ thymuses, exposed to 1 μM TBT showed marked increase of caspase-8, -9 and -3 activities, as well as appearing induction of the caspase-activated DNase (CAD) and consecutive DNA fragmentation. The suggested mitochondrial involvement in the programmed cell death by the significant caspase-9 activity increase was confirmed by the findings of increased cytochrome c expression and loss of the mitochondrial function within 10 minutes of TBT exposure [43]. In rat hepatocytes TBT-induced apoptosis, mainly observed as nuclear chromatin condensation and DNA fragmentation, was found to be governed by two caspase-dependent mechanisms: the mitochondrion-driven and the death receptor-driven pathways [44]. Actually, after treatment with 2.5 M TBT a gradual increase of the adaptor protein FADD was detected in both cell membrane and cytosol and, after 75 min, a 6-fold increase in caspase-8 activity and a 19 fold-increase in caspase-9 activity and cytosolic cytochrome c level. A gradual decrease of Bid in the cytosol and increase of tBid in the non cytosolic fraction were also recorded. Both initiator caspases activate the effector caspase-3. Procaspase-3 maximal activation (107-fold increase) occurring 75 minutes after hepatocytes exposure to 2.5 M TBT was accompanied by PARP cleavage and disappearance of ICAD/DFF45 protein.


Željko Jakšić

Oxidative stress and Modes of Cell Death In organotin induced apoptosis the oxidative stress seems to play a significant role. The enhanced [Ca2+]i uptake leads to harmful effects for the mitochondrion itself, such as loss of ∆Ψm, and enhanced ROS production, and it is followed by MPT and membrane depolarization. The ultimate ∆Ψm breakdown by MPT triggers enhanced cytochrome c release and caspase activation. ROS (particularly H2O2) as important signaling molecules, play a key role in the modulation of gene expression, cell cycle progression, and programmed cell death [45]. ROS are mitochondrion-derived molecules and at the same time, they can target the mitochondrial membrane directly or indirectly (via ceramide generation), increase the mitochondrial membrane gating potential and promote cytochrome c release as well as programmed cell death [46]. Apoptosis induced by 0.01 - 0.1 M trimethyltin (TMT) in cerebellar granule cells was associated with increase in the level of ROS, especially of hydrogen peroxide. ROS generation could be inhibited not only by catalase or nitric oxide synthase inhibitors, but by the selective protein kinase C inhibitor and by glutamate receptor agonists which significantly diminished ROS production and oxidative stress. Therefore in cerebellar granule cells death mechanisms were shown to be finely modulated by glutamate receptor, protein kinase C and catalase activation [47]. TBTC preferentially induces apoptosis via H2O2 generation in T helper 1 (Th1) rather than T helper 2 (Th2) cells, probably because the Th1 cells have a lower content of the major cellular antioxidant glutathione (GSH). Accordingly the HP100 cells, derived from HL-60 cells (human promyelocytic leukemia), exhibited an 18-fold higher catalase activity and were 340-fold more resistant to H2O2 than HL-60 cells. They displayed a significantly lower induction of DNA leader formation, no ∆Ψm loss and resistance to TBTC-induced apoptosis too. This H2O2induced effect could be attenuated by the inhibition of catalase in HP100 cells, or by replenishing glutathione in Th1 cells [48]. Furthermore, a link between organotin induced apoptosis and oxidative stress has been observed in fish nervous system. Dong and coworkers [49] proposed the association between TBT induced apoptosis and oxidative stress in early-stage zebrafish Danio rerio retinal neuronal cells, because of the findings of marked inhibition of TBT induced apoptosis by antioxidants. The increased ROS in the brain cells of cuvier Sebastiscus marmoratus were accompanied with increased level of caspase-3 activity and decrease of Na+/K+-ATPase activity in a dose dependent manner after exposure to TMT and TBT, respectively [50, 51]. Thus, findings suggesting the inducibility of oxidative stress, apoptosis and neurotoxicity in fish by organotins were strongly shouldered. As we saw, the organotin-induced apoptotic process involves [Ca2+]i increase, mitochondrial failure, and leads cells to death. Even if the dose or impulse intensity determines the manner or the type of cell death, intracellular ATP level plays an important role in organotin-induced cell death. Organotins bind to the ATP synthase complex and inhibit ATP synthesis, altering the proton gradient by blocking or decreasing proton translocation through the FO subunit of ATP synthase, and redirect electrons towards ROS synthesis. Actually, organotin inhibition of ATP production is induced by promoted exchanges of halide for hydroxide ions across the inner mitochondrial membrane and is followed by matrix swelling and organotin binding to the mitochondrial FOF1-ATP synthase [52, 53]. TBT binding to mitochondrial FOF1-ATP synthase complex resulted in depletion of intracellular ATP, the reduced state of the mitochondrial respiratory chain, and subsequent production of O2-• which underwent spontaneous or superoxide dismutase-catalyzed dismutation to H2O2 [44]. Accordingly, Jurkat T cells, exposed to TBTC and under conditions of insufficient glycolytic ATP production, died by necrosis while the maintenance of ATP levels accelerated ∆Ψm decrease and allowed efficient release of mitochondrial intermembrane proteins such as cytochrome c to cytosol. Furthermore, caspase activation only occurred in ATP containing cells, thus confirming that the mechanisms of cell death induced by organotins were ruled by ATP availability [54]. Cytoskeletal Modifications Cytoskeletal modifications present another indication of the ongoing apoptotic process. Even if TBT disrupts macromolecular synthesis and cellular energetics (ATP production), TBT was shown to induce apoptosis in thymocytes independently of a requirement for protein synthesis and without requiring fully conserved cellular energetics [23]. In organotin (TBT, TPhT, DBT)-treated thymocytes, rapid cytoskeletal modifications were found. TBT induced depolymerization of total thymocyte F-actin up to 80%, within 10 min, an effect reduced by 25 % by addition



