Tiao Geng decoction inhibits tributyltin chloride-induced GT1-7 neuronal apoptosis through ASK1/MKK7/JNK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Tiao Geng (TG) decoction is a Chinese herbal medicine extract that has been utilized for the treatment of menopausal symptoms for a history of over 30 years. In our previous study, we suggest that TG decoction possibly exerts an anti-apoptotic effect on hypothalamic neurons of ovariectomized rats via the ASK1/MKK7/JNK pathway. Tributyltin chloride (TBTC) causes oxidative damage and induces apoptosis of primary hypothalamic neurons in rats. AIM OF THE STUDY: The present work aimed to explore the inhibition of TG decoction on TBTC-induced GT1-7 cell apoptosis and its possible molecular mechanism. MATERIALS AND METHODS: The GT1-7 cell line was exposed to TG decoction at diverse doses (31.25, 62.5, 125 µg/mL) for 24 h and later with TBTC (1 mg/L) for 1 h, with 17ß-E2 (100 nM) treatment being the positive control. Then, CCK8 assay was conducted to evaluate cell viability, while flow cytometric analysis was conducted to examine the apoptosis level. Related pathways and differentially expressed proteins were identified by tandem mass tag (TMT)-based quantitative phosphoproteomics. qRT-PCR was carried out to examine mRNA levels of Bax and B-cell lymphoma-2 (Bcl-2). Western blotting was performed to detect the levels of Bax, Bcl-2, c-Jun, c-Jun N-terminal kinase (JNK), Caspase-3 (Casp3), Mitogen-activated protein kinase kinase 7 (MKK7), and apoptosis signal-regulating kinase 1 (ASK1) . Finally, cells were pretreated with SP600125, an inhibitor of JNK, later the expression of JNK and Casp3 was measured. RESULTS: Application of TG decoction mitigated the GT1-7 cell apoptosis and injury caused by TBTC; besides, it inhibited the activation of the ASK1/MKK7/JNK pathway. Moreover, Bcl-2/Bax ratio became higher, and the MKK7, ASK1, Casp3 and c-Jun levels were inhibited. Besides, TG decoction combined with SP600125 (the JNK inhibitor) more significantly inhibited GT1-7 cell apoptosis caused by TBTC. CONCLUSION: As discovered from the experiment in this study, TG decoction has a neuroprotective effect, which is achieved through inhibiting the ASK1/MKK7/JNK signal transduction pathway to reduce GT1-7 cell apoptosis.

C-Phycocyanin: an effective protective agent against thymic atrophy by tributyltin.

Spirulina platensis, used worldwide as a food supplement, is a natural source of protein, vitamins, carbohydrates and polyunsaturated fatty acids. C-Phycocyanin (C-Pc), its major biliprotein, is known to possess anti-oxidant, anti-inflammatory and radical scavenging properties. Our present study showed that treatment with C-Pc protects the rats from Tributyltin (TBT) induced thymic atrophy. The results reveal TBT-induced oxidative stress mediated apoptosis in rat thymocytes in vivo and its attenuation by C-Pc. This ameliorative effect could be attributed to antioxidant activity of the biliprotein. C-Pc also increased TBTC reduced thymic weight and cellularity as well. TBTC-induced ROS generation and lowered GSH levels were restored by C-Pc, suggesting its radical scavenging properties. The various apoptotic determinants such as mitochondrial membrane potential, Bax/Bcl-2 ratio, caspase-3 activity and apoptotic cell population were effectively modulated by C-Pc treatment. We make this first observation to illustrate the effectiveness of C-Pc in reducing TBTC-induced thymic atrophy. The morphology of thymic tissue was restored to near normal by this biliprotein. The present study, therefore, suggests that C-Pc could serve as an effective natural antioxidant for efficient management of TBTC induced oxidative damage.

Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales.

Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT.

Estrogens counteract the masculinizing effect of tributyltin in zebrafish.

Recently, it has been demonstrated that the biocide tributyltin (TBT) can interfere with fish sex differentiation, leading to a bias of sex toward males. On the contrary, it is well known that estrogenic compounds can induce fish feminization. Yet, the combined effects of mixtures of androgenic and estrogenic compounds on fish sex differentiation have never been investigated before, even though in the environment animals are frequently exposed to both groups of xenobiotics. Therefore, in order to investigate whether exposure to estrogenic compounds can block the masculinizing effect of TBT, 5 days post-fertilization zebrafish (Danio rerio) larvae were exposed for a four month period to TBT and to the synthetic estrogen-ethinylestradiol (EE2). The fish were fed a diet containing TBT at nominal concentrations of 25 and 100 ng TBT/g, and two groups of animals were also dosed with TBT plus EE2 at nominal water concentration of 3.5 ng/L, using a flow-through design. As expected, fish exposed to TBT showed a bias of sex toward males (62.5% males in control tanks and 86% and 82% in TBT 25 and TBT 100 ng TBT/g, respectively). Co-exposure to EE2 completely blocked the masculinizing effect of TBT, with 7% males in the TBT 25 ng/g + EE2 treatment and 0% in the EE2 alone and in the TBT 100 ng/ + EE2 exposed groups. These results clearly indicate that EE2, at environmentally relevant concentrations, can block the TBT masculinizing effects in zebrafish, which suggests that in the aquatic environment the presence of estrogens may neutralize the fish masculinizing effect of TBT. Our findings highlight the need of testing the combined effects of contaminants, as single exposure studies may not be sufficient to predict the effects of mixtures of xenobiotics with antagonistic properties.

