Matjaž Bunc

Effects of equinatoxin II from Actinia equina (L.) on isolated rat heart: The role of direct cardiotoxic effects in equinatoxin II lethality

Equinatoxin II is a lethal basic protein isolated from the sea anemone Actinia equina (L.) with LD50 in mice 35 microg/kg. The putative cause of death is cardiorespiratory arrest, but the mechanism of cardiotoxicity is poorly understood. It is not clear whether the toxin injected intravenously into an experimental animal reaches the heart in a concentration sufficient to cause direct effects on the heart. Therefore experiments were performed on rats and on isolated rat hearts in order to investigate the possible direct cardiotoxic effects of the toxin. For this reason the hearts were perfused with different concentrations of the toxin and with the effluent from the lungs collected during perfusion of the lungs with equinatoxin II. The results revealed the clear dose-dependent, direct cardiotoxic effects of the toxin and of the effluent from the lungs on Langendorffs heart preparations. The threshold concentration of equinatoxin II causing a drop in the perfusion rate, decreased left ventricular pressure, arrhythmia and increased LDH release, was found to be around 0.1 to 1 nM. With 10 nM equinatoxin II the left ventricular pressure dropped to 14+/-11% of the control, and the coronary flow to 9+/-3%. These effects were followed by arrhythmia and cardiac arrest. The concentration of equinatoxin recovered from the lungs after the perfusion with 100 nM equinatoxin II ranged between 0.8 and 5 nM. The results indicate that direct cardiotoxic effects of equinatoxin II play an important role in the lethal effects of the toxin.

Acute poisoning with autumn crocus (Colchicum autumnale L.).

SummaryIntroductionColchicum autumnale, commonly known as the autumn crocus or meadow saffron, contains the antimitotic colchicine, which binds to tubulin and prevents it forming microtubules that are part of the cytoskeleton in all cells.Case reportA 71-year-old woman ate a plant she thought to be wild garlic (Allium ursinum). Ten hours later she arrived at the emergency department complaining of nausea, vomiting and watery diarrhea. Ingestion of a poisonous plant was suspected and she was treated with gastric lavage, oral activated charcoal and an infusion of normal saline. Toxicology analysis with gas chromatography and mass spectrometry revealed colchicine in the patient’s gastric lavage, blood (5 μ/l) and urine (30 μ/l). She developed arrhythmias, liver failure, pancreatitis, ileus, and bone marrow suppression with pancytopenia. Alopecia began in the third week. Treatment was supportive only. Five months later she had no clinical or laboratory signs of poisoning.DiscussionThe patient mistakenly ingested autumn crocus instead of wild garlic because of their great similarity. Colchicine primarily blocks mitosis in tissues with rapid cell turnover; this results in gastroenterocolitis in the first phase of colchicine poisoning, bone marrow hypoplasia with pancytopenia in the second and alopecia in the third, all of which were present in our patient. Colchicine toxicity in tissues without rapid cell turnover caused arrhythmias, acute liver failure and pancreatitis.ConclusionColchicine poisoning can result in gastroenterocolitis followed by multi-organ dysfunction syndrome. In unexplained gastroenterocolitis after ingestion of wild plants as a salad or spice, especially when wild garlic is mentioned, we should always consider autumn crocus. Diagnosis could be confirmed only by toxicology analyses. Management of colchicine poisoning is restricted to supportive therapy.

