Tri-n-butyltin Research Articles

Synthesis, characterization and in vitro antibacterial activity of di- and tri n-butyltin(IV) complexes of 3-amino-4 -chloridobenzohydroxamic acid

New di-and tri- n-butyl tin(IV) hydroxamate complexes of composition [n-Bu2Sn(C6H3(3-NH2)(4-Cl)CONHO)2](I) and [n-Bu3Sn(C6H3(3-NH2)(4-Cl)CONHO)] (II) have been synthesized by the reactions of n-Bu2SnCl2 and n-Bu3SnCl with potassium 3-amino-4-chlorido benzohydroxamate(KHL) in predetermined metal:ligand 1:2 and 1:1 molar ratios respectively in MeOH+THF solvent medium and characterized by physiochemical, spectroscopic (IR,1 H and 13 C NMR) studies and mass spectrometry. The bidentate nature of hydroxamate ligand involving bonding through carbonyl and hydroxamic oxygen atoms (O,O coordination) has been inferred from IR spectra. The distorted octahedral and trigonal bipyramidal geometry around tin for (I) and (II) respectively have tentatively been proposed. The electrochemical behavior of complexes studied by cyclic voltammetric technique has shown two electron tin metal centered reductions. The in vitro antibacterial activity of (I) and (II) assayed against pathogenic gram -ve bacteria E.coli , P.aeruginosa; gram +ve bacteria S.aureus, B.cereus by MIC method has revealed these to be promising antibacterial agents relative to parent ligand.

Studi Eksperimen Laju Pertumbuhan Biofouling Pada Pelat Baja Dan Aluminium Dengan Lapisan Pelindung Lilin Dan Minyak Di Pantai Boom Banyuwangi

Indonesia is a maritime country with a tropical climate and has various biofouling species. The existence of fouling in a marine structure has a large impact on losses if it is not addressed immediately. Antifouling is needed to overcome the presence of fouling. Various development efforts to inhibit, prevent and eliminate biofouling continue to be made. Generally, the method used by the public is by using commercial antifoulant protective paints with heavy metal main components such as TBT (tri-n-butyl tin). However, currently various studies have proven that TBT compounds are not only toxic to biofouling biota but also harmful to other marine organisms. Further research is needed to find antifouling alternatives that do not contain toxic compounds and are more environmentally friendly. This study was designed to determine the growth rate of marine growth in steel and aluminum plate materials at Boom Banyuwangi Beach by using a protective coating in the form of oil and wax. From the results of the research designed, it can be analyzed the efficiency of using protective layers of oil and wax with steel and aluminum materials on the growth rate of marine growth according to the conditions of the waters of Boom Banyuwangi Beach. The use of oil protectors on steel and aluminum test specimens resulted in more efficient results in inhibiting the growth rate of biofouling than the use of wax protectors

Organophosphazenes 27: the Synthesis and Polymerization of (4- Ethenylphenyl)Pentafluorocyclotriphosphazene

The synthesis and homopolymerization of a fluorocyclotriphosphosphazene with a directly bonded styrene moiety is presented. A multistep sequence was employed wherein commercially available p-bromostyrene is first converted to 1-(4-bromophenyl)-1-methoxyethene, 4-BrC6H4C(OCH3)CH2 which in turn is lithiated and added to N3P3F6 to give (4-(1-methoxyethenyl)phenyl)pentafluorocyclotri phosphazene, N3P3F5(4-C6H4C(OCH3)˭CH2)(2). Addition of tri(n-butyl)tin hydride to 2 followed by elimination of tri(n-butyl)tin methoxide over silica produced the target monomer, (4-ethenylphenyl)- pentafluorocyclotriphosphazene, and N3P3F5(4-C6H4CH˭CH2)(3). The new phosphazene derivatives were characterized by mass spectrometry, NMR (1H,13C,19F,31P) and IR spectroscopy. Radical addition polymerization of 3 resulted in the formation of poly(p-pentafluorocyclotriphosphazenylstyrene)(4). Molecular weights of the new polymer were determined by GPC and membrane osmometry. The GPC data suggested the occurrence of chain transfer in the polymerization. TGA studies showed that 4 exhibits high thermal stability compared to other organophosphazene polymers. The Tg value is significantly greater than that of polystyrene.