of [Ca2+]i chelating agents. These findings led to conclude that two mechanisms were involved, namely [Ca2+]i increasedependent mechanism and a [Ca2+]i-independent mechanism [55]. During the exposure of human promyelocytic leukemia cells to 5 M TPhTC, release of tumor necrosis factor- (TNF-) was detected. Additionally, TPhTC– exposed cells showed a prompt [Ca2+]i increase and actin depolymerization within 1 min. These events were followed by NF-B activation after 15 min, formation of apoptotic bodies after 3 h, and finally DNA fragmentation 6 h after the treatment. Pre-treatment of cells with antibodies against human TNF-stoppedTPhTC-induced DNA fragmentation, while the inhibition of NF-B activation by pyrolidine dithiocarbamate (antioxidant, ROS scavenger) prevented actin depolymerization. NF-B activation may generate TNF- that could play an essential role in TPhTC-induced apoptosis in human promyelocytic leukemia cells [56]. Similarly, a dose dependent loss of actin cytoskeletal organization characterized by F-actin depolymerization, increase in number of apoptotic cells, significant induction of caspase activity and increase of Bax/Bcl-2 ratio were described in TBT-treated human amnion cells [57]. The co-occurrence of caspase activation and F-actin depolymerization and consequent degradation of cytoskeletal microfilament-associated proteins such as gelosin, paxillin and vimentin by caspase action during TBT-promoted apoptosis was also observed in human neutrophils [58]. Gene Expression Regulation Gene expression analysis enables deeper insight into the molecular mechanisms and effect of organotins induction of apoptotic pathway. Thereby, a modulation of gene expression in DBTC-treated rat thymocytes was identified by the cDNA microarray technology [59]. The induction of the nur77 orphan steroid receptor, which is considered to be a required transcription factor for apoptosis initiation in thymocytes [60, 61], was elicited by RT-PCR. Pre-treatment of thymocytes with nur77 antisense nucleotides followed by exposure to DBTC resulted into a significant decrease in apoptotic cells thus confirming the nur77 involvement in organotin-induced apoptotic cell death. These in vitro results were confirmed by the in vivo induction of nur77 expression in rat thymocytes treated with single doses of DBTC. The same DBTC dose was able to both induce the apoptotic morphology in rat thymocytes in vitro and significant expression of nur77 in vivo. nur77 transcription is regulated by calcium signals [62] and, in response to apoptotic stimuli, nur77 is translocated from nucleus to cytoplasm and targets mitochondria to induce cytochrome c release and apoptosis [63]. Rat primary thymocytes exposed in vitro to different doses of tributyltin oxide (TBTO) showed changes in the expression profiles of genes related to lipid metabolism (e.g. up-regulation of Srebf1, the sterol regulatory element binding factor 1) as early sign of cell function disturbances. Accordingly up-regulation of these genes was induced by 0.3 M TBTO before the early apoptotic signs were recorded. Up-regulation of Nr3c1 (Nuclear receptor subfamily 3, group C, member 1) was promoted by in vitro exposure of rat thymocytes to 0.5 M TBTO [64] and in apoptotic thymus of rats exposed in vivo to dioctyltin chloride (DOTC) [65]. The up-regulated expression of cell survival-promoting genes was also observed in TBTO-treated laboratory mice [66]. Although a dose- and timedependent regulation of genes involved in apoptosis could be detected, the up- and down-regulation, respectively of pro- and anti-apoptotic genes, was not confirmed by gene expression profiles in microarray studies, probably because it occurred in a limited number of cells while other cell populations underwent another fate or protected themselves against programmed cell death. The observed up-regulation of nur77 seems to be the earliest marker of organotininduced apoptotic cell death in rat thymocytes in vivo and in vitro [59]. Modulation of MAP Kinase Cascades The Mitogen-Activated Protein (MAP) kinase is a family of serine/threonine kinases which phosphorylates transcription factors, co-activators, repressors and chromatin-remodeling molecules, thus modulating gene transcription in response to cell environmental changes. By modulating signal transduction, MAP kinases control proliferation, differentiation, apoptosis, and play a role in pathological states [67]. Among several organotins, TBTC was shown to be the most potent activator of Extracellular signal-Regulated kinase (ERK), JNK and the p38 MAP kinase pathway. Human T lymphoblastoid cells exposed for 1 hour in vitro to 0.25 - 2.0 M TBTC behaved in a dose-dependent manner with increases of ERK, JNK, and p38 MAP kinases up to 4 hours following exposure, while no changes in individual total protein levels were found. The Ca2+ chelating agent BAPTA suppressed TBTC-induced MAP kinase phosphorylation, indicating the importance of Ca2+ mobilization from intracellular stores for MAP kinase phosphorylation in this cell type [68]. The TMT- and DBT-induced apoptosis in rat cerebellar granule cells includes activation of several MAP kinases. The cells were exposed to 3.0 M TMT and 0.3 M DBT, the concentrations which induced the apoptosis in about


