Main Toxic Component Research Articles

Kinetics of the continuous reaction crystallization of barium sulphate in BaCl2 - (NH4)2 SO4 - NaCl - H2O system - neural network model

Kinetics of the continuous reaction crystallization of barium sulphate in BaCl2 - (NH4)2 SO4 - NaCl - H2O system - neural network model One of the main toxic components of post quenching salts formed in large quantities during steel hardening processes is BaCl2. This dangerous ingredient can be chemically neutralized after dissolution in water by means of reaction crystallization with solid ammonium sulphate (NH4)2 SO4. The resulting size distribution of the ecologically harmless crystalline product - BaSO4 - is an important criteria deciding about its further applicability. Presence of a second component of binary quenching salt mixture (BaCl2-NaCl) in water solution, NaCl, influences the reaction-crystallization process kinetics affecting the resulting product properties. The experimental 39 input-output data vectors containing the information about the continuous reaction crystallization in BaCl2 - (NH4)2 SO4 - NaCl - H2 O system ([BaCl2]RM = 10-24 mass %, [NaCl]RM = 0-12 mass %, T = 305-348 K and τ = 900-9000 s) created the database for the neural network training and validation. The applicability of diversified network configurations, neuron types and training strategies were verified. An optimal network structure was used for the process modeling.

A New Anti-loxoscelic Serum Produced Against Recombinant Sphingomyelinase D: Results of Preclinical Trials

Envenomation by Loxosceles species (brown spider) can lead to local dermonecrosis and to serious systemic effects. The main toxic component in the venom of these spiders is sphingomyelinase D (SMase D) and various isoforms of this toxin are present in Loxosceles venoms. We have produced a new anti-loxoscelic serum by immunizing horses with recombinant SMase D. In the present study, we compared the neutralization efficacy of the new anti-loxoscelic serum and anti-arachnidic serum (the latter serum is used for therapy for loxoscelism in Brazil) against the toxic effects of venoms from spiders of the genus Loxosceles. Neutralization tests showed that anti-SMase D serum has a higher activity against toxic effects of L. intermedia and L. laeta venoms and similar or slightly weaker activity against toxic effects of L. gaucho than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy.

Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization

Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 min after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.

Prevalidation of in vitro continuous flow exposure systems as alternatives to in vivo inhalation safety evaluation experimentations: Outcome from MAAPHRI-PCRD5 research program

Diesel engine emission aerosol-induced toxicity patterns were compared using both in vitro (organotypic cultures of lung tissue) and in vivo experimentations mimicking the inhalation situation with continuous aerosol flow exposure designs. Using liquid media resuspended diesel particles, we show that toxic response pattern is influenced by the presence of tensioactive agent in the medium which alter particle-borne pollutant bioavailability. Using continuous aerosol exposure in vitro, we show that with high sulfur fuel (300 ppm) in the absence of oxidation catalysis, particulate matter was the main toxic component triggering DNA damage and systemic inflammation, while a very limited oxidant stress was evidenced. In contrast, with ultra-low sulfur fuel in the presence of strong diesel oxidation catalysis, the specific role of particulate matter is no longer evidenced and the gas phase then becomes the major component triggering strong oxidant stress, increased NO 2 being the most probable trigger. In vivo, plasma tumor necrosis factor alpha (TNFalpha), lung superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activity levels varied in agreement with in vitro observations. Diesel emission treatment with oxycat provokes a marked systemic oxidant stress. Again NO 2 proved to account for a major part of these impacts. In conclusion, similar anti-oxidant responses were observed in in vitro and in vivo experiments after diesel emission aerosol continuous flow exposures. The lung slice organotypic culture model-exposed complex aerosol appears to be a very valuable alternative to in vivo inhalation toxicology experimentations in rodents.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques—mouse bioassay, ELISA, HPLC and mass spectrometry—we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6±12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6±0 5 MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.