Prolonged psychosis after Amanita muscaria ingestion

ZusammenfassungAmanita muscaria ist ein Pilz mit einer leuchtend roten oder orangen Kappe mit kleinen weißen Flecken. Er enthält Isoxazol-Derivate, Ibotensäure, Muskimol und Muscazon und andere Toxine, wie etwa Muskarin. Die Dauer der klinischen Symptome nach dem Verzehr von Amanita muscaria ist üblicherweise nicht länger als 24 Stunden. Wir berichten über eine über fünf Tage anhaltende paranoide Psychose nach der Einnahme von Amanita muscaria. Ein 48-jähriger Mann mit völlig blander medizinischer Anamnese sammelte und aß Pilze, die er als Amanita caesarea agnostizierte. Eine halbe Stunde nach der Einnahme begann er zu erbrechen – anschließend schlief er ein. Er wurde komatös mit krampfartigen Zustand aufgefunden. Bei Eintreffen im Spital war er komatös – seine sonstige klinisch physikalische und neurologische Untersuchung ergab einen normalen Befund. Die Creatin-Kinase betrug 8,33 μkat/l. Die übrigen Laborbefunde und das Schädel-CT waren normal. Das toxikologische Screening ergab keinen Hinweis auf Medikamente im Blut und im Harn. Unser Pilzexperte identifizierte Amanita muscaria in den übrig gebliebenen Pilzen. Dem Patienten wurde aktivierte Tierkohle gegeben. 10 Stunden nach Einnahme des Pilzes erwachte er. Zu diesem Zeitpunkt schien er völlig orientiert. 18 Stunden nach der Einnahme verschlechterte sich der Zustand wieder; der Patient wurde verwirrt und zunehmend unkooperativ. Danach trat ein paranoid psychotisches Zustandsbild mit visuellen und akustischen Halluzinationen ein, welches fünf Tage lang anhielt. Ab dem sechsten Tag nach Einnahme begannen die psychotischen Symptome zu verschwinden. Ein Jahr später ist der Patient ohne jede Therapie und ohne Symptome psychiatrischer Erkrankung. Wir folgern daraus, dass eine paranoide Psychose mit visuellen und akustischen Halluzinationen noch 18 Stunden nach Einnahme von Amanita muscaria auftreten und bis zu fünf Tage lang anhalten kann.SummaryAmanita muscaria has a bright red or orange cap covered with small white plaques. It contains the isoxazole derivatives ibotenic acid, muscimol and muscazone and other toxins such as muscarine. The duration of clinical manifestations after A. muscaria ingestion does not usually exceed 24 hours; we report on a 5-day paranoid psychosis after A. muscaria ingestion. A 48-year-old man, with no previous medical history, gathered and ate mushrooms he presumed to be A. caesarea. Half an hour later he started to vomit and fell asleep. He was found comatose having a seizure-like episode. On admission four hours after ingestion he was comatose, but the remaining physical and neurological examinations were unremarkable. Creatine kinase was 8.33 μkat/l. Other laboratory results and brain CT scan were normal. Toxicology analysis did not find any drugs in his blood or urine. The mycologist identified A. muscaria among the remaining mushrooms. The patient was given activated charcoal. Ten hours after ingestion, he awoke and was completely orientated; 18 hours after ingestion his condition deteriorated again and he became confused and uncooperative. Afterwards paranoid psychosis with visual and auditory hallucinations appeared and persisted for five days. On the sixth day all symptoms of psychosis gradually disappeared. One year later he is not undergoing any therapy and has no symptoms of psychiatric disease. We conclude that paranoid psychosis with visual and auditory hallucinations can appear 18 hours after ingestion of A. muscaria and can last for up to five days.

Cardiovascular effects of equinatoxin III from the sea anemone Actinia equina (L.).

Equinatoxin III is the most hemolytic, and the least lethal of the three basic proteins isolated from the sea anemone Actinia equina (L.). Its LD50 in mice is 83 microg/kg. Preliminary results on Wistar rats have suggested cardiorespiratory arrest as a putative cause of death, but the mechanism of its action has not yet been studied. So far only equinatoxin II has been investigated more thoroughly. As equinatoxin II is less lythic, but more toxic, than equinatoxin III (its LD50 in mice=35 microg/kg), it may be assumed that haemolysis with a consequent rise in plasma potassium level is not the major factor in the lethality of equinatoxins. To assess the relative contribution of hyperkalemia in the lethality of the toxin in rat, the effects of equinatoxin III were compared to the effects of hyperkalemia caused by the injection of KCl giving the same final concentration of K+ in the plasma as that observed after an i.v. injection of 3LD50 of equinatoxin III. As coronary vasoconstriction may be an important mechanism of the cardiotoxic action of equinatoxins, the effect of EqT III on isolated porcine coronary arteries was studied by measurements of smooth muscle tension in the presence of 1-100 nM equinatoxin III. The results revealed that animals survive the elevated K+ plasma concentration caused by an i.v. application of KCl. This suggests that equinatoxin III induced haemolysis is not the major mechanism of equinatoxin III lethality. However, equinatoxin III increases the potassium induced contractions of coronary smooth muscle for 289+/-29%, suggesting that coronary vasoconstriction may be an important factor in the cardiotoxic effects of equinatoxin III.

