Dietrich Mebs

Toxicity in animals. Trends in evolution

Animals acquire toxicity either by metabolic synthesis of toxins (secondary metabolites), by expression of toxin genes or by the uptake, storage and sequestration of toxins produced by other organisms, i.e., microbes, plants or other animals. Variability of toxin structure and function is high. Peptide toxins in particular, although relying on a limited number of structural frameworks, often exhibit considerable structural hypervariability. An accelerated rate of evolution in the toxin gene structure (conserved introns, but high substitution rates in the exons) leads to the functional diversity of these peptides or proteins. The selective forces which may drive toxin evolution are unknown. Venomousness or the possession of toxins can be essential for survival, but the advantage of toxin biosynthesis may also be of minor importance or has been lost during evolution.

Survey of microcystins in environmental water by a highly sensitive immunoassay based on monoclonal antibody

By using a highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody, microcystin (MC) concentration was analyzed in environmental water samples (total, 134), collected in 1993-1995 from ponds, lakes, reservoirs, and rivers in Japan, Thailand, Germany, and Portugal. MCs detected in the water samples filtered over a glass filter were designated as free MCs, and those samples that were freeze-thawed twice before the filtration were designated as total MCs. MCs (> 50 pg/ml) were detected in 14 of 24 samples collected from the lakes that were used as recreation and water supply in Japan in different regions. In the MC-positive samples, the concentration of free MCs was only a few percentages of the total MCs, indicating that the most part of MCs found in the water samples was present in algal cells. An additional trial on 33 samples collected continuously from Lake Inbanuma, Japan, during June-September 1994-1995 revealed that the total MCs were in a range of 52-52,000 pg/ml. In Chiang Mai, Thailand, 6 of 10 samples were positive, with the mean and highest of 161 and 354 pg/ml, respectively. In the Frankfurt area. Germany, 4 of 10 and 7 of 8 samples collected in the same lakes for recreation in July 1993 and November-December 1994 showed the presence of MCs, with their mean and highest values of 257 and 407 pg/ml, respectively. Another survey of MCs in dense bloomed samples collected with plankton net revealed a contamination of MCs up to 36,000 pg/ml. In Portugal, 28 of 29 samples from 4 lakes, 20 rivers, and 5 reservoirs were positive for MCs, with the respective means of 13,664, 11,048, and 2,278 pg/ml. These data indicated that MCs contaminate environmental water in ponds, rivers, lakes, and reservoirs worldwide. The present ELISA is considered to be a reliable tool for the mass monitoring and risk assessment of MCs in water supplies.

Distribution and sequestration of palytoxin in coral reef animals.

In the reefs off the Colombian coast (Caribbean Sea) and around Lizard Island, Australia (Pacific), palytoxin (PTX), which has been detected in zoanthid species of the genus Palythoa, also occurred in various other marine organisms living in close association with zoanthid colonies, e.g. sponges (Porifera), soft corals (Alcyonaria), gorgonians (Gorgonaria), mussels, and crustaceans. Predators, e.g. polychaete worms (Hermodice carunculata), a starfish (Acanthaster planci) and fish (Chaetodon species) feeding on Palythoa colonies, accumulate high toxin concentrations in their organs, where PTX is stored in its active form. The high level of toxin tolerance observed in marine animals may enable the wide distribution of PTX in marine biota and its transport and sequestration in food chains.

Local necrotizing effect of snake venoms on skin and muscle: Relationship to serum creatine kinase

Twenty-five snake venoms were tested for their ability to induce an increase of serum creatine kinase (CK) level after i.m. injection (0.125-1.0 mg/kg) into rates. Of six Australian elapid venoms only those from Pseudechis colletti guttatus and P. australis produced a steep rise of CK-activity (30-70 times the normal value) 4 and 16 hr after injection (0.5 mg/kg). Viperid and crotalid venoms had only slight effects (2-5 times the normal value) even in doses of 1.0 mg/kg except for a sample of Crotalus adamanteus venom which caused a 20 fold increase in CK-level. From this venom a toxin of 5800 mol. wt. consisting of 50 amino acid residues was isolated. This toxin exhibited similarities in amino acid composition and in lethality to crotamine from Crotalus durissus terrificus and to a toxin from C. horridus horridus. The toxin from C. adamanteus induced some increase of CK-level in rats, but this does not account entirely for the activity of the crude venom, whereas crotamine and the toxin from C. horridus horridus were ineffective. Phospholipase A2 (fraction II) from Pseudechis colletti guttatus venom caused a dose-dependent increase of CK-level and myoglobinuria. Intradermal injection of snake venoms into mice is useful for testing hemorrhagic activity, but is too insensitive to measure necrotizing effects. Venom induced myonecrosis can be evaluated by assaying the CK-serum level and by histological examination.

