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Caspase Involvement in the Induction of Apoptosis by the Environmental Toxicants Tributyltin and Triphenyltin
Abstract
Organotin compounds such as tributyltin (TBT) and triphenyltin (TPT) can kill target cells by triggering apoptosis. The mechanism by which these environmental toxicants activate the apoptotic program is currently unclear. We have studied the effect of TBT and TPT in the human Hut-78 and Jurkat T-lymphocyte cell lines. Within 1 h there was a 30-fold increase in caspase activity, as measured by the cleavage of the fluorescent peptide DEVD-AMC. Morphological changes characteristic of apoptosis, such as membrane blebbing and nuclear fragmentation, were readily detectable. Blocking caspase activity with the peptide inhibitor z-VAD-fmk prevented all subsequent apoptotic changes. The optimal concentration range for induction of apoptosis was 0.5 to 5 microM TBT. TPT was also able to trigger caspase activity in the lymphocyte cell lines, but it took over 2 h to detect and occurred at a lower concentration range of 0.01 to 1 microM. Higher concentrations of TBT and TPT caused cell necrosis, and we showed that these concentrations were able to inhibit caspase activity in apoptotic cells. TBT and TPT were able interact with a vicinal thiol compound, similar to the known caspase inhibitor phenylarsine oxide, providing a potential mechanism for caspase inhibition. We propose that vicinal thiol proteins may be a general biological target of these organotin compounds, leading to the induction of caspase activity and apoptosis at low concentrations, and more extensive cell damage and necrotic cell death at higher concentrations.
... TBT has been shown to cause apoptosis in several cellular models. In vitro studies revealed the occurrence of apoptosis in rainbow trout hepatocytes [37], rat hepatocytes [38], rat thymocytes [39,40], human lymphoma cells (CCRF-CEM lymphoblastoid, Jurkat T lymphocytes and Hut-78 cell lines) [30,41,42], human neutrophils [43] and neuronal PC12 cells [44]. Studies in rainbow trout hepatocytes [37] revealed the potential implication of cysteine proteases in TBT-induced apoptosis. ...
... Studies in rainbow trout hepatocytes [37] revealed the potential implication of cysteine proteases in TBT-induced apoptosis. TBT has been found to increase activity of caspase-3 in Jurkat T-cell and HUT-78 lymphoma cell lines [41,45]. The precise apoptotic mechanism has not yet been determined. ...
... The broad-spectrum caspase inhibitor, z-VAD-fmk was a potent inhibitor of TBTinduced DNA fragmentation in rainbow trout hepatocytes [37]. In the T cell line HUT-78, DNA fragmentation and membrane blebbing was apparent following exposure to 2 AM TBT for 3 h [41]. Following pretreatment with z-VADfmk, these morphological changes were blocked. ...
Tri-n-butyltin (TBT), a biocide, is known for its immunotoxicity and hepatotoxicity and is a well-characterised mitochondrial toxin. This report investigates the mechanisms involved in induction of apoptosis by TBT in primary cultures of rat hepatocytes. Release of cytochrome c from mitochondria into the cytosol was apparent after 15 min of exposure to 2.5 μM TBT. In addition, activity of initiator caspase-9 increased after 30 min, representing activation of the mitochondrial pathway in hepatocytes. The death receptor pathway was also activated by TBT, as indicated by recruitment of the adaptor protein FADD from the cytosol to the membrane as soon as 15 min after treatment. In addition, levels of the pro-apoptotic protein Bid decreased in the cytosol, while there was an increase in levels of the cleaved form tBid, in TBT-treated hepatocytes. Activity of initiator caspase-8 increased after 30 min. The principal effector caspase-3 was activated following 30 min of treatment with TBT. Activation was confirmed by immunodetection of a 17-kDa cleaved fragment. Apoptotic substrates such as Poly(ADP-ribose) polymerase and DNA fragmentation factor-45 are cleaved by caspase-3 to ensure the dismantlement of the cell. Cleavage of Poly(ADP-ribose) polymerase into a 85-kDa fragment appeared after 30 min of TBT treatment. DNA fragmentation factor-45 disappeared in TBT-exposed rat hepatocytes. This is the first detailed study reporting the involvement of initiator and effector caspases, cleavage of their intracellular substrates and activation of both death receptor and mitochondrial pathways in TBT-induced apoptosis in rat hepatocytes. The comprehension of molecular events of apoptosis is important for the evaluation of the risk to humans and animals.
... The set of test substances, reportedly used as positive controls for apoptosis induction in in vitro cell systems, included cadmium chloride (CdCl 2 ), dithiothreitol (DTT), sodium chloride (NaCl), mercuric chloride (HgCl 2 ), tributyltin oxide (TBT-O), tributyltin chloride (TBT-Cl) and staurosporine [2][3][4][5][6][7][8][9][10][11][12][13][14]. ...
... NRK-52E and LLC-PK1 cells were exposed to seven different compounds ( Table 1). The concentrations and exposure durations were chosen based on literature data [2][3][4][5][6][7][8][9][10][11][12][13][14]. All exposures were performed and analyzed using standard conditions, i.e. in medium supplemented with 10% FBS. ...
... CdCl 2 , DTT, NaCl, HgCl 2 , TBT-O, TBT-Cl and staurosporine, but only the last three elicited apoptosis in LLC-PK1 cells when tested under standard conditions (in the presence of serum). TBT-O and TBT-Cl had been previously shown to induce apoptosis using the same methods and at similar concentrations for example in rat thymocyte cultures and Jurkat cells, respectively [7][8][9]. Staurosporine induced apoptosis after exposure to 100 nM for 4 hours. This is in good agreement with data from other investigators using human glioma cells (U251MG) or human kidney cells (HK-2) [12,13]. ...
Renal cell lines are frequently used models in toxicology. The aim of the experiments described here was to investigate the suitability of two of those renal cell lines, namely NRK-52E and LLC-PK1, as models for mid to late stage apop-tosis under standard cell culture conditions; the latter means that testing was performed in the presence of serum in the culture media. Seven known inducers of apoptosis already positively tested by other investigators were chosen as test substances using chromatin condensation (Hoechst staining) as endpoint. These substances were cadmium chloride (CdCl 2), dithiothreitol (DTT), sodium chloride (NaCl), mercuric chloride (HgCl 2), tributyltin oxide (TBT-O), tributyltin chloride (TBT-Cl) and staurosporine. From these, only TBT-O, TBT-Cl and staurosporine induced morphological features typical of apoptosis in LLC-PK1 cells. Morphologically discerned apoptosis was confirmed by DNA fragmentation (DNA laddering assay) analysis. LLC-PK1 cells, but not NRK-52E cells, were shown to be suitable models of mid to late stage apoptosis under the conditions employed. TBT-O, TBT-Cl and staurosporine were shown to be suitable positive controls for apoptosis in renal cells in vitro.
... Low levels of TBT induced cell death of lymphocytes. TBT has induced apoptosis and caspase activation in human leukemia T cells (14,15), peripheral T lymphocytes (16) and rat thymocytes (17,18). However, little is known about the immunotoxicity of TBT compounds for macrophages. ...
... In addition, apoptosis, one of the mechanisms of cell death, is closely related to TNFα (30). TBT induces apoptosis in several kinds of cells (13)(14)(15)(16)(17)(18). We hypothesized that TBT induces apoptosis in macrophages, and that the mechanism of the apoptosis induced by TBT is related to TNFα or c-jun. ...
... The increases in the percentage of TUNEL-positive cells and in caspase-3 activity strongly suggested that exposure to TBT at the level of 1 µM and over induces apoptosis in J774.1 cells. Stridh et al. (15) examined the induction of apoptosis in the human Hut-78 T cell line after exposure to TBT chloride. Hut-78 apoptotic cells appeared at 70% after exposure to 2 µM TBT for 3 hours. ...
Objective: Tributyltin (TBT) compounds have been widely used as antifouling agents for ship-bottom paint. The immune system is a target of TBT intoxication. We evaluated the effects of TBT chloride in macrophages, which have critical roles in the immune system, using a murine macrophage lineage cell line, J774.1, in vitro.
Methods: We examined tumor necrosis factor α (TNF α), interleukin-1β (IL-1β) and c-jun mRNA expression in J774.1 cells. The effects of TBT on the apoptosis of J774.1 cells were examined by determining the percentage of TUNEL-positive cells and caspase-3 activity.
Results: The mean values of the viabilities of J774.1 cells exposed to TBT decreased dose-dependently. The relative mRNA expression of TNFα increased dose-dependently, however, that of IL-1β was not significantly different among the groups. The mean percentage of TUNEL-positive cells increased dose-dependently. Increases in the caspase-3 activities of J774.1 cells were observed in the groups exposed to higher concentrations of TBT. The mean value of relative mRNA expression of c-Jun transcription factor increased dose-dependently.
Conclusions: The increases in the percentage of TUNEL-positive cells and in caspase-3 activity suggested that exposure to TBT induces apoptosis of J774.1 cells. The increases in the mRNA expression of TNFα and c-jun by TBT may be related to apoptosis in macrophages.
... Moreover, it has been described earlier that organotin compounds, especially TBT, have a clear immunotoxic effect in mammals [14][15][16], and this might be due to their exorbitant induction of apoptosis [17,18]. The effective concentration of TBT to induce apoptosis in the majority of treated cells is around or below 1 μM and has been shown in vitro [14,[18][19][20][21][22][23][24][25][26][27] as well as in vivo [28,29]. The question whether the disturbance of the intracellular calcium homeostasis is responsible for the onset of apoptosis [22,27,[30][31][32][33][34] or the direct effect on mitochondrial functions is the first event [18,20,24,32,33,35] is under discussion for a long time. ...
... The question whether the disturbance of the intracellular calcium homeostasis is responsible for the onset of apoptosis [22,27,[30][31][32][33][34] or the direct effect on mitochondrial functions is the first event [18,20,24,32,33,35] is under discussion for a long time. Stridh et al. [18,23] have shown a decade ago that TBT induces apoptosis via the activation of caspases in various human cells, the link for this caspase activation was not yet found. The most obvious players have been discussed to be the increase in calcium concentration or the opening of the permeability pore of the mitochondria. ...
... The most obvious players have been discussed to be the increase in calcium concentration or the opening of the permeability pore of the mitochondria. But induction of apoptosis has been demonstrated for very low concentrations of TBT which 2 Journal of Toxicology do not induce calcium influx [27], and caspases are often inhibited by high calcium concentrations [23]. Some years ago, evidence arose that mitochondria-independent mechanisms contribute to the induction of apoptosis and possibly death receptors or direct caspase activation are involved in the TBT induced effect [36][37][38][39]. ...
Tributyltin (TBT) is one of the most toxic compounds produced by man and distributed in the environment. A multitude of toxic activities have been described, for example, immunotoxic, neurotoxic, and endocrine disruptive effects. Moreover, it has been shown for many cell types that they undergo apoptosis after treatment with TBT and the cell death of immune cells could be the molecular background of its immunotoxic effect. As low as 200 nM up to 1
μ
M of TBT induces all signs of apoptosis in Jurkat T cells within 1 to 24 hrs of treatment. When compared to Fas-ligand control stimulation, the same sequence of events occurs: membrane blebbing, phosphatidylserine externalisation, the activation of the “death-inducing signalling complex,” and the following sequence of cleavage processes. In genetically modified caspase-8-deficient Jurkat cells, the apoptotic effects are only slightly reduced, whereas, in FADD-negative Jurkat cells, the TBT effect is significantly diminished. We could show that caspase-10 is recruited by the TRAIL-R2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines.
... 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. ...
... 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. ...