Paralytic toxicity in the ribbon worm Cephalothrix species (Nemertea) in Hiroshima Bay, Hiroshima Prefecture, Japan and the isolation of tetrodotoxin as a main component of its toxins

Paralytic toxicity of ribbon worms ‘himomushi’ in Japanese, identified as undescribed species of the genus Cephalothrix, found on the surface of the shells of cultured oysters in Hiroshima Bay, Hiroshima Prefecture was examined between April 1998 and December 2001. The toxicity study showed that all of specimens were found to contain toxins with strong paralytic action in mice; the highest toxicity (as tetrodotoxin, TTX) was 25,590 mouse units (MU) per gram for whole body throughout the monitoring period. The main toxic component of this himomushi toxin (HMT) was isolated from a pooled specimen (390 g; total toxicity 2,897,000 MU) by a method that consisted of treatment with activated charcoal, chromatography on Bio-Gel P-2 and Bio-Rex 70 (H + form), and finally crystallization from an acidified methanolic solution. The recrystallized toxin showed a specific toxicity of 3520 MU/mg. This toxin showed (M+H) + and (M+H–H 2O) + ion peaks at m/ z 320 and 302, respectively, by electrospray ionization–mass spectrometry (ESI–MS). The absorption band at 3353, 3235, 1666, 1612 and 1076 cm −1 were observed in infrared spectrum of this toxin. This spectrum was indistinguishable from that of TTX. The 1H-NMR spectrum for the recrystallized toxin was the same as that for TTX. The pair of doublets centered at 2.33 ( J=10.0 Hz) and 5.48 ppm ( J=10.0 Hz) which are characteristic of TTX, were shown to be coupled by double irradiation. Furthermore, by gas chromatography–mass spectrometry (GC–MS) of the alkali-hydrolyzate of this toxin indicated the presence of quinazoline skeleton (C9-base) specific to TTX.

Analysis of microcystins in cyanobacteria blooms and surface water samples from Meiliang Bay, Taihu Lake, China

Taihu Lake is the third largest freshwater lake in China. In recent years, the water pollution of cyanobacteria blooms has become a severe problem in this area. Microcystins (MCs) are an important group of toxic compounds mainly produced by some cyanobacteria species and have both acute and chronic hepatotoxic effects on animals and humans. This paper presents the first data on the identification and detection of MCs in both natural occurring cyanobacteria blooms and surface water samples (0−0.5 m), collected from Meiliang Bay, Taihu Lake, China. A conventional method for extraction and isolation of MCs from cyanobacteria blooms was applied. High-performance liquid chromatography (HPLC) analysis showed that the main toxic component in the cyanobacteria materials was MC-LR. The monoclonal antibody (mAb) against MC-LR produced by hybridoma technique was employed for direct competitive ELISA to detect the concentrations of MCs in bloom and water samples collected in 2001. The results not only revealed the presence of MCs but also temporal variations of MCs levels of three sampling stations in Meiliang Bay in 1 year. It is obvious that the MC contents were relatively higher during warm months and related with the status of eutrophication. Our study indicates the threat associated with MCs in water body of Taihu Lake. To prevent the MCs potential hazard on public health in this area, some necessary measures of monitoring and control of growth of cyanobacteria are urgently needed.

The influence of smoking on fertility, pregnancy and lactation

OBJECTIVE: To decribe the influence of smoking on different phases of the reproductive process, fecundation, pregnancy and lactation, drawing special attention to the mechanisms of action of the main toxic components found in cigarettes. To suggest prophylactic measures for environmental control and how to reduce childreńs exposure to tobacco smoke. METHODS: Nonsystematic literature review using MEDLINE database. RESULTS: Smoking has a negative effect on different phases of the reproduction process, by direct action of main toxic components, nicotine and carbon monoxide. Smoking reduces fertility, compromises the length of gestation and infant birthweight. Smoking mothers also have reduced breast milk production and shorter lactation, which affects infant weight gain through unclear mechanisms in which prolactin may probably be involved. CONCLUSIONS: Smoking compromises reproductive function quality in different phases, acting mainly on intrauterine and extrauterine development of infants. Since during pregnancy and lactation women are in closer contact with health professionals, antismoking campaigns should be especially addressed to this period.

Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library.

Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus.

Ontogenetic development of Loxosceles intermedia spider venom

Envenomation by Loxosceles spider has become a public health problem in the South region of Brazil, mainly due to high levels of domiciliary infestation by Loxosceles intermedia spiders. The toxic effects of L. intermedia venom are mostly associated with a 35 kDa protein (F35) which presents complement-dependent haemolytic and dermonecrotic activities. The aim of this study was to detect, through biological and immunochemical assays, the appearance of the main toxic component, F35, during the ontogenetic development of L. intermedia spiders. The toxin appeared in its fully active form in venom of third instar spiderlings; from then on its activity increased throughout development until adulthood. On the other hand, F35 was not detected in extracts of either eggs or spiderlings of the first and second instars.

A monoclonal antibody directed against the non-toxic subunit of a dimeric phospholipase A2 neurotoxin, crotoxin, neutralizes its toxicity.

Crotoxin is the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. It is a phospholipase A2 neurotoxin constituted by the association of two subunits: an acidic, non-toxic and non-enzymatic subunit (CA) and a basic, weakly toxic phospholipase A2 (CB). A murine monoclonal antibody directed to the non-toxic subunit CA, A-56.36, was shown to fully neutralize the toxicity of crotoxin. When the in vitro pharmacological properties of crotoxin were further tested, A-56.36 was shown to enhance the enzymatic activity on negatively-charged phospholipids and to increase the acetylcholine release triggered by crotoxin on Torpedo synaptosomes. These effects were explained by the fast dissociation of the crotoxin complex in the presence of the monoclonal antibody A-56.36 and the immunocomplexation of CA, with CB being released in solution. CB is less toxic than crotoxin, has a higher enzymatic activity and triggers a higher acetylcholine release than crotoxin, due to its strong enzymatic activity. A single-chain variable fragment antibody was prepared from monoclonal antibody A-56.36. It binds to CA with a similar affinity than the parental immunoglobulin and exhibits similar effects on the in vitro pharmacological properties of crotoxin.

Crystal structure of vipoxin at 2.0 Å: an example of regulation of a toxic function generated by molecular evolution

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A 2 (PLA 2) and a non-toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA 2 activity is of great pharmacological interest, and the present structure will be a model for structure-based drug design.

Biologically active human anti-crotoxin scFv isolated from a semi-synthetic phage library

Background: The display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. Objectives: Here we explored the use of this technology to find human antibodies with biological properties. Phage-scFv specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus, were isolated from a `single pot' repertoire of more than 108 clones made in vitro from human V gene segments [1]. The crotoxin molecule is composed of two noncovalently linked subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component B (CB) and an acidic, nonenzymatic and nontoxic subunit called component A (CA). CA is able to increase the toxicity as well as the specificity of action of CB simultaneously reducing its enzymatic activity. Study design: Two clones were isolated (4-21 and 5-3-1) which are specific of the basic subunit CB, but of a moderate affinity (about 10−7 M). Clones 4-21 and 5-3-1 have different amino acid sequences and different effects on CB properties suggesting that they are raised against different CB epitopes. Purely cholinergic synaptosomes isolated from Torpedo electric organs provide a suitable model to study the presynaptic effects of crotoxin. In this model, CB was shown to induce a larger acetylcholine release than crotoxin. Results: A dose-dependent increase of acetylcholine release was observed when crotoxin was incubated with increasing amounts of phage-scFv 4-21. This clone was also shown to increase the enzymatic activity of crotoxin. These observations suggest that phage-scFv might dissociate the complex CA-CB. It could be therefore a neutralizing antibody since CB is much less toxic than crotoxin. This shows that `single pot' libraries are capable of providing not only immunochemical reagents of high specificity but also biological reagents of high quality. The use of this library appears to open new possibilities for immune passive therapy.