Studies on the origin and distribution of palytoxin in a Caribbean coral reef.

In coral reefs of the Caribbean Sea (Colombia) palytoxin (PTX) has been detected in zoanthid species of the genera Palythoa and Zoanthus by assaying the delayed haemolysis in human erythrocytes produced by aqueous extracts, which is inhibited by ouabain pretreatment, and by HPLC. The toxin content of the polyps and colonies is highly variable and is not correlated with their reproductive cycle or with the amount of symbiotic algae. Sequestration of PTX has been observed in crustaceans (Platypodiella sp.) living in close association with Palythoa colonies and in polychaete worms (Hermodice carunculata) feeding on the zoanthids. Resistance of marine animals to the toxin may enable it to enter food chains.

A case of palytoxin poisoning due to contact with zoanthid corals through a skin injury.

A case of human poisoning by palytoxin after contact with zoanthid corals (Parazoanthus sp.) in an aquarium through skin injuries on fingers is reported. The clinical symptoms include swelling, paraesthesia and numbness around the site of the injury spreading over the arm, but also signs of systemic poisoning such as dizziness, general weakness and myalgia, irregularities in the ECG and indications of rhabdomyolysis. Symptomatic treatment consisted of infusion of physiological fluids. The patient recovered within 3 days. Analysis of the zoanthid coral involved revealed extremely high concentrations of palytoxin (between 2 and 3 mg/g).

Amino acid sequence of α-bungarotoxin from the venom of bungarus multicinctus

Summary The primary structure of α-bungarotoxin isolated from the venom of Bungarus multicinctus was determined. It composes of 74 amino acid residues including ten half-cystines. Comparing the sequence with those of cobras and sea snake species, striking similarities can be found, especially between α-bungarotoxin and Naja nivea α-toxin, indicating 50 % sequence homology.

Isolation and characterization of myotoxic phospholipases A2 from crotalid venoms.

Phospholipases A2 producing myonecrosis when injected i.m. into mice were isolated from venoms of Trimeresurus flavoviridis, Agkistrodon bilineatus, A. c. contortrix, A. c. mokeson, A. p. piscivorus and Bothrops asper by gels filtration on Sephadex G-75 followed by ion-exchange chromatography on CM-cellulose. They are basic enzymes with molecular weights between 14,000 and 15,000 containing 120-129 amino acid residues and exhibit relatively low enzymatic activity when tested on egg yolk suspension. Local myonecrosis is induced even at doses of 1.25 micrograms per mouse.

Purification, from Australian elapid venoms, and properties of phospholipases A which cause myoglobinuria in mice

Abstract Ten Australian snake venoms ( Oxyuranus s. scutellatus, Notechis s. scutatus, N. ater serventyi, N. a. humphreysi, Acanthophis antarcticus, Austrelaps superba, Pseudonaja textilis, Pseudechis australis, P. porphyriacus, P. colletti ) were fractionated by column chromatography on CM-Sephadex C-25 and the isolated fractions were tested in mice for ability to produce myoglobinuria after s. c. injection. Only venom fractions from A. superba, P. australis, P. porphyriacus and P. colletti produced this effect. Pure factors were obtained from the latter three venoms and shown to be neutral or basic phospholipases A containing 120–129 amino acid residues (formula wts between 13,400 and 14,200). The enzymes have acute LD 50 values (s.c. injection) of 4·3–7·7 mg/kg, however, the minimum doses necessary to produce myoglobinuria in mice are between 0·5 and 5·0 mg/kg. These venom components affecting skeletal muscle may play an important role in snakebite.

A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels.

Toxins from cone snail (Conus species) venoms are multiple disulfide bonded peptides. Based on their pharmacological target (ion channels, receptors) and their disulfide pattern, they have been classified into several toxin families and superfamilies. Here, we report a new conotoxin, which is the first member of a structurally new superfamily of Conus peptides and the first conotoxin affecting vertebrate K+ channels. The new toxin, designated conotoxin ViTx, has been isolated from the venom of Conus virgo and comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Its amino acid sequence (SRCFPPGIYCTSYLPCCWGICCSTCRNVCHLRIGK) was partially determined by Edman degradation and deduced from the nucleotide sequence of the toxin cDNA. Nucleic acid sequencing also revealed a prepropeptide comprising 67 amino acid residues and demonstrated a posttranslational modification of the protein by releasing a six-residue peptide from the C-terminal. Voltage clamp studies on various ion channels indicated that the toxin inhibits the vertebrate K+ channels Kv1.1 and Kv1.3 but not Kv1.2. The chemically synthesized product exhibited the same physiological activity and identical molecular mass (3933.7 Da) as the native toxin.