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.
... Caspase activation occurs after cytochrome c export from its mitochondrial intermembrane location to the cytosol, due to direct interaction of organotins at low concentrations with the vicinal thiols present on the adenine nucleotide translocator opening the permeability transitional pore [34,35]. Conversely, caspase inhibition, with consequent activation of necrotic cell death, occurs at higher organotin concentrations after interaction with vicinal thiols of an unknown caspase inhibitor [36]. The activated caspases finally cleave target proteins leading to irreversible DNA fragmentation via endonuclease activation [37,38]. ...
... Many in vitro studies have been performed in order to explain the mechanisms that are responsible for the toxicity of organotin compounds in whole organisms. TBT has been reported to cause apoptosis in cultured thymocytes [31,[68][69][70] and in leucemia T-cell lines [36,71] and an anti-proliferative effect has been suggested for both rat and fish lymphocytes [72,73] probably causing a decrease in circulating antibodies. DBT also decreases the survival rate of rat and human thymocytes [54]. ...
Leached from various sources, all the organotin compounds have an impact on natural aquatic environments. In both freshwater and seawater ecosystems, they are dangerous in that they can have deleterious effects on biocenoses already at low concentrations. All these compounds are known to be toxic at relatively low levels, not only for aquatic invertebrates, but also for fish and laboratory mammals. Moving easily along the trophic chains, organotins are also rapidly bioaccumulated in the tissues of non-target organisms living in the water-sediment interface, causing severe, long-term toxic effects on local epifauna, with repercussions on biodiversity and human health. Among toxic effects, genotoxicity and immunotoxicity are the most important affecting the capacity for survival of animals. Genotoxicity appearing in the form of chromosomal aberrations, increasing in frequency of micronuclei and induction of cytogenetic damage has recently been reported in mammals, fish and aquatic invertebrates. Organotins interfere selectively with the immune system of vertebrates, causing atrophy of the thymic cortex and lymphoid tissues with a consequent leucopoenia. Short-term in vitro exposures of haemocytes of various vertebrate and invertebrate organisms reveal inhibition of phagocytosis, cytolysis and/or apoptosis of leucocytes after inhibition of chemotaxis and respiratory burst, with resulting depression of cell-mediated immune responses. These immunosuppressive effects are dose- and time-dependent, and vary according to the number and type of organic moiety present. Both Ca2+-dependent and Ca2+-independent mechanisms of action have been proposed. They are linked and synergistic in triggering the cascade of secondary events that lead to toxic action.
... 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. ...
... 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 toxicities o f organotin compo unds in mammals (experimental animals and humans) were reviewed in several reports [1][2][3]5]. Various reports showed bis (tri-n-butyltin) oxide (TBTO), tributyltin (TBT) and triphenyltin (TPT) to be immunot oxic inducing thymic a trophy a nd apoptosis, and activation of caspases in Jurkat Tlymphocytes [6][7][8][9], and they are also known to be developmental and reproductive toxicants. The embryotoxicities of TBTO in vitro and in vivo were Accepted for publication: July 26, 2002 Correspondence: K. Kumasaka reported by Davis et al. [10] and Krowke et al. [11]. ...
... The body weight gain and other organs weights were not different between the control and treated mice. We administered TBTO with a low dosage and decreased frequency to mice to exclude the general toxicity of TBTO reported previously [1][2][3][5][6][7][8][9]. This result shows that TBTO administration affects spermatogenesis particularly differentiated germ cells in the mouse testes. ...
In this study, the toxicity of bis (tri-n-butyltin) oxide (TBTO), a marine pollutant, in premature mouse testes and the correlation of a total tin concentration with toxicity were examined. Mice were treated by oral administration twice a week for four weeks from five weeks of age at doses of 0 mg/kg (control), 0.4 mg/kg, 2.0 mg/kg, or 10 mg/kg, and sacrificed on the day following the final administration. The control mice were administered with distilled water containing 0.2% ethanol as vehicle. Removed testes were used for determination of the testicular sperm head counts and for histological study or determination of the total tin concentration. The sperm head count was significantly decreased in the 2.0 mg/kg and 10 mg/kg administration groups, although the testicular weights of both groups were unchanged compared to that of the control. The histological study revealed that TBTO caused vacuolization of Sertoli cells in several seminiferous tubules that failed to organize, however, the frequencies of occurrence were low. The total tin concentration in the testis increased in a dose-dependent manner in inverse proportion to the reduction of sperm head counts. These results suggest that TBTO is potentially an anti-testicular compound in mice.
... The mechanism of apoptosis by TBT of mNSCs is consistent with principal apoptotic pathway which is involved in disruption of the mitochondrial membrane potential and in the activation of caspase-3/7 (Figs. 4 and 5). It also has been shown in other cell lines such as human Hut-78 cells (Stridh et al., 1999a), Jurkat cells (Stridh et al., 1999a), and Th1/Th2 cells (Tada-Oikawa et al., 2008). ...
... The mechanism of apoptosis by TBT of mNSCs is consistent with principal apoptotic pathway which is involved in disruption of the mitochondrial membrane potential and in the activation of caspase-3/7 (Figs. 4 and 5). It also has been shown in other cell lines such as human Hut-78 cells (Stridh et al., 1999a), Jurkat cells (Stridh et al., 1999a), and Th1/Th2 cells (Tada-Oikawa et al., 2008). ...
Exposure to environmental neurotoxic chemicals both in utero and during the early postnatal period can cause neurodevelopmental disorders. To evaluate the disruption of neurodevelopmental programming, we previously established an in vitro neurosphere assay system, using rat mesencephalic neural stem cells (mNSC). Here, we examined the developmental neurotoxicity of tributyltin (TBT) in an in vitro neurosphere assay. A neurosphere was driven from rat E16 mesencephalon and seeded in a poly-l-ornithine/laminin-coated plate. Exposure to TBT increased the number of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL)-positive cells in time-dependent and dose-dependent manner: it was significantly detectable with treatment of 100pM TBT for 90min, or of 1μM TBT for 30min. Disruption of mitochondrial membrane potential and activation of caspase-3/7 were concomitantly observed. Furthermore, DNA microarray analyses using Affymetrix GeneChip revealed that as early as 0.5h after exposure to the metal (1μM), the expression levels of 71 genes were increased by more than 2-fold, whereas those of 8 genes were decreased by 2-fold or less: it was notably altered in expression of Ca(2+)-mobilizing-related genes, and retinoic-acid signal-related genes, as well as bifunctional apoptosis-related genes. The levels of gene expression of Wnt family were also significantly changed. Thus, we established transcriptome of TBT-induced apoptosis of mNSC. This would help to evaluate developmental neurotoxicity of TBT in vivo, contributing to the risk assessment methods based on infant physiology.
... Furthermore, mitochondrial susceptibility to damage by exposure to toxicants justifies investigating the impact of environmental chemicals on the organelle 29 . The effects of inhaled toxicants can include inhibition of ATP synthesis due to uncoupling of oxidative phosphorylation 30 , increased oxidative mitochondrial damage 31 , and mitochondria-initiated apoptosis 32 . ...
Toxins present in cigarette and e-cigarette smoke constitute a significant cause of illnesses and are known to have fatal health impacts. Specific mechanisms by which toxins present in smoke impair cell repair are still being researched and are of prime interest for developing more effective treatments. Current literature suggests toxins present in cigarette smoke and aerosolized e-vapor trigger abnormal intercellular responses, damage mitochondrial function, and consequently disrupt the homeostasis of the organelle’s biochemical processes by increasing reactive oxidative species. Increased oxidative stress sets off a cascade of molecular events, disrupting optimal mitochondrial morphology and homeostasis. Furthermore, smoking-induced oxidative stress may also amalgamate with other health factors to contribute to various pathophysiological processes. An increasing number of studies show that toxins may affect mitochondria even though exposure to secondhand or thirdhand smoke. This review assesses the impact of toxins present in tobacco smoke and e-vapor on mitochondrial health, networking, and critical structural processes including mitochondria fission, fusion, hyperfusion, fragmentation, and mitophagy. The efforts are focused on discussing current evidence linking toxins present in first, second, and thirdhand smoke to mitochondrial dysfunction
... This was observed in the case of lead, mercury/methylmercury ( Burlando et al., 2003;Wiemann et al., 1999;Schirrmacher, et al., 1998;Marty and Atchison, 1997;Nathanson et al., 1995;Trump et al., 1989), and ar- senite ( Shen et al., 2002;Zhang et al., 2000;Zhang et al, 1999;Alves, 1992). It was also shown that organotin compounds increase the Ca 2+ levels, however, more is known about tributyltin (TBT) ( Gennari et al., 2000;Oyama et al., 1993;Zazueta et al., 1994;Stridh et al., 1999a, Stridh et al., 1999b, Stridh et al., 1999cOrrenius et al., 1992;Gogvadze et al., 2002). ...
... Mitochondria may be damaged by exposure to toxicants, and therefore serve as an excellent model for investigation of environmental chemicals (Meyer et al., 2013). Effects produced by toxicants include inhibition of ATP synthesis due to uncoupling of oxidative phosphorylation (Wallace and Starkov, 2000), increased oxidative stress (Jia et al., 2007), and mitochondriainitiated apoptosis (Stridh et al., 1999). Sometimes in the presence of environmental stress, such as UV irradiation or actinomycin, mitochondria fuse to form a highly connected network through a process called stress-induced mitochondrial hyperfusion (SIMH) (Tondera et al., 2009). ...
Thirdhand cigarette smoke (THS) was recently recognized as an environmental health hazard; however, little is known about
it effects on cells. Mitochondria are sensitive monitors of cell health and report on environmentally-induced stress. We tested
the effects of low levels of THS extracted from terry cloth on mitochondrial morphology and function using stem cells with
well-defined mitochondria. Concentrations of THS that did not kill cells caused stress-induced mitochondrial hyperfusion (SIMH),
which was characterized by changes in mitochondrial morphology indicative of fusion, increased mitochondrial membrane potential
(MMP), increased ATP levels, increased superoxide production, and increased oxidation of mitochondrial proteins. SIMH was
accompanied by a decrease in Fis1 expression, a gene responsible for mitochondrial fission, and a decrease in apoptosis-related genes, including Aifm2, Bbc3 and Bid. There was also down regulation of Ucp2, Ucp4 and Ucp5, genes that decrease MMP thereby reducing oxidative phosphorylation, while promoting glycolysis. These effects, which collectively
accompany SIMH, are a pro-survival mechanism to rescue damaged mitochondria and protect cells from apoptosis. Prolonged exposure
to THS caused a reduction in MMP and decreased cell proliferation, which likely leads to apoptosis.
... Several studies have also discovered a strong connection between TBT and apoptosis. TBT can cause apoptosis in several cellular models (Reader et al., 1999;Stridh et al., 1999;Lavastre and Girard, 2002). TBT-induced apoptosis has also been found in ovaries, brains and tails of embryos in rockfish (Zhang et al., 2007(Zhang et al., , 2008. ...
... In addition, DNA does not appear the primary target for IST-FS 29 but, as previously hypothesized, perturbation of homeostasis, impairment of mitochondrial functions and inhibition of protein synthesis [5,11] may hold primary responsibility for cytotoxicity. A recent paper by Stridh et al. reported that organotin compounds such as tributyltin and triphenyltin can kill target cells triggering apoptosis by caspase activation at low drug concentrations, and by leading to necrotic cell death at higher concentrations [16]. This evidence suggests that apoptosis and necrosis are not totally distinct pathways but some overlap between them probably occurs. ...