Tri-n-butyltin-induced cytotoxicity on rat thymocytes in presence and absence of serum.

Influence of fetal bovine serum (FBS) on cytotoxicity induced by tri-n-butyltin (TBT), an environmental pollutant, on rat thymocytes was examined to reveal how FBS modifies TBT cytotoxicity. As the medium FBS concentration was increased from 0 to 10%, the cytotoxicity of TBT was dose-dependently reduced when the cells were incubated with 1 microM TBT for 3 h. Almost complete inhibitions of TBT-induced changes in cell viability and population of cells with exposed phosphatidylserine (cells undergoing apoptosis) were observed when the FBS concentration was 10%. Thus, the cytotoxicity induced by 3 h incubation with TBT in FBS-free medium may be different from that in medium containing 10% FBS. However, even in presence of 10% FBS, TBT at concentrations ranging from 10 to 300 nM exerted cytotoxic action on rat thymocytes when the cells were incubated with TBT for 24 h. TBT dose-dependently increased the population of shrunken cells, of which more than 30% were stained with propidium. TBT at 30 nM or more significantly increased the population of cells with hypodiploid DNA, indicating TBT-induced apoptotic cell death. Thus, in the presence of 10% FBS, the prolonged incubation (24 h) of rat thymocytes with TBT at nanomolar concentrations induced apoptosis rather than necrosis.

Effect of Several Solutions Including Mineral-encaging Zeolites on the Restoration of Cell Motility of Tributyltin-intoxicated Euglena gracilis Z.

To examine the detoxification effect of mineral-encaged zeolites on cells impaired by pollutant-intoxication, we used a bioassay system involving Euglena gracilis Z as the model organism and TBTCl as a pollutant. TBTCl exposure causes Euglena cells to quickly change shape from a spherical to spindle form, with the process being reversible by detoxification. Taking advantage of this morphological characteristic, we examined the restoration of motility by water containing zeolites encaging different minerals. TBTCl-intoxicated Euglena cells were incubated in processed water with different types of mineral-encaging zeolites for up to 3 hours. The restoration of motility was evaluated by observing the number of motile cells with a video microscope. Remarkable recovery was observed in the incubation systems with water containing Fe-, Zn-, and Mn-encaging zeolites. However, the effect was suppressed when the water species were treated with the chelator, Chelex-100(®). An equivalent concentration of FeCl3 to that in the Fe-encaging zeolite processed water did not show significant restoration effect.

Tributyltin and dexamethasone induce apoptosis in rat thymocytes by mutually antagonistic mechanisms.

The nuclei of apoptotic thymocytes can be identified by flow cytometry as a subpopulation exhibiting reduced DNA content. We observed that rat thymocyte cultures exposed to 1.0-2.5 microM tri-n-butyltin methoxide (TBT) exhibited a rapid time- and concentration-dependent induction of apoptosis, with > 85% of cells exhibiting reduced DNA content within 1 hr after exposure to 2.0-2.5 microM TBT. In contrast, exposure to 1.0 microM dexamethasone phosphate (DEX) resulted in a gradual time-dependent increase to approximately 45% induction of apoptosis by 6 hr versus approximately 15% spontaneous induction in controls. However, simultaneous exposure to TBT and DEX resulted in a decreased response: although TBT concentrations between 0.1 and 0.5 microM did not induce apoptosis, they reduced the ability of DEX to initiate apoptosis; while at TBT concentrations > or = 1.0 microM, simultaneous exposure to DEX substantially decreased the extent of TBT-induced apoptosis and cytotoxicity. Furthermore, while treatment with the protein synthesis inhibitor cycloheximide or the protein kinase C inhibitor H-7 completely blocked DEX-induced apoptosis, neither significantly reduced induction of apoptosis by TBT. Taken together, the toxicant-specific differences in the timing and extent of apoptotic induction and the dissimilar responses to CHX and H-7 suggest that TBT and DEX initiate endonuclease-mediated apoptotic cell death through different mechanisms. Moreover, the ability of each agent to retard the action of the other suggests that these mechanisms are directly or indirectly antagonistic.

Catalytic and activating protons follow different pathways in the H(+)-ATPase of potato tuber mitochondria.

The effect of some F0F1 inhibitors on the activation of the H(+)-ATPase by the electrochemical proton gradient was investigated in mitochondria extracted from potato tubers. Transient activated state of the ATPase was revealed by addition of ATP and of the detergent lauryldimethylamine oxide (LDAO) to energized mitochondria. Venturicidin, tri-n-butyltin and aurovertin at high concentrations did not affect the process of delta mu H(+)-activation, whereas oligomycin fully blocked it. The results support the idea of separate pathways or binding sites for catalytic and activating protons.