The mitochondrial F1FO-ATPase desensitization to oligomycin by tributyltin is due to thiol oxidation

The antibiotic oligomycin is known to inhibit mitochondrial F-type ATP synthases. The antibiotic inhibits both ATP synthesis and hydrolysis by blocking the H(+) translocation through FO which is coupled to the catalytic activity of F1. The amphiphilic organotin tri-n-butyltin (TBT), a known mitochondrial poison, can penetrate into biological membranes and covalently bind to electron-donor atoms of biomolecules such as sulfur. This study aims at exploring the mechanism(s) involved in the enzyme desensitization to oligomycin which occurs at concentrations >1 μM TBT. This poorly known effect of TBT, which only appeared at temperatures above the break in the Arrhenius plot of the enzyme activity, was found to be accompanied by the oxidation of isolated thiol groups of the mitochondrial complex. The oligomycin sensitivity was restored by the reducing agents glutathione and dithioerythritol and not influenced by antioxidants. The whole of data is consistent with the hypothesis that thiol oxidation is caused by TBT covalent binding to cysteine residues in a low-affinity site on FO and not by other possible oxidative events. According to this putative model, the onset of tin-sulfur bonds would trigger conformational changes and weaken the oligomycin interaction with FO.

Tri-n-butyltin binding to a low-affinity site decreases the F1FO-ATPase sensitivity to oligomycin in mussel mitochondria

In mussel digestive gland mitochondria the environmental pollutant tri-n-butyltin (TBT), other than strongly inhibiting ATPase activity at <1.0 μ m, at ≥1.0 μ m concentration was previously found to desensitize F1FO-ATPase to the antibiotic oligomycin. While F1FO-ATPase inhibition is widely known as one of the main mitochondrial damages caused by TBT, the enzyme's desensitization to oligomycin was quite unexpected. The possible mechanisms involved are here stepwise approached, aiming at enlightening the molecular mechanism(s) of TBT toxicity and the still poorly investigated oligomycin interaction with FO. The findings strongly suggest that the oligomycin desensitization directly stems from the covalent binding of TBT to monothiols of the F1FO-ATPase. This binding implies sulfur oxidation, irrespective of the possible formation of radical species in mitochondria, a mechanism which does not seem to be involved here. It is hypothesized that TBT interacts with the enzyme complex in at least two sites distinguished by different affinities: TBT binding to the high-affinity site would lead to ATPase inhibition, while TBT binding to monothiols in the low-affinity site could mirror the decrease in F1FO-ATPase oligomycin sensitivity at ≥1.0 μ m TBT. Experiments carried out on inside-out submitochondrial particles hint that TBT binding destabilizes the oligomycin-blocked FO conformation, allowing proton flux recovery within FO, without uncoupling the catalytic function from proton channeling. Copyright © 2012 John Wiley & Sons, Ltd.

Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland

The toxicity of organotins and especially tri- n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze–thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellman’s reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues.

On the effect of anorganic tin compound, Bis (trinbutyl tin) oxide, and 5-2′-Dichloro-4′ nitro-salycyl-anilide-ethanolamine to the marine organisms

On the effect of anorganic tin compound, Bis (trinbutyl tin) oxide, and 5-2′-Dichloro-4′ nitro-salycyl-anilide-ethanolamine to the marine organisms

Tri- n-butyltin increases intracellular Zn 2+ concentration by decreasing cellular thiol content in rat thymocytes

Effect of tri- n-butyltin (TBT), an environmental pollutant, on intracellular Zn 2+ concentration was tested in rat thymocytes to reveal one of cytotoxic profiles of TBT at nanomolar concentrations using a flow cytometer and appropriate fluorescent probes. TBT at concentrations of 30 nM or more (up to 300 nM) significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn 2+ concentration, under external Ca 2+- and Zn 2+-free condition. Chelating intracellular Zn 2+ completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn 2+ from intracellular store site. Oxidative stress induced by hydrogen peroxide also increased the FluoZin-3 fluorescence intensity. The effects of TBT and hydrogen peroxide on the fluorescence were additive. TBT-induced changes in the fluorescence of FluoZin-3 and 5-chloromethylfluorescein, an indicator for cellular thiol content, were correlated with a coefficient of −0.962. Result suggests that the intracellular Zn 2+ release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn 2+ potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn 2+ concentration may be a type of stress responses to protect the cells.