... In addition, DNA does not appear the primary target for IST-FS 29 but, as previously hypothesized, perturbation of homeostasis, impairment of mitochondrial functions and inhibition of protein synthesis [5,11] may hold primary responsibility for cytotoxicity. A recent paper by Stridh et al. reported that organotin compounds such as tributyltin and triphenyltin can kill target cells triggering apoptosis by caspase activation at low drug concentrations, and by leading to necrotic cell death at higher concentrations [16]. This evidence suggests that apoptosis and necrosis are not totally distinct pathways but some overlap between them probably occurs. ...
... Electrophoretic data and ultrastructural observations such as profound subcellular degeneration suggested that exposure to 1mM concentrations of PSP and TOTP modulated a necrotic reaction. Similar patterns of high concentration evoked necrosis and lower concentration induced apoptosis have been described for many toxicants including N-methyl-D-aspartate or peroxynitrite (Bonfoco et al., 1995), antimycin A , tributlytin and triphenyltin (Stridh et al., 1999), and sulfur mustard (Dabrowska et al., 1996). ...
... * p < 0.05. ** p < 0.001 (48 vs 24, 72 vs 24). 1 p < 0.05 (TBT-Cl vs TPT-Cl). 2 p < 0.001 (48 vs 72). 3 p < 0.001 (72 vs 24, 72 vs 48). at higher concentrations (Stridh et al., 1999). In vivo triorganotin compounds undergone metabolic disproportionation, the loss of one alkyl or aryl group may occur through the intervention of enzymes such as aromatase (Lee, 1985). ...
... Despite the heterogeneity observed in the TBT resistance profile of Gram negative and Gram positive bacteria, the former are generally more resistant to TBT (Mendo et al., 2003;Cruz et al., 2007), which can be related to the different architecture of the bacterial cell wall. According to the literature, the highest TBT concentration tested was about (Whalen et al., 1999) (Fent, 1996) (Antizar-Ladislao, 2008) (Hoch, 2001) (Grun and Blumberg, 2006) (Ohtaki et al., 2007) (Stridh et al., 1999) (Mizuhashi et al., 2000) (Liu et al., 2006) ATCC7966 was isolated from "a tin of milk with a fishy odor" (Seshadri et al., 2006). ...
Tributyltin (TBT) is one of the most toxic xenobiotics ubiquitous in the aquatic environment. Several reports have described the negative impact of TBT in living organisms, from bacteria to mammals.
Over the world, TBT contamination has being described as a serious problem. Thus, it is imperative to econtaminate TBT polluted sites.
Bioremediation strategies may constitute an alternative to conventional decontamination methods, benefiting from the microorganisms potential to metabolize xenobiotics. Several microorganisms among bacteria, fungus, and algae have been reported to possess the ability to resist and, in certain cases, degrade TBT in their simple
and less toxic derivatives. Due their characteristics, some of those microorganisms have been used for bioremediation studies and to construct bioreporters to detect TBT in the environment. This review
provides an overview regarding microbial TBT resistance, while focusing on TBT degradation and bioremediation. A comprehensive revision on the several applications of organotin compounds, adverse
biological effects on living organisms, and information regarding the available TBT bioreporters is also included.
... Tributyltin (TBT) was used to induce mitochondrial apoptosis since this compound has been shown to effectively induce apoptosis via the mitochondrion (117)(118)(119)(120). ...
... The result of their inhibition is activation of Ca 2+ influx pathways, leading to massive accumulation of cytosolic Ca 2+ and subsequent death of the cell by apoptosis (Chow et al., 1992;Corsini et al., 1997;Kass and Orrenius, 1999). Several thymocyte studies have shown classic apoptotic events leading to the death of thymocytes and include (but are not limited to) an increase in intracellular Ca 2+ concentration, cytochrome c release from mitochondria, caspase activation and chromatin condensation (Aw et al., 1990;Chow et al., 1992;Stridh et al., 1999;Yu et al., 2000). ...
11 th October 2004 ii Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; and any editorial work, paid or unpaid, carried out by a third party is acknowledged.
... In this study, we uncover the mechanism that TBT induces DNA damage, increases irreparable DSBs during meiosis and thus may cause errors in synapsis and crossover recombination, leading to sterility and embryonic lethality in C. elegans. TBT interacts with mitochondria directly or indirectly by disturbance of [Ca2+], causing elevation of reactive oxygen species (ROS) and oxidative stress (Stridh et al., 1999b;Gennari et al., 2000;Nakatsu et al., 2007) which are suggested to in turn induce DNA damage (Liu et al., 2006). Wang et al. (2012) found that TBT induced expression of oxidative stress-response proteins, e.g. ...
Tributyltin (TBT), one of the environmental pollutants, has been shown to impact the reproduction of animals. However, due to the lack of appropriate animal model, analysis of the affected molecular pathways in germ cells is lagging and has been particularly challenging. In the present study, we investigated the effects of tributyltin chloride (TBTCL) on the nematode Caenorhabditis elegans germline. We show that exposure of C. elegans to TBTCL causes significantly elevated level of sterility and embryonic lethality. TBTCL exposure results in an increased number of meiotic DNA double-strand breaks in germ cells, subsequently leading to activated DNA damage checkpoint. Exposing C. elegans to TBTCL causes dose- and time-dependent germline apoptosis. This apoptotic response was blocked in loss-of-function mutants of hus-1 (op241), mrt-2 (e2663) and p53/cep-1 (gk138), indicating that checkpoints and p53 are essential for mediating TBTCL-induced germ cell apoptosis. Moreover, TBTCL exposure can inhibit germ cell proliferation, which is also mediated by the conserved checkpoint pathway. We thereby propose that TBT exhibits its effects on the germline by inducing DNA damage and impaired maintenance of genomic integrity. Index Descriptors and Abbreviations: TBTCL, tributyltin chloride; C. elegans, Caenorhabditis elegans; NGM, nematode growth medium; DMSO, dimethyl sulfoxide; DAPI, 4', 6-diamidino-2-phenylindole; DSBs, DNA double-strand breaks.
... A similar phenomenon of impairment of egg production has been reported in the copepod Acarita tonsa (Johansen & Møhlenberg 1987) and in the sea urchin Paracentrouts lividus (Girard et al. 1997(Girard et al. , 2000 in response to TBT exposure. The cytotoxicity of TBT often results in an arrest of cellular dynamics, leading to apoptosis (Stridh et al. 1999) or a blocking of cell division (Girard et al. 1997) primarily occurring through an alteration of macromolecular syntheses (Snoeij et al. 1988, Girard et al. 1997 or membrane-mediated processes controlling cell signaling. These processes consist primarily of a disruption of calcium homeostasis (Chow et al. 1992, Matsuoka & Igisu 1996 or calcium signaling (Corsini et al. 1997, Girard et al. 1997. ...
In order to examine the biological effects of tributyltin (TBT) exposure, the caprellid amphipod Caprella danilevskii was exposed to 5 levels (0, 10, 100, 1000 and 10000 ng l(-1)) of TBT during the embryonic stage (5 d), The male and female ratios changed dramatically in the hatched juvenile with increases in TBT concentrations; i.e. the proportion of females was found to increase to 55.6% at 10 ng l(-1), 85.7% at 100 ng l(-1) and 81.8% at 1000 ng l(-1). All specimens died in 10 000 ng TBTCl l(-1) within 5 d after spawning due to the acute toxic concentration for the species, No significant differences were observed to occur in the sex ratio in response to the exposure after hatching (50 d) in a previous study. Sex disturbance might therefore be induced during the embryonic stage in the caprellid. Reproductive inhibitions such as brood loss and oogenesis inhibition occurred even at 10 to 100 ng TBTCl l(-1) exposures in the short term in both parental females and their offspring females. The survival rate decreased drastically as the TBT concentrations increased, with the decrease being observed at TBT concentrations as low as 10 ng l(-1) (69%) in the offspring. In parental females, the survival rate also decreased at more than 100 ng TBTCl l(-1), despite movement after 5 d into the. no-TBT-added seawater. Therefore, our data suggest that nanogram concentrations of TBT similar to those encountered in coastal waters can directly affect sex ratio, reproduction and survival in the caprellid, and this phenomenon occurs at environmentally realistic concentrations in the coastal ecosystem.
... For example, immunotoxic action of organotins could partly be caused by cytoskelton modification in addition to perturbation of thymocyte Ca 2+ homeostasis that may be linked to apoptosis of thymus cells caused at 5 µM level by TBT or DBT (Chow and Orrenius 1994). Apoptotic changes in human and rat T-lymphocytes (Stridh et al. 1999) or rat thymocytes (Gennari et al. 2000) were induced at less than or around 1 µM of TBT or TPT. ...
Tributyltin and triphenyltin (TBT and TPT) are biocides that have been used to prevent fouling of boats, preserve wood, kill molluscs, and other uses. Due to observed effects on oysters and snails, their use in boat paints has been banned in many nations. However, use on ships and some uses other than as antifouling paints continue. These uses, the relative persistence of these compounds, their tendency to bioaccumulate, and their toxicity cause lingering concerns about risks to humans and non-human organisms. This paper outlines an integrated assessment of TBT and TPT. Based on prior human health and ecological assessments, it suggests that an integrated assessment that recognized common pathways of transport, fate and exposure, and common modes of action would be more efficient and complete than additional independent assessments. The presentation of risks in an integrated manner could also lead to better decisions by defining the various benefits of any management action.
... In mammals, different levels of TBT change the functions of a variety of endocrine tissues such as the pituitary, pancreas, gonads, and thyroid glands (Oberdorster et al. 1998;Vos et al. 2000;Wada et al. 1982). Others studies showed that TBT induced apoptosis in hepatocytes (Reader et al. 1999) in human Hut-78 and Jurkat T-lymphocyte cells (Stridh et al. 1999). An imbalance between development of ovarian follicles with cystic follicles, apoptotic cells in corpus luteum, and granulosa cells was noted, as well as a rise in number of atretic follicles after TBT treatment. ...
Triorganotins are environmental contaminants, commonly used in antifouling agents for boats, that bioaccumulate and thus are found in mammals and humans due to ingestion of contaminated seafood diets. The importance of triorganotins as environmental endocrine disruptors and consequent reproductive toxicity in different animal models is well known; however, the adverse effects on reproductive cycle are less well understood. The potential reproductive toxicity of tributyltin (TBT) on regular reproductive cycling of female rats was examined. Wistar female rats (12 wk old, weighing approximately 230 g) were divided into two groups: control (vehicle, ethanol 0.4%) and tributyltin (100 ng/kg/d, 7 d/wk, for 16 d by gavage). Tributyltin significantly decreased the cycle regularity (%), duration of the reproductive cycle, the proestrus and diestrus phases, and number of epithelial cell in proestrus phase. TBT also increased the duration of metestrus and the number of cornified cells in this phase. Ovary weight and serum 17β-estradiol levels decreased markedly, accompanied by a significant increase in progesterone levels. Histological analysis showed apoptotic cells in corpus luteum and granulosa cells layer, with cystic follicles after TBT exposure. Tributyltin also elevated number of atretic follicles and corpoa lutea. The micronucleus (MN) test, using Chinese hamster ovary cells, demonstrated a concentration-dependent mutagenic effect of TBT, and at 2.0 × 10(-2)ng/ml most of the cells were nonviable. The toxic potential of TBT over the reproductive cycle may be attributed to changes found in the ovarian weight, unbalanced levels of sexual female hormones, and number of ovarian follicles and corpora lutea.
... Cytotoxicity of TBT often results in an arrest of cellular dynamics, leading to apoptosis [13] or blocking of cell division [9], occurring mainly through alteration of macromolecular syntheses [9,14] or membrane-mediated processes controlling cell signaling, which consist mainly in disruption of calcium * To whom correspondence may be addressed (girard@unice.fr.). homeostasis [15,16] or calcium signaling [9,17]. ...