Željko Jakšić

50 % of those cells. Following the 6 hours exposition to TMT the increased levels of phosphorylated JNK and total JNK as well as phospho-p38 was observed, while the DBT exposition showed very little effect on those MAP kinases. However, treatment of rat cerebellar granule cells with both TMT and DBT resulted in the activation and phosphorylation of anti-apoptotic ERK1/2 in the absence of changes in total ERK1/2. Furthermore, the inhibition of JNK and p38 MAP kinases partially attenuated apoptosis in cells exposed to TMT, and has no effect on DBTinduced apoptosis. The cerebellar granule cell apoptosis has not been affected by the inhibition of ERK1/2 activation in both cases. These results indicate no involvement of MAP kinase cascade in DBT-induced apoptotic mechanism. Conversely in TMT-treated cells a pro-apoptotic mode of action of JNK and p38 kinases was shown [69]. Accordingly, the pharmacologic blockade of p38 reduced TMT-induced apoptosis observed in rat adrenal medulla pheochromocytoma cells [70]. Both studies indicated a pro-apoptotic function of JNK and p38 MAP kinase in TMT treated cerebellar granule and pheochromocytoma cells and indicated different DBT-driven action mechanisms in apoptotic pathway [69, 70]. Furthermore, following the exposition of human melanoma cells A375 to organotin(IV)(sulfonatophenyil)prophinates, the induced programmed cell death as well as the activation of ERK1/2 and p38 MAP kinase were confirmed [71]. Resembling results following the exposition of rainbow trout Oncorhynchus mykiss RTG-2 fibroblast cell line to 0.125 to 1 μM TBT was achieved. The TBT exposure induced not only significant caspase activation but induced rapid and sustained accumulation of ERK, JNK and p38 MAP kinase, leading to programmed cell death [72]. ER Stress As previously mentioned, upon ER stress, [Ca2+]i homeostasis modulation and calpain activation take part in the apoptotic process. Treatment of human neuroblastoma SH-SY5Y cells with 0.6 µM TBT stimulated the expression of both GADD (growth arrest- and DNA damage-inducible gene) 153 mRNA and GADD153 protein levels as well as induction of caspase-3 activity. These changes followed the transient but marked phosphorylation of the JNK protein. Conversely, total JNK protein remained unchanged [73]. The JNK phosphorylation and GADD153 gene expression were inhibited by SP600125 inhibitor without any influence on total JNK level but with a significant consequence on GADD153 protein level suppression. As GADD153 expression plays an important role in ER stress mediated apoptosis [74], these results confirmed the TBT ability to induce ER stress in human neuroblastoma cells, although the regulation of GADD153 expression and its influence on the apoptotic process still remains to be fully explained. The TBTinduced ER stress and consequent pro-apoptotic signaling was found in rat hepatocytes. The rapid elevation of [Ca2+]i level in hepatocytes during the first 20 min of exposure to 3.5 M TBT was slowed by thapsigargin, an inhibitor of the calcium ATPase pump of the ER. Furthermore, the activation of Ca2+ dependent cysteine protease calpain, the cleavage of cytoskeletal protein vinculin and activation of procaspase-12, localized on the cytoplasmic side of ER, were inhibited by the Ca2+ chelating agent ethylene glycol tetraacetic acid or by calpain inhibitor 1 [75]. Novel and promising research in this field is oriented towards a deeper characterization and understanding of organotin-induced interactions with cell macromolecules, changes in signaling and functional genomics. p53-Dependent Mechanism Although studies on organotins in cancer chemotherapy confirmed interactions with DNA by different binding modes [76], the high DNA damage potential is related to ROS, generated by the organotin effect on mitochondrial respiration, which attack cell macromolecules and cause significant damage to DNA. Actually, highly reactive hydroxyl radicals (OH•), generated by electron transfer to H2O2 in a Fenton-like reaction, attack DNA bases or sugar residues, resulting in base mispairing, replication block, and strand breaks [77]. The DNA damage, like double strand breaks, evokes p53 phosphorylation which may trigger p21 expression and cell proliferation (G1→S arrest) and DNA repair. Alternatively, high levels of double-strand breaks can activate proapoptotic genes such as Bax, Fas receptor and p53 up-regulated modulator of apoptosis (PUMA) whose protein product binds to pro-survival Bcl-2 and Bcl-XL and promotes cytochrome c release from the mitochondria and apoptosis [78]. The genotoxic effect of TBTC and induced G0→G1 arrest, DNA repair and/or apoptotic progress was studied in vivo on mussels Mytilus galloprovincialis injected below the mantle with 0.1 – 10.0 g TBTC/g mussel, and incubated for up to 24 hours in laboratory basins. The gills cell cycle of control mussels was characterized by large diploid G0/G1, broad S and small G2/M peaks. An asymmetrical G1 peak appeared in gill cells just 90 min and 24 hours after