The SWIFT periphyton test for high-capacity assessments of toxicant effects on microalgal community development

The SWIFT periphyton test was developed as a simple and high-capacity approach for assessing toxicant effects on the succession of natural communities. Attached microbial communities (periphyton) were developed over a 7 to 9 day period on artificial glass substratum submerged at sea and then transferred to a controlled indoor environment for 4 day incubation with toxicants. Added nutrients and continuous light over the test phase facilitated periphyton growth and consequently accelerated the rate of ecological succession. The structure and physiology of the algal community can be analysed using any suitable method. In this paper we suggest HPLC-based analyses of community pigment profiles as a fast and less laborious alternative to e.g. microscopic species counts. Several crucial properties of the SWIFT test were evaluated: the biological complexity of the periphyton used for testing, the consequences of the 4 day incubation under artificial conditions, toxicant effects on community structure and the feasibility of using pigment profiles as descriptors of periphyton communities. The performance of SWIFT as an ecotoxicological test system was evaluated using the three antifouling agents irgarol 1051, Sea-Nine™ 211 (DCOIT) and TBT (tri- n-butyltin). Periphyton communities used in SWIFT were of high taxonomic richness and diversity, and there were no indications for adverse changes in these characteristics over the 4 day test phase. Community pigment profiles discriminated between different communities with a resolution equally good to microscopic species counts. Comparative concentration–response studies with a two-week microcosm experiment and SWIFT revealed similar concentration and toxicant-dependent shifts in the relative abundances of species. This shows that despite the methodological simplifications in SWIFT, crucial ecological features such as competition and species sensitivity profiles were still the major factors determining the ecological succession under toxicant exposure. The sensitivity of SWIFT was equal to that of the flow-through microcosms for irgarol and TBT and lower for DCOIT, probably because of the semi-static test regime in SWIFT. Species composition and pigment profiles over seasons, together with toxicant-induced effects on the diatom xantophyll cycle and the relative content of beta-carotene, shows that pigment profiles reflect both the taxonomy and the physiology of a community. Similar to encountered changes in species composition, pigment profiles were specifically influenced by the tested toxicants.

The Effect of Tri(n-butyl) Tin(IV) –2– [3–Benzoyl phenyl] Propionate on the Degradation and Stabilisation of PVC in Inert and Oxidative Atmospheres

Tri(n-butyl) tin(IV)–2–[3–benzoyl phenyl] propionate has been used to enhance the shelf life of rigid poly (vinyl chloride) (PVC). Studies of the thermal degradation of PVC have been a prime focus for a long time. Different substances have been tried to control and stabilise the process of thermal degradation. In the present work the aforementioned stabiliser has been used for this purpose. The samples of PVC containing different amounts of the stabiliser were prepared and treated at 190 ± 3 °C. The effect of the stabiliser on the degradation in nitrogen and air environments was studied through conductometric and spectroscopic measurements. The stabiliser was effective in slowing down the process of degradation. It also increased the initial induction period, i.e. the time during which no sign of degradation were observed. About 3% of stabiliser reduced the rate of the degradation of PVC by half, and almost doubled the induction period. It was more efficient in nitrogen than in air. These findings indicate that tri(n-butyl) tin(IV)–2–[3–benzoyl phenyl] propionate is a good inhibitor as well as an effective retarder.

Tri- n-butyltin-induced blockade of store-operated calcium influx in rat thymocytes

Tri- n-butyltin (TBT), one of environmental pollutants, disturbs intracellular Ca 2+ homeostasis by increasing intracellular Ca 2+ concentration ([Ca 2+] i). Effect of TBT on oscillatory change in [Ca 2+] i (Ca 2+ oscillation) of rat thymocytes was examined using a laser microscope with fluo-3-AM in order to further elucidate the TBT toxicity related to intracellular Ca 2+. The Ca 2+ oscillation was completely attenuated by 300 nM TBT. Since store-operated Ca 2+ channels are involved in the generation of Ca 2+ oscillation, the action of TBT on an increase in [Ca 2+] i by Ca 2+ influx through store-operated Ca 2+ channels was examined. The increase in [Ca 2+] i by the store-operated Ca 2+ influx was not affected by 3 nM TBT. However, TBT at 10 nM or more significantly reduced the increase in [Ca 2+] i. It is likely that TBT attenuates the Ca 2+ oscillation by reducing the Ca 2+ influx through store-operated Ca 2+ channels.