Nnomolar concentrations of tri- n-butyltin (TBT) from 5 × 10−10 M to 5 × 10−9 M, were assayed on sea urchin (Paracentrotus lividus) egg cleavage and on larval development. Preincubation enhanced TBT toxicity to first cleavage DNA and protein syntheses but not to intracellular calcium sequestration. Exposure to nanomolar TBT affected the larval development up to the ninth day by reducing arm length and increasing the diameter of the rudiment. Chromatographic analysis of TBT in eggs shows a dose-dependent biomagnification with a half-time of 5 min, which is much shorter than the length of preincubation necessary to provoke cytotoxicity at the same concentration (5 × 10−9 or 5 × 10−10 M). Our data suggest that nanomolar concentrations of TBT similar to those encountered in polluted waters could directly affect sea urchin egg development after fertilization and the larval cycle, these effects being independent of bioaccumulation.
... We have analyzed the PI fluorescence of subpopulations defined by the Ca 2ϩ -related gates (Figs. 9, 10). After addition of various concentrations of TBT, PI fluorescence rose only slightly in Ca-TR cells (Fig. 10B) even after a high TBT concentration of 10 M that is characterized by a high necrotic potency (32). On the contrary, a steady increase in the PI fluorescence of Ca-SST cells was found (Fig. 10A). ...
Background
Programmed cell death, also termed apoptosis, is the main focus of interest in a variety of scientific and clinical areas. For a better understanding of the mechanisms of apoptosis, from the onset of the cellular death program to the late stages of apoptosis or apoptotic necrosis, very early functional events have to be quantified because they might be involved in temporal and causal relationships between apoptosis-related key processes.Methods
We have established a flow cytometric technique to quantify time-dependent signals simultaneously with high temporal resolution (Δt = 1 s) in living cells. With this technique, the response of cells to apoptosis-stimulating agents can be analyzed over 15 min. For this purpose, a thermostatted sample tube holder for repeatable interruption-free injection of substances into the cell suspension was developed. Early detectable fluorescence and scatter parameters were related to intracellular free Ca2+ concentration, [Ca2+]i (Indo-1 fluorometry), membrane permeability (propidium iodide [PI] influx), and cell volume (forward scatter).ResultsA T-cell line (Jurkat) served as a model system. Apoptosis was induced by the biozid Tri-n-butyltin (TBT). Dependent on the TBT concentration (0.3–10 μM), the mean free [Ca2+]i increased by a factor of 1.2–6 during a short time interval of just 2 min. Especially after low TBT concentrations (<0.5 μM), this [Ca2+]i increase was nearly transient during the observation time of 15 min. Higher TBT concentrations (0.5–10 μM), however, induced a transient increase of [Ca2+]i (Ca-TR) only in a fraction of the cells; in another subpopulation, a steady-state Ca2+ signal (Ca-SST) was observed. The analysis of the simultaneously registered PI signals of the Ca-SST cells showed a shift to increasing PI fluorescence (by a factor of about 4) with increasing Ca2+ concentrations. In Ca-TR cells, the PI fluorescence remained nearly unchanged. These apoptosis-related changes (increase in [Ca2+]i and membrane permeability) could be confirmed by the additional observation of a TBT concentration-dependent decrease in cell volume measured during the same early time period.Conclusions
The simultaneously analyzed parameters (i.e., [Ca2+]i, membrane permeability, and cell volume) suggested that, in our model system of Jurkat T-cells treated with TBT, an apoptotic cell fate was indicated very early (within 15 min) by the steady-state [Ca2+]i level. Cytometry 44:45–56, 2001. © 2001 Wiley-Liss, Inc.
... In this system, TBT promotes cellular suicide by activating cysteine proteases (called caspases), which selectively cleave vital cellular substrates and this results in internucleosomal fragmentation of DNA by selectively activated DNases. 36,37 TBT is also known to induce a rapid increase of intracellular Ca 2 levels (vide supra) and N,N,N',N'-tetra-acetic acid], and can block caspase activation and TBT-induced apoptosis. In this pathway, the rise in Ca 2 content is a prerequisite for postmitochondrial events involved in caspase activation leading to induction of apoptosis, events which TBT-exposed cells grown in a Ca 2 -free medium are able to evade, dying by necrosis. ...
Considerable attention has been given in recent years to the possibility that xenobiotics in the environment may affect reproduction in animals. In this study, the relative impact of tributyltin(IV) (TBT) chloride, one of the most toxic environmental pollutants, was investigated using Ciona intestinalis ovary as a model system. The pleiotropic effects of TBT exposure are concentration dependent and include a decrease of ATP levels, lipid content and nucleic acid content and synthesis. In contrast, a marked increase in calcium (Ca2+) and glucose content is observed. Furthermore, TBT alters enzymatic activity, inhibiting creatine kinase and stimulating alkaline phosphatase and cholinesterase (at concentrations higher than 10−5M in sterile sea water solution). The implications of these effects on reproduction and embryonal development are discussed, along with the possibility that they reflect an extreme cellular defence mechanism triggered to avoid deleterious consequences for the survival of the species.Copyright © 2001 John Wiley & Sons, Ltd.
... Organotins, including TPT, are highly toxic to all sorts of aquatic primary producers, invertebartes and vertebrates (Fargasová 1998; Jak et al. 1998; Petersen and Gustavson 2000; Rehage et al. 2002). Organotins have been reported to inhibit mitochondrial oxidative phosphorylation and consequently energy transfer, Ca 2+ homeostasis, protein and DNA synthesis in the cell (Chandra et al. 1989; Girard et al. 1997; Tiano et al. 2003 ), to cause immunosuppression and premature apoptosis (programmed cell death) in both vertebrates and invertebrates (Stridh et al. 1999; Cima et al. 2002), and have photosynthesis inhibiting properties (Mooney and Patching 1995). This variety of fundamental processes are not immediately visible and may take time before effects can be observed. ...
The study objectives were to shed light on the types of freshwater organism that are sensitive to triphenyltin acetate (TPT) and to compare the laboratory and microcosm sensitivities of the invertebrate community. The responses of a wide array of freshwater taxa (including invertebrates, phytoplankton and macrophytes) from acute laboratory Single Species Tests (SST) were compared with the concentration–response relationships of aquatic populations in two types of freshwater microcosms. Representatives of several taxonomic groups of invertebrates, and several phytoplankton and vascular plant species proved to be sensitive to TPT, illustrating its diverse modes of toxic action. Statistically calculated ecological risk thresholds (HC5 values) based on 96h laboratory EC50 values for invertebrates were 1.3μg/l, while these values on the basis of microcosm-Species Sensitivity Distributions (SSD) for invertebrates in sampling weeks 2–8 after TPT treatment ranged from 0.2 to 0.6μg/l based on nominal peak concentrations. Responses observed in the microcosms did not differ between system types and sampling dates, indicating that ecological threshold levels are not affected by different community structures including taxa sensitive to TPT. The laboratory-derived invertebrate SSD curve was less sensitive than the curves from the microcosms. Possible explanations for the more sensitive field response are delayed effects and/or additional chronic exposure via the food chain in the microcosms.
... Exposure to low concentration of TBT activates caspase-3-like enzymes in the human T lymphocyte lines Jurkat and HUT-78 (Stridh et al., 1999) causing the rapid loss of the mitochondrial membrane potential, that blocks oxidative phosphorylation, with a time course appropriate for a role in the signalling of apoptosis (Stridh et al., 1998). Detection of KRS _ SD, with high homology to human KRS/MST, that is phosphorylated and proteolytically cleaved by caspase-3-like protease following TBT stress in sponge, propose a mechanism of oxidative stress response that is conserved from sponges to human cells. ...
Marine sponges as sessile filter feeders are inevitably under a constant influence of changes in their environment. Mediation of extracellular signals and regulation of cellular response to environmental stress is a key function of cellular protein kinases. Expression, proteolytical cleavage and phosphorylation of stress-responsive KRS_SD protein kinase, in control and tributyl-tin (TBT) treated sponges were investigated. In control sponge, two KRS_SD proteins were expressed: KRS_SD1 (54 kDa) corresponding to KRS_SD calculated molecular weight, and KRS_SD2 (50 kDa). Exposure of sponges to TBT resulted in alteration of KRS_SD1 and KRS_SD2 expression levels and their phosphorylation state. KRS_SD1 band disappearance after 4 h coincides with the appearance of additional 35 kDa immunoreactive protein band (p35) and decrease in number of viable cells. Incubation of recombinant KRS_SD with protein extracts from TBT stressed sponges resulted in cleavage of recombinant KRS_SD protein. The size of the resulting protein fragment suggests caspase-3 recognition site, present in both recombinant KRS_SD and KRS_SD, as a possible site of cleavage. Phosphorylated KRS_SD bands appear at the same time as p35 and remain after p35 band intensity reached control level. KRS_SD induction, its phosphorylation and proteolytical cleavage during TBT stress suggest that in sponge cells exist mechanism similar to one present in human cells where KRS/MST protein kinase is involved in promotion of apoptosis following oxidative stress.
... TBT and TPT are well known to be toxic to several cells or cause apoptosis [5,22,32]. We attempted to investigate whether or not reporter gene transactivation decreases because the yeast growth was inhibited by organotin compounds. ...
In aquatic invertebrates, particularly marine gastropods, organotin compounds induce irreversible sexual abnormality in females, which is termed imposex, at very low concentrations. Organotin compounds are agonists for nuclear receptors such as RXRs and PPARgamma. However, the imposex phenomenon has not been reported to act as an antagonist on estrogen receptors in other species, including vertebrates and invertebrates. In order to gain insights into the antagonistic activity of organotin compounds on estrogen receptors (ERs), we examined the inhibitive effect of these compounds on estradiol-dependent beta-galactosidase activity using the yeast two-hybrid detection system consisting of a combination of the human estrogen receptor (hERbeta) ligand-binding domain and the co-activator steroid receptor co-activator-1 (SRC1). Tributyltin-hydroxide (TBT-OH) and triphenyltin-chlorine (TPT-Cl) exhibited an inhibitive effect on E₂-dependent transcriptional activity, similar to antagonistic chemicals such as 4-hydroxytamoxifen (OHT) or ICI 182,780, at a very low concentration of 10⁻¹⁴ M TBT or 10⁻¹⁰ M TPT, respectively. The yeast growth and transcriptional activity with transcriptional factor GAL4 did not exhibit any effect at the tested concentration of TBT or TPT. Moreover, the yeast two-hybrid system using the interaction between p53 and the T antigen of SV40 large did not describe any effect at the tested concentration of OHT or ICI 182,780. However, the interaction between p53 and T antigen was inhibited at a TBT or TPT concentration of 10⁻⁹ M, respectively. These results indicate that TBT and TPT act as inhibitors of ER-dependent reporter gene transcriptional activation and of the interaction between hERbeta LBD and the co-activator SRC1 in the yeast two-hybrid system. Consequently, our data could partly explain the occurrence of organotin compound-induced imposex on the endocrine system of mammals, including humans.
... Apoptosis, or programmed cell death, is a highly conserved cell death process involved in tissue remodeling and degeneration in a variety of cell types (Schwartzman and Cidlowski, 1993;Steller, 1995). It is reported that TBT causes apoptosis in several cellular models (Raffray and Cohen, 1993;Kawanishi et al., 1999;Reader et al., 1999;Stridh et al., 1999;Lavastre and Girard, 2002). In our previous study, the same TBT-exposed Sebastiscus marmoratus displayed an increased number of TUNEL positive ovarian cells compared to the control (Zhang et al., 2007). ...