Recording of electroneurograms from the nerves innervating the pancreas of a dog.

Electroneurograms (ENGs) from the vagus, splanchnic and pancreatic nerves innervating the pancreas of a dog, were recorded with chronically implanted silicone multi-electrode circular cuffs in an intact pancreas and in a pancreas partly disabled with alloxan. The cuffs contained 33 platinum electrodes (0.6x1.5 mm) arranged in three parallel circular groups integrated into the inner surface of the cuff. Each circular group contained 11 electrodes at a distance of 0.5 mm apart, with 6 mm between the circular groups. The cuffs had an inner diameter of 2.5 mm and the length of 18 mm. In a 2-year study, the cuffs were implanted into two adult Beagle dogs (one female and one male). In the vagus nerve, the cuff was installed on the nerve at the neck, whilst in the splanchnic nerve, the cuff was installed on the nerve before the celiac ganglion, and in the pancreatic nerve, the cuff was installed on the nerve just before it enters the pancreas. In each of the three implanted cuffs, the electrodes of the central circular group were connected to each other and this signal provided one input to a multi-channel ENG amplifying system. The electrodes of each of the two outer spiral groups were connected to each other and then both these groups were short-circuited. This signal then provided another input to the multi-channel ENG amplifying system. The ENG amplifying system was designed to amplify the ENGs 100000 times and to pass frequencies of between 500 and 10 kHz. In our study, three recordings in each animal were conducted. Recordings in the intact pancreas were conducted 2 and 6 months after the implantation, while the recording in the partly disabled pancreas, was conducted 10 months after the implantation and 10 days after the disablement. Due to the fact that the results obtained in both animals were actually quite similar, we present the results of the recordings obtained in one animal. In both animals the cuffs were left implanted for more than 1 year and were used for pancreatic stimulation, although this is not in this paper. The results show that cuffs implanted chronically on the nerves innervating the pancreas of a dog could reliably record the ENGs. This information could be used effectively in further study of pancreatic innervation and its function. Moreover, the results suggest that cuffs could also be useful in recording the ENGs from other nerves of the autonomic nervous system that innervate various glands and internal organs.

Toxic effects of head-to-tail 3-alkylpyridinium polymers isolated from the marine sponge Reniera sarai in rat

Toxic water soluble polymeric 3-alkylpyridinium salts (poly APS; MW 18900 and 5520Da) were isolated from the marine sponge Raniera sarai. In vitro they strongly inhibited acetylcholinesterase. In order to evaluate the role of acetylcholinesterase inhibition in toxin lethality, and to assess other possible lethal effects, in vivo experiments were performed on male Wistar rats, and ECG, blood pressure and breathing pattern were monitored. The results showed that none of the animals died due to the acetylcholinesterase inhibitory action of poly-APS. Doses lower than 1mg/kg caused only transient bradycardia and transient prolongation of expirium. At doses above 2.7mg/kg of poly-APS all treated animals died, but signs were not typical of acetylcholinesterase inhibition. Arterial blood pressure fell to mid-circulatory pressure, and breathing stopped after a few breaths with an increase of the residual volume. Autopsy of the experimental animals that died due to the effects of the toxin revealed that mid-size and small sized blood vessels in the heart and lungs were filled with granular brownish material with inclusions of red blood cells and platelets. Data obtained on blood samples from animals treated with poly-APS also revealed numerous thrombocyte aggregates. In vitro poly-APS induced thrombocyte aggregation in a dose dependent manner. The acetylcholinesterase-inhibitory effects were most pronounced only at lower doses of poly-APS. With higher doses those effects were masked or covered by other, more pronounced and faster developing lethal effects of the toxin such as platelet aggregation. Therefore it is reasonable to assume that acetylcholinesterase inhibitory effects are not responsible for the lethal activity of the toxin.