Transformation of Grayanotoxin III to 10-epi-Grayanotoxin III. Its X-Ray Crystallographic Analysis and Acute Toxicity in Mice.

Grayanotoxin (GTX) III, a tetracyclic diterpenoid, is the main toxic component obtained from the leaves of Leucothoe grayana MAX. In this paper, we report the correlation between toxicity and C10 stereostructure of GTX-III. 10-epi-GTX-III was derived from GTX-III in four steps. Oxidation of the GTX-II-3,6,14,16-tetraacetate with formic acid/hydrogen peroxide in chloroform gave 10,20-epoxy derivation in high yield. Reduction of the 10,20-epoxyacetate with LiAlH4 in tetrahydrofuran gave the two products, 10-epi-GTX-III and 10,20-epoxy-GTX-II. The absolute structure of the 10-epi-GTX-III was deduced from the results of X-ray crystallographic analysis. Dosage level of acute toxicity of 10-epi-GTX-III in mice was estimated at about half of natural GTX-III.

Haemoperfusion in Amanita phalloides Poisoning

Amanita phalloides is responsible for about 90 per cent of all fatal cases of mushroom intoxication. The amatoxins, the main toxic component of these fungi, are responsible for gastro-intestinal symptoms as well as hepatic and renal failure. Three brothers with Amanita phalloides poisoning were admitted with gastro-intestinal symptoms beginning 12 h after ingestion. Jaundice, hepatomegaly and neurological symptoms were not present, but liver enzymes were moderately increased. Alfa-amanitin was detected in sera of all patients. All patients underwent charcoal hemoperfusion and two of them had additional hemodialysis along with conservative therapy. Liver enzymes that showed a marked increase on the second day of therapy decreased to normal levels on the 28th day. All of our patients survived. This life saving role of early haemoperfusion in Amanita phalloides poisoning is emphasized.

Amino acid sequences of a heterodimeric neurotoxin from the venom of the false horned viper ( Pseudocerastes fieldi)

The main toxic component of the venom of the false horned viper, Pseudocerastes fieldi, is a heterodimeric neurotoxin composed of a basic subunit, Cb II, and one of two acidic subunits, either Cb Iα or Cb Iβ. The nontoxic acidic subunit increases the toxicity of the basic subunit. Both subunits have phospholipase A 2 (PLA 2) amino acid sequences. Cb Iα and Cb Iβ themselves are inactive towards phosphatidylcholine and when complexed with Cb II promote a delay in the onset of phospholipase activity of Cb II. Cb Iα and Cb Iβ do hydrolyze the synthetic substrate, 3-octanoyloxy-4-nitrobenzoic acid, but at < 1% the rate of Cb II. Comparisons of the amino acid sequences of Cb II and Cb Iα with the corresponding acidic and basic subunits of other heterodimeric neurotoxins show high amino acid sequence identity. Some of the amino acids which are different between the acidic and basic subunits are in highly conserved sequences in their respective types of PLA 2. This suggests that these amino acid changes in the conserved regions are important for the structure and function of the heterodimeric proteins.

Reversal of toxicity using avidin-based hemoperfusion: a model system in rats using biotinylated melittin.

The high-affinity interaction between avidin and biotin (Kd = 10(-15)M) can be exploited to develop specific protocols for retrieval of biotinylated drugs and toxicants from biological fluids. Melittin, the main toxic component of bee venom, was biotinylated and used as a model toxicant to determine whether avidin-based extracorporeal hemoperfusion could remove biotinylated melittin and thus alter the severity of the toxic response in rats. Melittin was biotinylated using N-hydroxysuccinimide-long-chain biotin. Biotinylated melittin produced 100% lethality in rats by 120 min following four sequential intravenous injections of 1.7 mg/kg biotinylated melittin (0, 5, 20, and 35 min). An avidin hemoperfusion column was constructed (10 mg avidin/1 ml gel) and connected via the femoral vasculature to rats intoxicated with biotinylated melittin. Controls rats were hemoperfused using avidin columns blocked with d-biotin. None of the 6 rats hemoperfused using the biotin-blocked avidin column control survived, whereas 5 of 9 of the experimental rats survived to 120 min. The difference between the two survival rates was statistically significant (p < 0.0048). Thus, avidin-based hemoperfusion improved survival following biotinylated melittin toxicity and strengthens the concept that avidin-based hemoperfusion can reverse the toxicity of biotinylated toxicants.