Tributyltin (TBT) is a ubiquitous marine environmental contaminant characterized primarily by its reproductive toxicity. However, the neurotoxic effect of TBT has not been extensively described, especially in fishes which have a high number of species in the marine environment. This study was conducted to investigate the neurotoxic effects of TBT at environmental levels (1, 10, and 100ngl(-1)) on female Sebastiscus marmoratus. The results showed that TBT exposure induced apoptosis in brain cells of three regions including the pallial areas of the telencephalon, the granular layer of the optic tectum, and the cerebellum. In addition, the increase of reactive oxygen species and nitric oxide levels, and the decrease of Na+/K+-ATPase activity were found in the brain. The results strongly indicated neurotoxicity of TBT to fishes. According to the regions in which apoptosis was found in the brain, TBT exposure might influence the schooling, sensory and motorial functions of fishes.
... Our results showed that the activities of Caspase-3 in the tails were increased after TBT exposure. It is reported that TBT causes apoptosis in several cellular models (Raffray and Cohen, 1993;Kawanishi et al., 1999;Reader et al., 1999;Stridh et al., 1999;Lavastre and Girard, 2002). TBT-induced apoptosis has also been reported for invertebrate hemocytes exposed to the biocide (Cima and Ballarin, 1999). ...
Tributyltin (TBT) is a ubiquitous marine environmental contaminant characterized primarily by its reproductive toxicity. However, the embryotoxicity of TBT has not been extensively described, especially in fishes. The aim of this study was to investigate the developmental toxicity of waterborne TBT at environmental levels (0, 0.1, 1, and 10 ng L(-1) as Sn) on Sebastiscus marmoratus embryos. Our study showed that TBT reduced the hatchability and caused apparent morphological abnormalities including dorsal curvature, severely twisted tails and pericardial edema. In addition, localized apoptosis was found in the tail regions of embryos after TBT exposure. The study provided a possible mechanistic link between apoptosis and TBT-induced twisted tails abnormality. TBT exposure induced retinoid X receptor α expression in S. marmoratus embryos at the 0.1 and 1 ng L(-1) group, which would be responsible for the increasing apoptotic cells induced by TBT. The results of the present study have widespread implications for environmental ecological assessment, management and the etiology of developmental defects.
Toxins present in cigarette and e-cigarette smoke constitute a significant cause of illnesses and are known to have fatal health impacts. Specific mechanisms by which toxins present in smoke impair cell repair are still being researched and are of prime interest for developing more effective treatments. Current literature suggests toxins present in cigarette smoke and aerosolized e-vapor trigger abnormal intercellular responses, damage mitochondrial function, and consequently disrupt the homeostasis of the organelle’s biochemical processes by increasing reactive oxidative species. Increased oxidative stress sets off a cascade of molecular events, disrupting optimal mitochondrial morphology and homeostasis. Furthermore, smoking-induced oxidative stress may also amalgamate with other health factors to contribute to various pathophysiological processes. An increasing number of studies show that toxins may affect mitochondria even through exposure to secondhand or thirdhand smoke. This review assesses the impact of toxins present in tobacco smoke and e-vapor on mitochondrial health, networking, and critical structural processes, including mitochondria fission, fusion, hyper-fusion, fragmentation, and mitophagy. The efforts are focused on discussing current evidence linking toxins present in first, second, and thirdhand smoke to mitochondrial dysfunction.
Tributyltin (TBT) is a persistent organotin pollutant widely used as agricultural and wood biocides, exhibiting well‐documented toxicity to reproductive functions in aquatic organisms. However, the effect of TBT on early pregnancy and placental development has been rarely studied in mice. Pregnant mice were fed with 0, 0.2, and 2 mg/kg/day TBT from gravid day 1 to day 8 or 13. TBT exposure led to an increase in the number of resorbed embryo and a reduction in the weight of fetus at gestational days 13. Further study showed that TBT significantly decreased placental weight and area, lowered laminin immunoreactivity and the expressions of placental development‐related molecules including Fra1, Eomes, Hand1, and Ascl2. Moreover, TBT treatment markedly inhibited the placental proliferation and induced up‐regulation of p53 and cleaved caspase‐3 proteins, and down‐regulation of Bcl‐2 protein. In addition, TBT administration increased levels of malondialdehyde and H2O2 and decreased activities of catalase and superoxide dismutase. Collectively, these results suggested TBT‐induced adverse pregnancy outcomes during early pregnancy might be involved in developmental disorders of the placenta via dysregulation of key molecules, proliferation, apoptosis, and oxidative stress.
The organotin compounds, tributyltin (TBT) and triphenyltin (TPT), are widely used as stabilizers for polyvinyl compounds, agricultural fungicides and antifouling paints for ships and fishing nets. Along with the rapid expansion in the use of these compounds, there is concern for environmental hazards. In humans and animals, TBT and TPT are known to cause various neurotoxic symptoms, such as tremors, convulsions, ataxia and spontaneous involuntary movement. There are, however, no or only minor histopathological changes in neuronal tissues and cells. Therefore, functional impairment of neurons seems to underlie the organotin neurotoxicity. Recently, we investigated the effects of four organotin compounds on tetrodotoxin-resistant, voltage-dependent Na channel, which plays a crucial role in the generation of action potentials, using whole-cell patch clamp techniques in dorsal root ganglion neurons. In this review, we will focus on the action of organotin compounds to modify the Na channel activity and discuss the mechanisms underlying organotin neurotoxicities.
During the past several decades, butyltin compounds (BTs), one of the representative groups of organotin compounds (OTs), have been widely used as an antifouling agent in paints for boats, ships, and aquaculture nets (Fent 1996, Champ and Seligman 1996), thus these compounds have been found in a variety of marine organisms, often at concentrations exceeding acute or chronic toxicity levels (Bryan and Gibbs 1991; Alzieu 1996). The hazardous effects of antifouling paints containing BTs in marine ecosystem have become a significant environmental issue all over the world (Champ and Wade 1996; Bosselmann 1996). To prevent the destruction of marine ecosystems, BT application to small boats and fish farming equipment has been banned or regulated in developed countries since the late 1980s (Champ and Wade 1996; Bosselmann 1996). Nevertheless, significant accumulation of BTs has been noted at various trophic levels in the marine food chain including plankton, algae, crustaceans, fishes and cetaceans, indicating that BTs impact continues to be felt in marine ecosystems.
Oxidative phosphorylation (oxphos) is the primary process by which the energy derived from the catabolism of carbohydrates, fats, and proteins is used to synthesize ATP in virtually every cell of eukaryotic organisms. Compounds that have a primary action on oxphos have a long history of use to control pests. Today, compounds that disrupt oxphos in the target species are widely used as fungicides and have important uses as insecticides and acaricides. This chapter discusses the toxicology of compounds that have their most important primary toxic effect on oxphos in vertebrates or have a primary effect on mitochondrial oxphos in target species with a probability of similar action in vertebrates. It describes the cell structures and mechanisms involved in oxphos and ATP synthesis, and the different ways by which pesticides can disrupt oxphos and hence cause damage to cells and tissues. Mechanisms by which oxphos is disrupted by pesticides include: inhibiting the operations of the electron transport chain; preventing the electrochemical potential gradient from being coupled to ATP synthesis; blocking the ATP synthase machinery; diverting electrons from the electron transport chain and generating reactive oxygen species (ROS); and arsenolysis. The chapter describes the toxicological consequences of disrupting oxphos by pesticide action and their relevance for toxicity, and provides details of the general properties, uses, and toxicological profiles of several pesticides.
The interaction of the organotin compounds trimethyltin(IV) and tributyltin(IV) chlorides with the calcium pump from sarcoplasmic reticulum membranes was studied. It was found that the presence of calcium fully protects against the inhibitory effect of both organotin compounds. However, the apparent affinity of the protein for tributyltin chloride is two orders of magnitude higher than for trimethyltin chloride (K 0.5 values of 14 µ m and 1.4 m m, respectively). Studies of intrinsic fluorescence of the Ca2+‐ATPase and enzyme phosphorylation by ATP and Pi support the hypothesis that the inhibitory properties of trialkyltin compounds are due to the inhibition of calcium binding to the high‐affinity binding sites of the Ca2+‐ATPase. This suggests that there is a specific interaction between the trialkyltin compounds and the calcium binding sites of the protein. The effect of trialkyltin compounds on Ca2+‐ATPase was also addressed by differential scanning calorimetry to assess the thermal transition of the protein denaturation, and by infrared spectroscopy in the absorption region corresponding to the amide I band (1600–1700 cm−1) to observe changes in the secondary structure of the protein. We conclude that the interaction of trialkyltin compounds with Ca2+‐ATPase reduces the affinity and cooperativity for calcium binding and, consequently, the inhibition of ATPase activity. These events are accompanied by changes in the secondary structure of the protein, including loss of α‐helix structure and a concomitant increase in protein aggregation or unfolding. The activity of trialkyltin compounds on the Ca2+‐ATPase is discussed in relation to their solubility in water and in the lipid phase. Copyright © 2012 John Wiley & Sons, Ltd.
To examine the biological effects of tributyltin (TBT) exposure, the caprellid amphipod, Caprelladanilevskii, was exposed to five levels (0, 10, 100, 1000 and 10 000 ng l1, 85·7% at 100 ng l1. All specimens died in 10 000 ng TBTCl l100 ng TBTCl l1 (69%) during the five days. In parental females, the survival rate also decreased at more than 100 ng TBTCl l−1, despite movement after five days into seawater with no TBT added. Data suggest that nanogram concentrations of TBT similar to those encountered in coastal waters around the developed countries can directly affect sex proportion, reproduction, and survival in the caprellid.
A new arenetelluronic triorganotin ester, namely (Me3Sn)4[o-Me-PhTe(μ-O)(OH)O2)]2 () has been prepared by the reaction of o-tolyltelluronic acid and Me3SnCl in the presence of potassium hydroxide. The complex was fully characterized by elemental analysis, FT-IR, NMR ((1)H, (13)C, (119)Sn) spectroscopy and X-ray crystallography. Structure analysis revealed that the complex crystallized as Sn4Te2 units and a 1D linear chain was formed by intermolecular C-HO interactions. Cytotoxic assessments showed that the complex can induce apoptotic cell death via accumulation of ROS, collapse of the MMP and activating caspase-3. The results indicated that ROS is crucial to the cytotoxicity induced by the complex.
Organotin compounds are widely distributed toxicants. They are membrane-active molecules with broad biological toxicity. We have studied the interaction of tributyltin and triphenyltin with phosphatidylserine model membranes using differential scanning calorimetry, infrared spectroscopy and X-ray diffraction techniques. Organotin compounds produced a broadening of the gel to the liquid-crystalline phase transition of the phospholipid and a shifting of the phase transition temperature to lower values. Infrared spectroscopy experiments showed that tributyltin exerted a fluidizing effect on the apolar part of the bilayer, and that both tributyl- and triphenyltin interact with the interfacial region of the bilayer, making the carbonyl groups less accessible to water. As seen by X-ray diffraction experiments, organotin compounds were unable to change the bilayer macroscopic organization of the phospholipid, but they were able to reduce the long-range order of the multibilayer system and to disorder the packing of the phospholipid molecules. The observed interaction between organotin compounds and phosphatidylserine membranes promotes physical perturbations that could affect membrane function and may mediate some of their toxic effects. Copyright © 2004 John Wiley & Sons, Ltd.