Reversibility of tributyltin-chloride-induced protein synthesis inhibition after ATP recovery in HEL-30 cells.

The effect of tributyltin chloride (TBT) on ATP levels and protein synthesis was investigated in a murine epidermal cell line (HEL-30). Five minutes after exposure to 10(-5) M TBT, cellular levels of ATP decreased by 57% and protein synthesis was significantly inhibited. The effect of ATP was stable up to 2 h incubation, whereas inhibition of protein synthesis increased with time of treatment. Partial (1 h) or complete (2 h) recovery of the two cell functions was observed when dithiothreitol (DTT) was added to the medium, 5 min after the damage and in the absence of TBT. The sequence of the observed effects suggests that TBT inhibition of protein synthesis is due to impairment of ATP production.

Effects of tricyclohexylhydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents.

Tricyclohexylhydroxytin, commonly known as Plictran, inhibited Na+, K+-ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 microM. Both K+-stimulated para-nitrophenylphosphatase and [3-H]-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 microM, respectively. Altered pH and Na+, K+-ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na+, K+-ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na+, K+-ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K+ activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na+, K+-ATPase, K+-para-nitrophenylphosphatase, and [3-H]-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na+, K+-ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.

Inhibition of tributyltin mediated hemolysis by mercapto compounds.

Hydrophobic tributyltin (TBT) compounds at concentrations greater than 10 microM caused hemolysis of human erythrocytes and formed structures in plasma membranes. The mercapto compounds, beta-mercaptoethanol (beta MER), 2,3-dimercaptopropanol (BAL), 2,3-dimercapto-1-propane sulfonate (DMPS), DL-dithiothreitol (DTT), and meso-2,3-dimercaptosuccinic acid (DMSA) were examined for their ability to inhibit TBT mediated hemolysis. The relative order of effectiveness for inhibition of TBT mediated hemolysis was BAL greater than DTT greater than DMSA greater than DMPS greater than beta MER. A four-fold excess of BAL over TBT prevented hemolysis for 4 hrs and addition of BAL 0.5 hr after TBT reduced the rate of hemolysis. The number of membrane associated TBT aggregates observed per cell profile decreased as the BAL concentration increased from 0 to 100 microM. However, the mean diameter of TBT aggregates nearly doubled in erythrocyte suspensions at 100 microM BAL. Reactions of dimercapto compounds with lipophilic TBT aggregates may depend on their relative lipid solubilities. Also, conversion of the weak Lewis acid, TBT, from a four to a five or six-coordinate tin adduct by the dimercapto Lewis bases used could also be a factor slowing hemolysis rates.

Does phenobarbital protect against trimethyltin-induced neuropathology of limbic structures?

Because of the similarity in the pattern of limbic sites damaged by both compounds, it has been suggested that trimethyltin (TMT) may be an excitotoxin like kainic acid (KA). KA produces seizures which eventually result in neuronal damage similar to that found in epilepsy. Anticonvulsants reduce both the seizures and pathology associated with KA. Because TMT may also produce seizures, we undertook to determine whether or not some of the TMT-induced limbic neuropathology could result from seizure activity. To do this, a single dose of TMT chloride (either 7.5 or 15 mg/kg) was given per os to rats, and then phenobarbital (30 mg/kg) was administered subcutaneously in repeated doses. Treatment with phenobarbital did not prevent pathologic changes in the hippocampus, dentate gyrus, and pyriform or prepyriform cortex. Since phenobarbital did not protect against TMT-induced neuronal damage, as it has been reported by others to protect against KA-induced damage, the present findings suggest that these two toxicants probably produce hippocampal pathology via different mechanisms and that the TMT-induced pathologic changes do not require sustained electrical seizure activity.

Antagonism by d-amphetamine of trimethyltin-induced hyperactivity evidence toward an animal model of hyperkinetic behavior.

In male rats of the Long-Evans strain, either 7.0 mg/kg of trimethyltin (TMT) or 0.9% NaCl was administered by intragastric gavage. After a period of recovery from the typical signs of trimethyltin toxicity, each rat was tested at 72-hr intervals for its locomotor activity in an open field apparatus, the floor of which was divided into square grids. The baseline activity of each of the trimethyltin-treated rats was significantly greater than the saline-treated controls. d-Amphetamine, injected intraperitoneally in a dose of 0.5 or 2.0 mg/kg, augmented the hyperactivity of the trimethyltin-treated animals. However, a 4.0 mg/kg dose of d-amphetamine markedly attenuated the hyperactivity of trimethyltin-treated rats while elevating that of the controls. Since trimethyltin produced an autism-like behavioral disorder involving hyperactivity, preservation, aggressiveness and impairment in problem-solving and memory function, the placating effect of amphetamine supports the proposition that the pathology due to trimethyltin may represent an experimental analogue to the hyperkinetic syndrome in children.