Toxic principle of selva ant venom is a pore-forming protein transformer

Ectatomin (Ea) is a newly isolated main toxic component of Ectatomma tuberculatum ant venom. Structural and electrophysiological studies were performed with purified Ea. The protein consists of two homologous polypeptide chains (37 and 34 residues) and forms a four α-helix bundle in aqueous solution. On insertion into artificial bilayer membranes, two Ea molecules form an ion pore. Our results suggest that the ‘inside-out’ mechanism of pore formation requires a significant movement of Ea helical parts. The pore formation in the cell membrane might well explain the toxic activity of Ea, not excluding at the same time its intracellular activities.

Crotoxin induces aggregation of human washed platelets

Crotoxin induces aggregation of human washed platelets. Toxicon 32, 217–225, 1994.—Crotoxin, the main toxic component isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, is a reversible protein complex composed of a non-toxic non-enzymatic acidic polypeptide (crotapotin) and a toxic basic phospholipase A 2 (PLA 2). In this study, we have evaluated the ability of crotoxin to induce aggregation in human washed platelets. Human washed platelet aggregation was monitored in a Payton aggregometer and thromboxane B 2 (TXB 2) release measured by direct radio-immunoassay (RIA). Crotoxin (15–50 μg/ml) produced dose-dependent and irreversible human washed platelet aggregation, which was inhibited by pre-incubation of the platelets with sodium nitroprusside (50–500 μM) or iloprost (8–80 nM). Crotoxin also induced TXB 2 release (207 ± 8 ng/ml, n = 6), and although indomethacin significantly reduced the release of TXB 2 (to 23.5 ± 5 ng/ml, P <0.001, n = 6), it did not inhibit crotoxin-induced aggregation. Our results clearly demonstrate that crotoxin induces human washed platelet aggregation and that this phenomenon is independent of the formation of pro-aggregatory arachidonic acid metabolites.

Pharmaceutical applications

Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). It has been used in treatment of various chronic inflammatory diseases. However, the cellular mechanism and the anti-apoptotic effect of melittin in Propionibactierium acnes (P. acnes)-induced THP-1 cells have not been explored. In the present study, we investigated the anti-inflammatory and anti-apoptotic mechanism by examining the effect of melittin on P. acnes-induced THP-1 monocytic cells. THP-1 monocytic cells were stimulated by heat-killed P. acnes in the presence of melittin. The expression levels of pro-inflammatory cytokines, NF-κB signaling, caspase family, and PARP signaling were measured by ELISA or Western blot analysis. The number of apoptotic cells and changes of cell morphology were examined using fluorescence microscopy and flow cytometry. Heat-killed P. acnes increased the secretion of pro-inflammatory cytokines and cleavage of caspase-3 and -8 in heat-killed P. acnes-induced THP-1 cells. However, treatment with melittin inhibited the pro-inflammatory cytokines and cleavage of the caspase-3 and -8. Moreover, the cleaved PARP appeared after 8 h of heat-killed P. acnes treatment and its cleavage was reduced by melittin treatment. These results demonstrate that 1.0×107 CFU/ml of heat-killed P. acnes induces THP-1 cell apoptosis and secretion of inflammatory cytokines. Also, administration of melittin significantly decreases the expression of various inflammatory cytokines in heat-killed P. acnes-treated THP-1 monocytic cells. In particular, melittin exerts anti-apoptotic effects against 1.0×107 CFU/ml of heat-killed P. acnes injury to THP-1 cells.