Programmed cell death, or apoptosis, plays a central role in animal development and tissue homeostasis. Since its discovery interest increases in the biological as well as in medical aspects of this genetic program. On the one hand, missing or low induction of apoptosis is connected to an increased cancer risk, on the other hand, an excessive occurrence of cell death results in serious consequences for the whole organism. An early death of immune competent cells may weaken the immune defense. Additionally, the acceptance of transplanted organs or synthetic prostheses is decreased when surrounding tissues are induced to undergo apoptosis. Particularly metallic ingredients or impurities in alloys or plastic materials may interfere with cellular signal transduction pathways that induce apoptosis leading to detrimental effects. The review summarises recent publications on apoptosis induced by metals and metal compounds with the aim of providing a better understanding of the connection between the apoptotic machinery and the toxic effects of metals in medicine.
For Abstract see ChemInform Abstract in Full Text.
The superimposition of male sex organs (penis and vas deferens) in a female gastropod, called imposex, is widely attributed to the exposure to tributyltin (TBT) compounds, used world-wide in antifouling paints for ships. It has been hypothesized that the TBT-induced imposex is mediated by an increasing androgen level relative to the estrogen level, namely a decreased conversion of androgens to estrogens (i.e., aromatization). In the present study, we tested this hypothesis by examining the effects of TBT or triphenyltin (TPT) on the aromatase activity in a cultured human granulosa-like tumor cell line, KGN, which was recently established by our group. Treatment with more than 1000 ng/ml TBT compounds was very toxic to the cells and caused immediate cell death within 24 h, while 200 ng/ml was found to cause apoptosis of the cells. Treatment of the KGN cells for more than 48 h with 20 ng/ml TBT or TPT, which is a concentration level reported to cause imposex in marine species, did not affect cell proliferation but significantly suppressed the aromatase activity determined by a [3H]H2O release assay. Treatment with 20 ng/ml TBT compounds for 7 days also resulted in a reduction of the E2 production from Δ4-androstenedione stimulated by db-cAMP. The changes in the aromatase activity by TBT compounds were associated with comparable changes in P450arom mRNA assessed by RT-PCR. The luciferase activity of the P450arom promoter II (1 kb) decreased after the addition of 20 ng/ml TBT compounds in transfected KGN cells either in a basic state or in states stimulated by db-cAMP. The Ad4BP-dependent increase in the luciferase activity of P450arom promoter II was also downregulated by such treatments. These results indicate that TBT compounds inhibited the aromatase activity and also decreased the P450arom mRNA level at the transcriptional level in KGN cells. The direct inhibitory effect of TBT compounds on the aromatase activity may therefore partly explain the induction of imposex by these compounds in female species.
The effects of tributyltin (TBT) on cytosolic Ca(2+) concentration ([Ca(2+)](c)) and cell viability were investigated in nerve growth factor-differentiated PC12 cells. TBT concentration dependently increased [Ca(2+)](c) with an EC(50) value of 0.07μM. This effect was markedly reduced by removal of the extracellular Ca(2+) or membrane depolarization with a high K(+) medium, but unaffected by thapsigargin causing depletion of intracellular Ca(2+) stores. The L-type voltage-dependent Ca(2+) channel (VDCC) blocker nicardipine blocked the effect of TBT, but the N-type VDCC blocker ω-conotoxin did not. TBT decreased the number of viable cells with an EC(50) value of 0.09μM. The TBT-induced cell death was prevented by nicardipine or by chelating the cytosolic Ca(2+) with BAPTA-AM, but not by ω-conotoxin. The results show that TBT causes an increase in [Ca(2+)](c) via activating L-type VDCCs, and support the idea that the organotin-induced cell death arises through Ca(2+) mobilization via L-type VDCCs.
The signaling pathways leading to cellular protection or cell death following exposure to heavy metals have not been fully clarified. Mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK transmit extracellular signals into the nucleus, and have been shown to participate in a diverse array of cellular functions such as cell growth, differentiation and apoptosis. Treatment with cadmium, inorganic mercury or tributyltin can activate ERK, JNK and p38 MAPK, and induces the expression of c-fos and c-jun genes prior to the development of apoptosis. However, the members of the MAPK family appear to be differentially activated depending on the heavy metal and the cell type exposed. Consequently, various cellular responses may be caused by the distinct pattern of MAPKs activation. MAPKs may be one of the important cellular signal transduction pathways affected by various environmental pollutants, including heavy metals.
Tributyltin (TBT) compounds have been widely used as antifouling agents for shipbottom paint. The immune system is a target of TBT intoxication. We evaluated the effects of TBT chloride in macrophages, which have critical roles in the immune system, using a murine macrophage lineage cell line, J774.1,in vitro.
We examined tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) andc-jun mRNA expression in J774.1 cells. The effects of TBT on the apoptosis of J774.1 cells were examined by determining the percentage of TUNEL-positive cells and caspase-3 activity.
The mean values of the viabilities of J774.1 cells exposed to TBT decreased dose-dependently. The relative mRNA expression of TNFα increased dose-dependently, however, that of IL-1β was not significantly different among the groups. The mean percentage of TUNEL-positive cells increased dose-dependently. Increases in the caspase-3 activities of J774.1 cells were observed in the groups exposed to higher concentrations of TBT. The mean value of relative mRNA expression of c-Jun transcription factor increased dose-dependently.
The increases in the percentage of TUNEL-positive cells and in caspase-3 activity suggested that exposure to TBT induces apoptosis of J774.1 cells. The increases in the mRNA expression of TNFα andc-jun by TBT may be related to apoptosis in macrophages.
The FG cell line derived from the gills of flounder paralichthys olivaceus was used to determine the acute cytotoxic effects of the organotin compound bis-(tri-n-butyltin)oxide (TBTO). Its cytotoxic effects were initially measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay, and cell protein assay. Results indicated that the doses of TBTO ranging from 1.7 x 10(- 10) to 1.3 x 10(- 7) M were all toxic, and no difference in cytotoxicity was found between the three test systems. The transmission electron microscopic examination of TBTO-exposed cells revealed that their ultrastructures were markedly altered, as evidenced by dilation of mitochondria, breakdown of rough endoplasmic reticulum, nuclear membrane dissolution, and increased level of lysosomes. It is clear that the cells are highly susceptible to TBTO. This renders FG cells one of several choices for rapidly evaluating the acute toxicities of organotin compounds like TBTO. The mode of action of TBTO leading to the cytotoxicity, including the ultrastructural alteration in FG cells, is also proposed.
The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme, named apopain, is composed of two subunits of relative molecular mass (M
r) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-lβ-converting enzyme (ICE) and CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro, suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death.
The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.
The inner membrane of liver and heart mitochondria possesses an anion uniport pathway, known as the inner membrane anion channel (IMAC). IMAC is inhibited by matrix Mg2+, matrix H+, N,N'-dicyclohexycarbodiimide, mercurials and amphiphilic amines such as propranolol. Most of these agents react with a number of different mitochondrial proteins and, therefore, more selective inhibitors have been sought. In this paper, we report the discovery of a new class of inhibitors, triorganotin compounds, which block IMAC completely. One of the most potent, tributyltin (TBT) inhibits malonate uniport via IMAC 95% at 0.9 nmol/mg. The only other mitochondrial protein reported to react with triorganotins, the F1F0ATPase, is inhibited by about 0.75 nmol/mg. The potency of inhibition of IMAC increases with hydrophobicity in the sequence trimethyltin much less than triethyltin much less than tripropyltin less than triphenyltin less than tributyltin; which suggests that the binding site is accessible from the lipid bilayer. It has long been established that triorganotins are anionophores able to catalyze Cl-/OH- exchange; however, TBT is able to inhibit Cl- and NO3- transport via IMAC at doses below those required to catalyze rapid rates of Cl-/OH- exchange. Consistent with previous reports, the data indicate that about 0.8 nmol of TBT per mg of mitochondrial protein is tightly bound and not available to mediate Cl-/OH- exchange. We have also shown that the mercurials, p-chloromercuribenzene sulfonate and mersalyl, which only partially inhibit Cl- and NO3- transport can increase the IC50 for TBT 10-fold. This effect appears to result from a reaction at a previously unidentified mercurial reactive site. The inhibitory dose is also increased by raising the pH and inhibition by TBT can be reversed by S2- and dithiols but not by monothiols.
Reaction of isolated mitochondria with a variety of agents that lead to oxidation or cross-linking of sulfhydryl groups leads to an increased "open" probability of the permeability transition pore, a cyclosporin A-sensitive channel. We have investigated the mechanism by which the pore is induced by menadione, diamide, arsenite, and tert-butylhydroperoxide. We find that these inducers increase the probability of pore opening by shifting its gating potential to higher values. Furthermore, the induced shift was prevented by treatment with N-ethylmaleimide or dithiothreitol. At moderate levels of depolarization an apparent I50 for N-ethylmaleimide of bout 5 microM can be defined, while the N-ethylmaleimide or dithiothreitol effects are overcome by maximal depolarization. We conclude that the oxidation-reduction state of vicinal thiols in cysteinyl residues plays a critical role in tuning the voltage sensor of the transition pore, with an increase of gating potential (i.e. an increase in the probability of pore opening despite a high transmembrane potential difference) as the couple is poised to a more oxidized state. These findings may have implications for the mechanism of cell damage under oxidative stress.
Treatment of rat thymocytes with the immunotoxic environmental pollutant tributyltin (TBT) caused a rapid decrease in the F-actin content resulting in the depolymerization of 80% of the total thymocyte F-actin in 10 min. Removal of extracellular Ca2+ and pretreatment of the thymocytes with the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N',N',N',N',-tetraacetic acid, abolished the TBT-induced increase in cytosolic free Ca2+ concentration but reduced the depolymerization of F-actin by only 25%. Thus, the data suggest that 75% of the decrease in F-actin content in the thymocytes was due to other effects of TBT. Pretreatment of rat thymocytes with the alkylating agent N-ethylmaleimide completely inhibited TBT-induced F-actin disruption, suggesting that thiol group modification is involved. The TBT-induced decrease in thymocyte F-actin was not specific for any particular subpopulation of thymocytes. Furthermore, triphenyltin and the metabolite of TBT, dibutyltin, were also found to induce depolymerization of thymocyte F-actin, whereas nonimmunotoxic organotin compounds like trimethyltin and triethyltin had no effect. In conclusion, TBT was found to induce rapid depolymerization of F-actin in thymocytes through both Ca(2+)-dependent and Ca(2+)-independent mechanisms, suggesting that the potent immunotoxic effect of TBT may involve cytoskeletal modifications in addition to the perturbation of thymocyte Ca2+ homeostasis reported previously.
There is compelling evidence that members of the caspase (interleukin-1β converting enzyme/CED-3) family of cysteine proteases
and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a
novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities.
The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity
of caspases 2, 3, and 7 andCaenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of
apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components
in a proteolytic cascade that amplifies the death signal.
REVIEW
Apoptosis, an evolutionarily conserved form of cell suicide, requires specialized machinery. The central component of this
machinery is a proteolytic system involving a family of proteases called caspases. These enzymes participate in a cascade
that is triggered in response to proapoptotic signals and culminates in cleavage of a set of proteins, resulting in disassembly
of the cell. Understanding caspase regulation is intimately linked to the ability to rationally manipulate apoptosis for therapeutic
gain.