Application of a Gas Chromatography/Mass Spectrometric Method for the Determination of Butyltin Compounds in Sediment

A gas chromatography/mass spectrometric (GC/MS) method has been developed for the determination of trace mono-n-butyltin (MBT), di-n-butyltin (DBT), and tri-n-butyltin (TBT) compounds in sediments. Samples were extracted by 10% acetic acid in methanol containing 0.03% tropolone and were then derivatized for GC/MS analysis. Ethylation by sodium tetraethylborate and phenylation by sodium tetraphenylborate were evaluated as a derivatization reaction of the organotins in sample extract. n-Hexane was added into reaction media in the beginning of the reaction for the continuous extraction of derivatized organotins. Ethylation requires less than 2 hours to get proper derivatization yields for MBT, DBT, and TBT altogether and produces relatively low amounts of side reaction products. Compared to ethylation, phenylation requires much longer time but provides relatively lower yield and produces considerable amounts of side reaction products. Therefore, the ethylation reaction was applied for the analysis of organotin compounds in sediment. An isotope dilution mass spectrometric (IDMS) method based on GC/MS has been applied to the accurate determination of DBT compounds in the sediments. The IDMS results from the analyses of sediment samples showed a reasonable repeatability and a good agreement with the values obtained by IDMS based on liquid chromatography/induced coupled plasma/mass spectrometry.

Involvement of mitochondrial and death receptor pathways in tributyltin-induced apoptosis in rat hepatocytes

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.

Involvement of mitochondrial and death receptor pathways in tributyltin-induced apoptosis in rat hepatocytes

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.

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 μ m 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 n m 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 n m 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.

Effects of A23187 and CaCl 2 on tri- n-butyltin-induced cell death in rat thymocytes

As tri- n-butyltin (TBT), one of the environmental pollutants, is accumulated in wild animals, concern regarding the toxicity of TBT in both wildlife and human is increasing. TBT has been reported to increase intracellular Ca 2+ concentration in several types of cells. In order to examine how Ca 2+ is involved in TBT-induced cell death, the effect of TBT on rat thymocytes has been compared with that of A23187, a calcium ionophore, under various concentrations of external Ca 2+ using a flow cytometer and fluorescent probes. Although both TBT and A23187 were toxic to cells under normal Ca 2+ condition, under external Ca 2+-free condition the cytotoxic action of TBT was potentiated without changing the threshold concentration while that of A23187 was completely abolished. A23187 attenuated the TBT-induced descent in cell viability under normal Ca 2+ concentration despite intracellular Ca 2+ concentration was increased. As external Ca 2+ concentration increased, the TBT-induced increase in number of dead cells gradually decreased whereas the number of cells in an early stage of apoptosis increased. Results suggest that Ca 2+ has contradictory actions on the process of TBT-induced cell death in rat thymocytes.

Relationship between lysosomal membrane destabilization and chemical body burden in eastern oysters (Crassostrea virginica) from Galveston Bay, Texas, USA

Lysosomal destabilization was measured by using hemocytes of eastern oysters (Crassostrea virginica) collected along a chemical concentration gradient in Galveston Bay, Texas, USA. Results of the lysosomal response were compared to concentrations of organic compounds and trace elements in oyster tissue. Concentrations (on a dry-wt basis) ranged from 288 to 2,390 ng/g for polycyclic aromatic hydrocarbons (PAHs), 38 to 877 ng Sn/g for tri-n-butyltin (TBT), 60 to 562 ng/g for polyclorinated biphenyls (PCBs), and 7 to 71 ng/g for total DDT. Trace element concentrations (on a dry-wt basis) ranged from 1.1 to 4.0 microg/g for Cd, 105 to 229 microg/g for Cu, 212 to 868 microg/g for Al, and 1,200 to 8,180 microg/g for Zn. The percentage of destabilized lysosomes ranged from 34 to 81%. A significant positive correlation (p < 0.05) was observed between lysosomal destabilization and body burden of organic compounds (PAHs, PCBs, TBT, and chlorinated pesticides). No significant correlation was found between metal concentrations and lysosomal destabilization. Based on lysosomal destabilization, the study sites in Galveston Bay can be placed in one of three groups: healthy (Hanna Reef and Confederate Bay), moderately damaged (Offats Bayou and Todd's Dump), and highly damaged (Yacht Club and Ship Channel). Lysosomal destabilization that is consistent with toxic chemical body burdens supports previous observations that lysosomal membranes are damaged by toxic chemicals and indicates that this method can serve as an early screening tool to assess overall ecosystem health by using oysters.