The potential value of the protein S-100B level as a criterion for hyperbaric oxygen treatment and prognostic marker in carbon monoxide poisoned patients.

Carbon monoxide (CO) poisoning resulting in diffuse tissue hypoxia. Cerebral hypoxia is a major cause of morbidity and mortality after CO poisoning. There are some clinical criteria that could help a physician to make a decision concerning the application of hyperbaric oxygenation therapy. However, it would be convenient to discover an objective biochemical serum marker that could help in the grade evaluation of CO poisoning and indication of therapy in CO-poisoned patients. We present two case reports where the established criteria for the CO poisoning were not optimum for the decision regarding therapy. It seems that the S-100B protein could be used as a biochemical marker of CO induced brain injury. S-100B values could perhaps help us to select patients for hyperbaric oxygen therapy and to predict the short and long term outcome.

In vivo effects of head-to-tail 3-alkylpiridinium polymers isolated from the marine sponge Raniera sarai.

Abstract Water soluble polymeric 3-alkylpyridinium salts (poly APS; MW 18900 and 5520 Da) were isolated from the marine sponge Raniera sarai. In vitro it strongly inhibited acetyl cholinesterase (AChE) from different species (electric eel, horse serum, human erythrocytes). In our experiments the importance of anti AChE activity in the toxin lethality was evaluated. In vivo experiments were performed on male Wistar rats and ECG, blood pressure and breathing pattern were monitored. After i.v. application of lethal doses of the toxin ECG showed signs of hypo perfusion. Arterial blood pressure fell to mid-circulatory pressure, and breathing stopped after a few breaths At sublethal doses the toxin caused an increase of residual volume, prolongation of expiration, and bradycardia. Patho-anatomical examination revealed that the plugs in lung circulation may cause the death of experimental animals due to cardiorespiratory failure.

Evaluation of the strength of elbow flexors in patients with neuromuscular diseases

In planning the optimum treatment for patients with neuromuscular diseases (NMD), it is essential to know as much as possible about their functional state. Assessment of the strength of certain muscles is the most direct measure of motor deficiency. In the development of normative data needed for patients with NMD, the use of torque measurements is required. Forty-nine patients (31 men and 18 women),f rom 18 to 54 years (mean age 33 +/- 8.9 years), were included in the study. Five groups of patients, each having one of five different NMDs, were formed. We tested unilaterally the biceps brachii muscle that normally generates the highest torque. For this purpose an eletronic brace enabling isometric measurements of torque during elbow flexion was designed. The patients produced three maximum voluntary elbow flexions that lasted about 3 s and separated by a pause of about 3 s. Force development was rapid with continuous build-up and isometric. About 15 s later the patients produced the last maximum voluntary elbow flexion, keeping it as stable aspossible for a period of 30 s. Patients with mitochondrial myopathy (MM), having the shortest mean half fatigue time (4.3 s), elicited the highest mean torque in both short maximum voluntary elbow flexions (1.34 Nm) as well as in the 30 s-long maximum voluntary elbow flexions. In contrast, patients with facioscapulohumeral muscular dystrophy (MD-FSH), having the longest mean half-fatigue time (15.4 s), elicited the lowest mean torque in both the short maximum voluntary (0.29 Nm) as well as in 30 s-long maximum voluntary elbow flexions. Patients with Becker muscular dystrophy (MD-B), having a mean half-fatigue time (11.1 s) slightly shorter than the patients with MD-FSH, elicited a higher mean torque in both the short (0.82 Nm) and the 30 s-long elbow flexions. Finally, patients with limb-girdle muscular dystrophy (MD-RM) and spinal muscular atrophy type 3 (SMA3), having a similar mean half-fatigue time (6.9 s for patients with MD-RM and 7.4 s for patients with SMA3), also elicited similar torque in both short (0.45 Nm for patients with MD-RM and 0.65 Nm for patient with SMA3) and 30 s-long elbow flexions. The results of the study show that the methodology developed to quantitative measure the torque of elbow flexions in patients with NMD enables the characteristics and natural course of NMD to be more objectively documented. Accordingly, the optimum treatmentforpatients with NMD could be restored.In planning the optimum treatment for patients with neuromuscular diseases (NMD), it is essential to know as much as possible about their functional state. Assessment of the strength of certain muscles is the most direct measure of motor deficiency. In the development of normative data needed for patients with NMD, the use of torque measurements is required. Forty-nine patients (31 men and 18 women), from 18 to 54 years (mean age 33 - 8.9 years), were included in the study. Five groups of patients, each having one of five different NMDs, were formed. We tested unilaterally the biceps brachii muscle that normally generates the highest torque. For this purpose an electronic brace enabling isometric measurements of torque during elbow flexion was designed. The patients produced three maximum voluntary elbow flexions that lasted about 3 s and separated by a pause of about 3 s. Force development was rapid with continuous build-up and isometric. About 15 s later the patients produced the last maximum voluntary elbow flexion, keeping it as stable as possible for a period of 30 s. Patients with mitochondrial myopathy (MM), having the shortest mean halffatigue time (4.3 s), elicited the highest mean torque in both short maximum voluntary elbow flexions (1.34 Nm) as well as in the 30 s-long maximum voluntary elbow flexions. In contrast, patients with facioscapulohumeral muscular dystrophy (MD-FSH), having the longest mean half-fatigue time (15.4 s), elicited the lowest mean torque in both the short maximum voluntary (0.29 Nm) as well as in 30 s-long maximum voluntary elbow flexions. Patients with Becker muscular dystrophy (MD-B), having a mean half-fatigue time (11.1 s) slightly shorter than the patients with MD-FSH, elicited a higher mean torque in both the short (0.82 Nm) and the 30 s-long elbow flexions. Finally, patients with limb-girdle muscular dystrophy (MD-RM) and spinal muscular atrophy type 3 (SMA3), having a similar mean half-fatigue time (6.9 s for patients with MD-RM and 7.4 s for patients with SMA3), also elicited similar torque in both short (0.45 Nm for patients with MD-RM and 0.65 Nm for patients with SMA3) and 30 s-long elbow flexions. The results of the study show that the methodology developed to quantitatively measure the torque of elbow flexions in patients with NMD enables the characteristics and natural course of NMD to be more objectively documented. Accordingly, the optimum treatment for patients with NMD could be restored.