Recent evidence indicates that a profound alteration in mitochondrial function constitutes an obligatory early event of the apoptotic process. The molecular mechanism accounting for this alteration is mitochondrial permeability transition (PT). PT is both sufficient and necessary for apoptosis to occur. Experiments performed in cell-free systems of apoptosis demonstrate that mitochondria undergoing PT release protease activators that can trigger nuclear manifestations of apoptosis. Bcl-2 and its homologs are endogenous regulators of PT. It appears that some types of necrosis, those inhibited by Bcl-2, involve PT. If PT is a rate-limiting event of both apoptosis and necrosis, then downstream events including caspase activation and the bioenergetic consequences of PT must determine the choice between both modes of cell death. PT without caspase activation would cause necrosis. These findings have important implications for the comprehension of the apoptotic process, for the dichotomy between apoptosis and necrosis, and for the phylogeny of programmed cell death. Apoptosis may have evolved together with the endosymbiotic incorporation of aerobic bacteria (the precursors of mitochondria) into ancestral unicellular eukaryotes.
To evaluate the functional significance of di-n-butyl (DBTC)- and di-n-octyltin dichloride (DOTC)-induced lymphoid depletion various immune function studies were carried out. The delayed type hypersensitivity reaction, a parameter of cell-mediated immunity was decreased in rats fed 50 or 150 ppm of DOTC for 6 weeks. This decrease was dose-related. Allograft rejection, another cellular immune response, was significantly delayed by DBTC and DOTC. The antibody response against E. coli LPS, probably a T cell-independent antigen, was not affected by DBTC. However, the humoral immune response against sheep red blood cells (SRBC), which needs the co-operation of T helper cells and B cells, was distinctly depressed by DBTC. Hemagglutination and hemolysin titers as well as the number of direct plaque-forming cells against SRBC per spleen were decreased in a dose-related manner by DBTC. The phagocytic capacity of macrophages of rats was not affected by DOTC as was shown in the carbon clearance test. Altered immune functions were never found in mice or guinea pigs exposed to DBTC or DOTC. From this study it is concluded that both DBTC and DOTC induce immune suppression in rats by a selective inhibition of T-lymphocyte activity. Immune suppression was most pronounced in animals exposed to the chemicals during the developmental phase of the lymphoid system.
Rats were fed di-n-octyltindichloride (DOTC) for 6 weeks at dietary levels of 0, 50, or 150 ppm. At 150 ppm, slight but significant growth retardation associated with slightly decreased food efficiency was noted. Serum alkaline phosphatase (AP) activity was significantly increased both at 50 and 150 ppm. This increase in serum activity was accompanied by increased activity of this enzyme in kidney and liver homogenates. The main effect of DOTC was on the thymus. Thymus weights of rats fed 50 or 150 ppm DOTC for 4 weeks were only 48 and 16% of the control weights. Histologically, lymphocyte depletion was observed in thymus and in thymus-dependent areas of spleen and lymph nodes. In other organs no treatment-related histopathological changes were noted. The number of nucleated thymocytes and the viability of these cells exhibited a prominent and dose-related decrease with 4 weeks feeding of 50 and 150 ppm DOTC, reducing the total number of thymocytes by 67 and 94%, respectively. The effect on spleen cell counts and cell viability was less pronounced. No effect was found on bone marrow cell counts and cell viability. It is unlikely, therefore, that the primary effect of DOTC is on lymphoid stem cells. Based on the histology of the adrenals and on the results of an adrenalectomy study, it is concluded that the selective effect of DOTC on thymus is not induced by stress-related release of glucocorticoids.
Treatment of rat thymocytes with micromolar concentrations of tributyltin caused a rapid increase in the cytosolic free Ca2+ concentration that was inhibited by Ni2+, which blocks Ca2+ influx through membrane channels. The elevation of cytosolic Ca2+ was associated with extensive DNA fragmentation, which was prevented by pretreatment of the cells with either of the intracellular Ca2+ chelators quin-2 or 1,2-bis(2-amino-phenoxy)ethane-N',N',N',N',-tetraacetic acid. Loss of thymocyte viability, which followed DNA fragmentation, was also prevented by the two Ca2+ chelators or by removing extracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The pattern of DNA fragmentation was characteristic of that produced by agents which activate a Ca2(+)- and Mg2(+)-dependent endogenous endonuclease during apoptosis or programmed cell death. Additional studies showed that other organotin compounds, including trimethyltin, triphenyltin, and dibutyltin had minimal effects on cytosolic Ca2+, DNA fragmentation, and cell viability. These results are consistent with a greater susceptibility of thymocytes to tributyltin and provide a basis for understanding its selective immunotoxicity in vivo.
The inhibitor of oxidative phosphorylation tri-n-butyltin chloride (TBTC) causes membrane damage and disintegration of isolated rat thymocytes at concentrations higher than 1 microM. From a concentration of 0.1 microM, TBTC disturbs energy metabolism as indicated by an increase in methylglucose uptake, glucose consumption and lactate production and by a decrease in cellular ATP levels. Over the same TBTC concentration range, the incorporation of DNA, RNA and protein precursors are markedly reduced. Moreover the production of cyclic AMP upon stimulation of the cells with prostaglandin E1 is effectively inhibited. These effects cannot be explained by an inhibition of nucleoside kinase activity, amino acid uptake or adenylate cyclase activity. The effects of TBTC on macromolecular synthesis and cyclic AMP production are possibly due to a disturbance of the cellular energy state.
Di-n-butyltin dichloride (DBTC) or tri-n-butyltin chloride (TBTC) given in the diets of rats have previously been shown to cause atrophy of the thymus and subsequently suppression of the T-cell-dependent immune responses. To study the mechanism of the immunotoxic effects, the dose-effect relationships and the kinetics of the thymus atrophy caused by DBTC and TBTC were investigated in detail. A single oral dose of DBTC or TBTC to rats induced a dose-related reduction of relative thymus weight, which was maximal 4 days after intubation. The log dose-effect relationships for both compounds were linear and ran parallel over a dose range of 5-60 mg/kg. Dose levels calculated to cause 50% reduction of relative thymus weight were 18 mg DBTC and 29 mg TBTC per kg body wt. A single oral dose of mono-n-butyltin trichloride (MBTC), however, did not cause thymus atrophy at dose levels up to 180 mg/kg. The kinetics of the dibutyltin- and tributyltin-induced thymus atrophy in rats were investigated by measuring thymus weight, total thymic cell count, number of small, intermediate and large cells and the incorporation of DNA, RNA and protein precursors into isolated thymocytes during a period of 9 days after a single oral dose. DBTC and TBTC caused atrophy of the thymus due to a selective reduction in the number of rapidly proliferating lymphoblasts in the first 2 days after dosing. As a consequence the large pool of small lymphocytes declined in the following 2 days. On the fourth day, when atrophy was most pronounced, the frequency of the lymphoblasts increased above the controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkyltin compounds are used as stabilizers and antifouling agents. Food chain accumulation and bioconcentration have been demonstrated in crabs, oysters and salmon exposed to tributyltin oxide. In mammalian species, tributyltin compounds may be metabolized to dibutyltin derivatives and related metabolites. Di- and tributyltins appear to be less potent neurotoxicants than trimethyltins and triethyltins. Dibutyltins and tributyltins produced bile duct damage in rats, mice and hamsters. Tributyltin oxide and dibutyltin and dioctyltin compounds are potent thymolytic and immunotoxic agents in rats. Tributyltin oxide at 5 ppm in the rat diet produced immunotoxicity in a 2-year feeding study, and at 50 ppm increased the incidence of tumors of endocrine origin. In preliminary reports, 5 ppm tributyltin produced no detectable increase in tumor incidence, and 0.5 ppm produced no detectable immunotoxicity in long-term studies. Tributyltin oxide and dibutyltin acetate did not appear to be mutagenic in a large battery of mutagenicity assays but produced base-pair substitutions in one of the bacterial strains tested. Tributyltin oxide produced mutations in Chinese hamster ovary cells, increased the incidence of micronuclei in the erythrocytes of exposed male BALB/c mice, and was highly embryotoxic in vitro. Embryotoxic and teratogenic effects in mice exposed to tributyltin oxide in vivo may have been due either to direct tributyltin oxide action or responses secondary to maternal toxicity. More information is needed to determine the applicability to human risk assessments of the immunotoxicity data derived from rat studies and to establish a definitive tolerable daily intake for tributyltin oxide.
Studies of the effects of trimethyltin, tripropyltin, tributyltin and triphenyltin chlorides on the permeability of the inner mitochondrial membrane by the osmotic swelling technique in NaCl and NH4Cl media show that these compounds mediate transport of chloride ions. Studies in KCl media show that these compounds mediate a strictly coupled chloride-hydroxide antiporter transport. This exchange allows rapid titration of mitochondrial contents by pulses of HCl. Similar results on erythrocytes and smectic mesophases indicate that this is a direct action of the tin compounds and is not due to modification of a pre-existing anion carrier in the membrane.
1. The binding of trimethyltin and triethyltin to rat liver mitochondria was determined and the results were analysed by the method of Scatchard (1949). 2. One binding site (site 1) has the correct characteristics for the site to which trimethyltin and triethyltin are attached when they inhibit oxidative phosphorylation. For each compound the concentration of site 1 is 0.8nmol/mg of protein and the ratios of their affinity constants are the same as the ratio of the concentrations inhibiting oxidative phosphorylation. 3. Binding site 1 is present in a fraction derived from mitochondria containing only 15% of the original protein. In this preparation ultrasonication rapidly destroyed site 1. 4. Dimethyltin and diethyltin do not prevent binding of triethyltin to rat liver mitochondria, whereas triethyl-lead does. 5. Trimethyltin and triethyltin bind to mitochondria from brown adipose tissue and the results indicate a binding site 1 similar to that in rat liver mitochondria. 6. The advantages and limitations of this approach to the study of inhibitors are discussed.
To evaluate the functional significance of bis(tri-n-butyltin)oxide (TBTO)-induced thymus atrophy, lymphocyte depletion in spleen and lymph nodes, lymphopenia, and increased serum IgM and decreased IgG concentrations, in vivo and in vitro function studies were performed for specific and nonspecific resistance. Weaned male rats were fed diets containing 0, 20, or 80 mg TBTO/kg for at least 6 weeks. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin as well as tuberculin were significantly depressed at both dietary concentrations. Resistance to the nematode Trichinella spiralis was significantly suppressed as shown by a retarded expulsion of adult worms from the small intestine, increased counts of muscle larvae, reduced inflammatory reaction in parasitized musculature, and suppressed serum IgE titers. Also the secondary mercaptoethanol-resistant (presumably IgG) hemagglutinating antibody titer to sheep red blood cells was significantly reduced, while no significant alterations were found in IgM and IgG titers to T. spiralis, ovalbumin, and tetanus toxoid. TBTO exposure reduced the response of thymocytes in both treatment groups and of spleen cells in the 80-mg/kg group upon stimulation with T-cell mitogens and increased the response of spleen cells to B-cell mitogens. When calculated per whole spleen, the response to T-cell mitogens was strongly impaired but unaltered by B-cell mitogens. This difference can be explained by a relative increase of splenic B cells as a result of reduced numbers of T cells, as shown by cell surface marker analysis using monoclonal antibodies. Reduced splenic T-cell numbers appeared equally due to a decreased number of T helper and to T suppressor cells. From these data and from results of a time-sequence study in which effects of TBTO on cell count and cell viability of thymus, spleen, and bone marrow were investigated, it is concluded that TBTO-induced immunodeficiency was primarily due to its direct toxic action on thymocytes. When cultured in vitro in the presence of TBTO, viability of thymus and bone marrow cells was equally reduced, while after in vivo treatment viability of bone marrow cells was unaffected. Thus, the in vitro situation does not mimic the in vivo one. Concerning the nonspecific resistance, TBTO reduced macrophage function as shown by impaired splenic clearance of Listeria monocytogenes bacteria. From in vitro studies it is concluded that impaired in vivo splenic clearance was due to a reduction in both the number of adherent cells in the spleen and bacterial digestion on a cell for cell basis.(ABSTRACT TRUNCATED AT 400 WORDS)
The effect of triphenyltin on mitochondrial Ca2+ content was studied. It was found that this trialkyltin compound induces an increase in membrane permeability that leads to Ca2+ release, drop of the transmembrane potential, and efflux of matrix proteins. Interestingly, cyclosporin A was unable to inhibit triphenyltin-induced Ca2+ release. Based on these results it is proposed that the hyperpermeable state is produced by modification of 2.25 nmol of membrane thiol groups.