Modification of NMDA responses by tri‐n‐butyltin in rat brain neurons

1. The effects of the organotin, tri-n-butyltin (TBT), on N-methyl-D-aspartate (NMDA) induced membrane currents were investigated in order to evaluate possible neuronal actions of this toxic environmental pollutant. Experiments were conducted on neurons acutely dissociated from the rat dorsal motor nucleus of vagus (DMV) using the nystatin-perforated patch clamp recording technique. 2. In Mg(2+)-free physiological recording solutions, the application of NMDA to single DMV neurons held at a holding potential (V(H)) of -40 mV evoked an inward current which rapidly reached a peak before declining to a steady-state inward current. This was followed, immediately after NMDA washout, by a transient outward current. TBT (100 nM) reversibly caused a slight reduction in the inward currents and greatly increased the amplitude of the outward currents. 3. The reversal potential of the NMDA-induced outward current in the presence of TBT was -86.7 mV, close to the theoretical K(+) equilibrium potential of -85.7 mV. 4. The NMDA-induced outward current was completely blocked when the K(+) in the internal solution was replaced with equimolar Cs(+). Under these conditions, the NMDA induced current was more sustained and was unaffected by TBT. 5. The NMDA-induced outward current was markedly inhibited by 5 mM tetraethylammonium chloride and 300 nM charybdotoxin, and it was abolished by removal of extracellular Ca(2+), suggesting that the outward current was due to the activation of Ca(2+)-activated K(+) channels by Ca(2+) influx through NMDA receptors. 6. In conclusion, in rat DMV neurons, TBT potentiates the Ca(2+)-activated K(+) current induced by NMDA application without having any direct effects on the NMDA-induced inward current. Given the significant role of NMDA receptor mediated excitation in various physiological and pathological processes, the modulation of this response by TBT may have an important influence on neuronal function.

Genotoxic, cytotoxic and ontogenetic effects of tri- n-butyltin on the marine worm, Platynereisdumerilii (Polychaeta: Nereidae)

The genotoxic, cytotoxic and ontogenetic (embryo-larval) or developmental effects of tri- n-butyltin (TBT), were investigated in Platynereis dumerilii. Following the determination of maximum tolerated dose with regard to ontogenetic effects and mortality, early life stages of P. dumerilii were exposed to a range of TBT concentrations. Subsequently, the embryo-larvae were analysed for evidence of genotoxicity and cytotoxicity. Genotoxicity was assessed using cytogenetic endpoints that included the frequency of sister chromatid exchanges and chromosomal aberrations from metaphase spreads. Cytotoxicity was evaluated by determining the proliferative rate index of the growing embryo-larval cells using 5-bromodeoxyuridine labelling of the chromosomes or fluorescence plus Giemsa staining technique. TBT-exposed embryo-larvae of P. dumerilii exhibited sensitivity similar to that of other invertebrates, indicating that P. dumerilii is a suitable ecotoxicity test species. The results also suggested dose-dependent effects for genotoxic and cytotoxic end points in relation to TBT exposure. The present study highlights the need to elucidate the relative importance of direct genotoxic and indirect effects through production of genotoxic hormonal derivatives.

Induction of apoptosis in the blue mussel Mytilus galloprovincialis by tri- n-butyltin chloride

Induction of apoptosis by tri- n-butyltin (TBT) in gill tissue of the mussel Mytilus galloprovincialis was investigated. The terminal dUTP nick-end labeling technique (TUNEL) was used to detect cells displaying DNA fragmentation within gill structures. Genomic DNA fragmentation was detected as characteristically ladder-like pattern of DNA fragments induced by single injection of different doses of TBT (1–5 μg/g) below the mantle, directly into the pallial fluid, after 24 h of incubation. DNA degradation of higher order DNA structure, as well as reduced G 0/G 1 cell cycle region (the sub-G 1 region) was detectable after 1.5 h of TBT incubation. Presence of apoptotic cells in mussels’ gills was indicated by the selective loss of G 2/M cells concomitant with the appearance of cells with decreased DNA content in S and G 0/G 1 cell cycle regions. The effect of the TBT on cell cycle in a mussel gill was a dose related and exposure time depending. The possible mechanism of induction of apoptosis in vivo in gill tissue of mussel treated with TBT is discussed.