Modulation of hormone secretion by functional electrical stimulation of the intact and incompletely dysfunctional dog pancreas

The purpose of the present study was to modulate the secretion of insulin and glucagon in Beagle dogs by stimulation of nerves innervating the intact and partly dysfunctional pancreas. Three 33-electrode spiral cuffs were implanted on the vagus, splanchnic and pancreatic nerves in each of two animals. Partial dysfunction of the pancreas was induced with alloxan. The nerves were stimulated using rectangular, charge-balanced, biphasic, and constant current pulses (200 micros, 1 mA, 20 Hz, with a 100-micros delay between biphasic phases). Blood samples from the femoral artery were drawn before the experiment, at the beginning of stimulation, after 5 min of stimulation, and 5 min after the end of stimulation. Radioimmunoassay data showed that in the intact pancreas stimulation of the vagal nerve increased insulin (+99.2 microU/ml) and glucagon (+18.7 pg/ml) secretion and decreased C-peptide secretion (-0.15 ng/ml). Splanchnic nerve stimulation increased insulin (+1.7 microU/ml), C-peptide (+0.01 ng/ml), and glucagon (+50 pg/ml) secretion, whereas pancreatic nerve stimulation did not cause a marked change in any of the three hormones. In the partly dysfunctional pancreas, vagus nerve stimulation increased insulin (+15.5 microU/ml), glucagon (+11 pg/ml), and C-peptide (+0.03 ng/ml) secretion. Splanchnic nerve stimulation reduced insulin secretion (-2.5 microU/ml) and increased glucagon (+58.7 pg/ml) and C-peptide (+0.39 ng/ml) secretion, and pancreatic nerve stimulation increased insulin (+0.2 microU/ml), glucagon (+5.2 pg/ml), and C-peptide (+0.08 ng/ml) secretion. It was concluded that vagal nerve stimulation can significantly increase insulin secretion for a prolonged period of time in intact and in partly dysfunctional pancreas.