Three patients who developed acute nephropathy following ingestion of triphenyltin acetate (TPTA) are described. All of them had significant proteinuria, azotemia, and polyuria. Mild neurological manifestations in all patients were also noted. Hematuria and pyuria were noted in 1 severely poisoned patient. Evidence for hepatitis was present in 2 patients, and for pancreatitis in 1. Renal biopsy showed focal fusion of glomerular cell processes and proximal tubular damage with cellular necrosis. Two patients survived with complete recovery of renal functions. One old patient died of aspiration pneumonia. Acute nephropathy following organotin intoxication appears to result mainly from proximal renal tubular damage with a benign and reversible clinical course.
Recent in vitro studies have suggested that activation of apoptosis could account for the profound depletion of cortical thymocytes, which characterizes tributyltin (TBT) immunotoxicity. However, it has also been shown that TBT disrupts macromolecular synthesis and cellular energetics to an extent that might be expected to interfere with the initiation of apoptosis. The purpose of these studies was to further evaluate the morphological and biochemical characteristics of thymocyte killing by TBT and to relate this to key cellular processes. Ex vivo thymocyte cultures from immature rats were treated with bis(tri-n-butyltin) oxide (TBTO) at concentrations ranging from those which rapidly produced necrosis (5-10 microM), down to cytotoxic but subnecrotic concentrations (0.1-2 microM). In cells exposed to TBTO concentrations that caused a rapid and near maximal inhibition of protein synthesis, it remained possible to demonstrate the stereotypic internucleosomal DNA cleavage and morphological changes indicative of apoptosis. Further confirmation that apoptosis was occurring independently from protein synthesis was provided by the absence of a protective effect following cycloheximide pretreatment. Apoptosis still occurred in TBTO-treated thymocytes although intracellular ATP levels were depressed to 20% or less of control values. Cytoprotective effects were noted with the intracellular Ca2+ chelators BAPTA-AM and Quin-2 AM, and also with zinc. Cell killing by TBTO occurred without overt disturbance of thymocyte cell cycle parameters. These results indicate that thymocyte apoptosis stimulated by TBT exposure occurs independently of a requirement for protein synthesis and does not require fully conserved cellular energetics.
A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.
Organotin compounds are ubiquitous contaminants in the environment. The high biological activity of some compounds toward aquatic organisms lead to deleterious impacts in aquatic ecosystems. Here, the aquatic ecotoxicology of organotins is reviewed based on a multidisciplinary approach involving environmental chemical, toxicological, and ecological aspects. Basic results were obtained both with field and laboratory studies, and some of the most important recent results and conclusions are critically reviewed. The contamination of and fate in aquatic systems is reported and linked with effects at different levels of biological organization. Major emphasis is placed on the development of a concept of ecotoxicology that encompasses not only effect assessment alone, but also integrates environmental chemistry with aquatic toxicology. Thereby, the influence of speciation for bioavailability, basic modes of toxic action, and aquatic toxicity are discussed. This case study on organotins allows to a certain extent generalizations to ecotoxicology in general.
Programmed cell death (PCD), a genetically controlled cell deletion process, plays an important role in the regulation of cellular and tissue homeostasis. The requisite for proteolysis during PCD-induced apoptosis is well documented. The cellular proteolytic machinery includes numerous proteases localized in membranes, cytoplasm, and nucleus. This machinery may function to remove denatured or misfolded protein from the cytoplasm on a routine basis and may also cleave proteins thereby implementing their activation. The well established role of some proteases is to maintain fundamental cellular processes; however, the precise cellular location and function of other proteases which make a contribution to a unique unidirectional process such as apoptosis remains unclear. The functional overlap between 'scheduled' and 'unscheduled' proteolysis may potentially lead to confusion in this research area. In this review we will discuss certain cellular proteolytic systems and highlight the possible involvement of each in apoptosis.
Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents
cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation
of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the
mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol
and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria
and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release
from mitochondria.
Biochemical analyses of nuclear apoptosis in vitro have revealed the existence of multiple active interleukin-1beta-converting enzyme-related proteases (caspases) with distinct substrate recognition properties in extracts of preapoptotic chicken DU249 cells (S/M extracts). Previously we demonstrated that the activity of a caspase that cleaves lamins is required for the disintegration of nuclei in the late stages of apoptosis, despite the presence of a second active caspase that cleaves poly(ADP-ribose) polymerase (PARP). One simple explanation for this observation was that the lamin-cleaving caspase is sufficient to drive the nuclear events of apoptotic execution. Here, we report that phenylarsine oxide (PAO) inhibits the protease activities of recombinant human caspases as well as endogenous chicken caspases that are active in S/M extracts. PAO at 100 microM blocks the morphological changes of nuclear apoptosis in vitro and internucleosomal DNA fragmentation in S/M extracts without interfering with PARP or lamin A cleavage. Thus, lamin cleavage is not sufficient to drive the changes in nuclear morphology characteristic of apoptosis. Affinity labeling with YV(bio)KD-aomk shows that the degree of sensitivity to PAO differs among active caspases in S/M extracts. These results suggest that a PAO-sensitive caspase that is distinct from the PARP- or lamin-cleaving enzymes is required for the initiation of apoptotic morphological changes and for the activation of endonuclease(s). Taken together, our results suggest that two or more caspases are required for proteolytic events that are essential for the initiation and completion of nuclear apoptotic changes. The observation that PAO is an inhibitor of caspases and nuclear apoptotic events should be useful for the biochemical dissection of apoptosis in vitro and in vivo.
Tributyltin (TBT) salts are potent skin irritants both in humans and rodents. Data in the literature indicate mitochondria as target of TBT effects. Here, we investigate the early intracellular molecular events that follow TBT treatment and the relevance of calcium ions and mitochondria in gene-regulatory signaling pathways. Confluent HEL30 cells were treated with increasing doses of TBT (0-5 microM). At different times thereafter, the level of intracellular Ca2+, the cellular oxidative activity, nuclear factor-kappaB (NF-kappaB) activation, and IL-1alpha production were measured. TBT induced a dose-related increase of intracellular Ca2+ that reached the plateau 4 min following treatment. The increase of intracellular Ca2+ was followed by an increase in cellular oxidative activity as measured by DCFH oxidation (15 min) that preceded NF-kappaB activation (30 min) and IL-1alpha production (4 hr). All these events can be almost completely abrogated by BAPTA, an intracellular Ca2+ chelator. Furthermore, the modulation of cellular oxidative activity induced by TBT observed with rotenone, an inhibitor of the electron entry from complex I to ubiquinone, or after prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, indicates mitochondria as an important intracellular source of reactive oxygen species. These findings indicate the rise in intracellular Ca2+ as the starting event and indicate the role of mitochondria as the source of second messenger molecules essential for TBT-induced NF-kappaB activation and IL-1alpha production.
The induction of apoptosis in Jurkat T-lymphocytes with 50 microM hydrogen peroxide was associated with caspase activation. Caspase activity was first detected 3 h after treatment, and the morphological features of apoptosis were apparent by 6 h. At higher concentrations of hydrogen peroxide there was no detectable caspase activity, and the cells died by necrosis. Cells treated with hydrogen peroxide were impaired in their ability to undergo Fas-mediated apoptosis. This appeared to be the result of direct inhibition of the cysteine-dependent caspases. The cells were able to recover and undergo apoptosis at later times. Therefore, hydrogen peroxide has two distinct effects. It initially inhibits the caspases and delays apoptosis. Then, depending on the degree of the initial oxidative stress, the caspases are activated and the cells die by apoptosis, or they remain inactive and necrosis occurs. We discuss the physiological implications of cells having to maintain a reducing environment during apoptosis to allow the caspases to function.
The ability of H2O2 and tributyltin (TBT) to trigger pro-caspase activation via export of cytochrome c from mitochondria to the cytoplasm was investigated. Treatment of Jurkat T lymphocytes with H2O2 resulted in the appearance of cytochrome c in the cytosol within 2 h. This was at least 1 h before caspase activation was observed. TBT caused cytochrome c release already after 5 min, followed by caspase activation within 1 h. Measurement of mitochondrial membrane potential (delta psi(m)) showed that both H2O2 and TBT dissipated delta psi(m), but with different time courses. TBT caused a concomitant loss of delta psi(m) and release of cytochrome c, whereas cytochrome c release and caspase activation preceded any apparent delta psi(m) loss in H2O2-treated cells. Thus, our results suggest that different mechanisms are involved in triggering cytochrome c release with these apoptosis-inducing agents.
1. In animal studies, TPTA was found to be neurotoxic. In humans, variable CNS pictures have been described with or without significant EEG findings. Brain CT does not usually reveal any abnormalities. 2. Our patient presented with intermittent unique spontaneous involuntary movement of hands, facial twitching, silly smile and crying. Diplopia, drowsiness, giddiness, vertigo, bidirectional nystagmus, impairment of calculation ability, as well as disorientation to time, people and place also developed. EEG showed mild cortical dysfunction without seizures. MRI and Tc-99m HMPAO brain SPECT revealed no significant findings. TPTA may cause cellular dysfunction of brain without structural damage, which results in variable CNS clinical presentations. 3. Nadir of leucopenia was noted on the sixth day after consumption of TPTA. Liver impairment occurred on the ninth day. Borderline demyelinated neuropathy developed on the fifty-third day. CNS abnormalities, delayed peripheral neuropathy, hepatitis and leucopenia deserve monitoring for a prolonged period, even when the victim initially presents with GI upset only after consumption of TPTA.
The effect of triphenyltin on the activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated. Triphenyltin inhibits the hydrolysis of chromatophore membrane-bound pyrophosphatase in a pH-dependent pattern, being maximal at pH 9-10. At basic pH values, the inhibition produced by this organotin on membrane-bound pyrophosphatase is very similar to that produced on the chromatophore H+ATPase (I50 = 14.4 and 10 microM, respectively). Detergent-solubilized membrane-bound pyrophosphatase is also inhibited by triphenyltin, but the cytoplasmic enzyme of R. rubrum is inhibited only slightly. The inhibitory effect of triphenyltin on membrane-bound pyrophosphatase is the same with Mg-PPi or Zn-PPi, and is dependent on the chromatophore membrane concentration. Triphenyltin modified mainly the Vmax of the enzyme, and only slightly its Km. Free Mg2+ does not reverse the inhibition. Reducing agents prevent triphenyltin inhibition of the membrane-bound pyrophosphatase, but their effect is due to an alteration of the inhibitor, and not to a modification of thiol groups of the enzyme. The most likely site for triphenyltin inhibition in chromatophore membrane-bound pyrophosphatase is a component either within or closely associated with the membrane.
The release of cytochromec
- Kluck
Tributyltin increases cytosolic free Ca2+2+2+2+
- Chow
Role of mitochondria and Ca2+
- Corsini
Induction of apoptotic program in cell-free extracts: Requirements for dATP and cytochromec
- Liu
Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis
- Nicholson