exposure to 1 g TBTC/g. The mussels treated with 3 g TBTC/g and 90 min exposure showed no gill cells in G2/M phase and the appearance of a specific apoptotic peak representing the low DNA content cells. The highest applied dose resulted in the worst cell cycle scenario and the highest number of low DNA content cells. Moreover, the gill cells of mussel exposed to 1.0 and 3.0g TBTC/g for 24 hours showed characteristic internucleosomal DNA fragmentation. At the same time the haemocytes of mussels exposed to 1 g TBTC/g did not show internucleosomal DNA fragmentation but a significant increase of double-strand breaks were observed [79, 80]. In trimethyltin chloride (TMTC)-treated human hepatoma cells the concentration-dependent increase in DNA damage, release of cytochrome c from mitochondria and caspase-3 activation were accompanied by up-regulation of Bax expression as well as by the activation of p53 target genes p21 and PUMA [81]. The TMT concentrationdependent apoptosis and up-regulated Bax expression were previously described in hippocampal neurons and rat liver epithelial cells [82, 83]. According to the proposed model, Bax translocates to the mitochondrial membrane where it forms a protein complex with the mitochondrial voltage-dependent anion channel (VDAC) and opens the MPT pore which results in ∆Ψm loss and release of the apoptosis promoting factors to cytosol [84]. Other Proteins Affected In eukaryotes the majority of intracellular proteins are degradable via ubiquitin/proteasome-dependent pathway. The irreversible inhibition by TPhT of cellular proteasome chymotripsin (CT)-like activity (which cleaves proteins after the hydrophobic residues) was found to result in accumulation of ubiquitinated proteins and induced caspase-3 activity and programmed cell death in several human cell lines [85]. Among several investigated phenyltins and butyltins, TPhT and TBT showed the highest inhibition potential to purified 20S rabbit proteasome chymotripsinlike activity. In the intact human breast cancer cell line, MDA-MB-231 cells, TPhT and TBT chloride were the most potent proteasome inhibitors in vitro, followed by their corresponding mono-, di-, aryl and alkyl derivates. At the same time MDA-MB-231 cells treated with TPhT and TBT showed significant calpain-mediated decrease of p21/p18/Bax and increase in p36/Bax levels. Similarly, TPhT and TBT dose-dependent activated proteasome CTlike activities were accompanied by dose-dependent cell death in human peripheral blood Jurkat T cells. Evidence of direct TPhT binding to cellular proteasome was ultimately derived from immunoprecipitation studies where equal proteasome β5 subunit levels, but lower CT-like activity, were found in TPhT-treated MDA-MB-231 cells as compared to the control. In silico docking studies, confirmed with 47 % efficiency, gave rise to the hypothesis that Thr-1 Oγ atom of proteasome β5 subunit may perform a nucleophilic attack on JNK Sn atom in the organotin molecule, forming a coordinate bond, and resulting into irreversible inhibition of proteasome proteolytic activity [85]. Another protein, named stannin (Snn) was identified as high specific marker and mediator of apoptosis induced by low concentrations of organotins in neuronal cells [86]. Snn is a highly conserved 88 amino acid residue mitochondrial and other vesicular-membrane protein. Snn expression blockade by specific antisense oligonucleotides was shown to protect the neuronal cell primary culture against apoptosis [87]. Snn dealkylates TMT and irreversibly binds DMT, thus compromising membrane integrity and initiating apoptosis [88]. CONCLUSION As shown, apoptotic programmed cell death is modulated by complex pathways that involve a series of biochemical regulators and molecular interactions. Organotin compounds were shown to elicit apoptotic cell death in various cell types and organisms. The apoptotic mechanisms induced by organotins not only depend on cell type, specificity and sensitivity but also on organotin chemical reactivity (number and type of substitutes, alkyl chain length) and insult intensity (dose, concentrations, exposure time). As known mitochondrial toxins, organotins mainly trigger apoptosis via the mitochondrial pathway and by blocking mitochondrial ATP synthesis and promoting ROS production. Generally, the apoptotic pathway induced by organotin compounds starts with [Ca2+]i increase and consequent MPT, release of cytochrome c from mitochondria to cytosol and caspase activation, eventually leading to plasma membrane blebbing and DNA fragmentation. The particular features of organotin-induced apoptotic cell death mechanism and signaling can be summarized as follows:


Željko Jakšić



Changes in cellular Ca2+ distribution within the ER, cytoplasm and mitochondria play an important role in molecular mechanism of apoptotic cell death induced by organotins;



Ca2+ acts on the apoptotic process by stimulating cytochrome c release from mitochondria and participating in executioner caspase activation;



Cytochrome c export from mitochondria plays a central and critical step in organotin-induced caspase activation and apoptosis;



Two cytochrome c release mechanisms are known in mammals: a Ca2+-independent mechanism which involves a slow ∆Ψm loss, and a Ca2+-dependent and MPT inherited mechanism;



Lack of significant ∆Ψm loss, induction of MPT, and cytochrome c release prior to induction of 3caspase activity in some cell types exposed to organotins raises the possibility of mitochondrialindependent caspase-3 induction scenario;



The [Ca2+]i increase is followed by mitochondrial (as the primary source of) ROS generation;



Dual ATP control mode can be identified in programmed cell death pathways: promotion of necrosis by insufficient glycolytic ATP production, and accelerated ∆Ψm decrease and promotion of the caspase by maintained ATP levels;



Distinct pathways and mechanisms of programmed cell death may be activated depending on the toxicant dose and the duration of organotin exposure,



DNA damage activates the p-53 dependent mechanism which may promote the repair of DNA or contribute to the apoptotic process by activating the proapoptotic genes,



Induction of pro-apoptotic functions of JNK and p38 MAP kinase by some organotin compounds have been noted in several cell types,



Modulation of the ubiquitin/proteasome-dependent pathway by organotins may result in accumulation of ubiquitinated proteins, induction of caspase-3 activity and programmed cell death.

In spite of numerous studies, the mechanisms of organotin induced apoptosis are still not fully explained and described. The regulation of genes, the role and function of proteins involved in apoptosis such as MAP kinase, proteasome, Snn, as well as the involvement of mitochondrial death receptor, ER stress, p53 pathways and related signaling mechanisms require deeper investigations in the future. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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