原矛头蝮蛇毒毒性及恢复规律

目的:对原矛头蝮(P.mucrosquamatus)的毒性及其恢复规律进行考察,增进对该蛇毒性的认识。方法:用该蛇连续攻击小鼠,用梯度剂量蛇毒对小鼠进行腹腔注射,统计死亡率,以折算绝对毒性和半数致死量。对毒性耗竭个体,在梯度时间间隔采集蛇毒,以考察毒性恢复规律。结果:原矛头蝮在2 h内可导致6只小鼠死亡。蛇毒对小鼠的半数致死量为12.204μL/Kg。毒性耗竭后,在间隔21天所获得蛇毒的容积达到最大值即190μL,湿重为149.7 mg。结论:毒蛇在攻击猎物时,并不一次性排完毒腺中的所有毒液,随着攻击次数的增多,毒腺中毒液量逐步减少直至耗竭。其毒液量的恢复需要约3周时间,期间,单次排毒量逐渐增加,干物质的含量也基本呈递增趋势。就单次最大排毒量而言,原矛头蝮高于竹叶青、蝮蛇和银环蛇。其单位剂量毒性高于五步蛇和竹叶青。     

Comparison of the biodistribution of free or liposome-entrapped Crotalus durissus terrificus (South American rattlesnake) venom in mice

The local absorption rate, clearance and tissue distribution of Crotalus durissus terrificus venom, (Cdt) were examined using a two-antibody sandwich ELISA assay. We compared the biodistribution of both free or encapsulated Cdt in mice. Following subcutaneous injection of 10 microg/mouse of free Cdt (0.8 LD50), venom was detected in serum after 15 min, showed its highest level at 30 min (45+/-5 ng/ml) and was cleared from the circulation after 6 h. After 2 h of inoculation, venom was detected in the kidney (57+/-9 ng/g of tissue), spleen (18+/-4 ng/g of tissue) and brain (14+/-6 ng/g of tissue). For both subcutaneous or intravenous injection of free Cdt, venom was firstly detected in the kidney. No Cdt appeared either in the kidney, spleen, brain, or other tissues after subcutaneous inoculation of encapsulated venom even though a higher dose was used, 25 microg/mouse (2 LD50). Venom remained at the site of injection for a period of 1 week. Following intravenous injection of encapsulated venom (5 microg/mouse, 2 LD50), venom was detected in liver and spleen tissues. The biodistribution of encapsulated venom is discussed in relation to the effects of reduction of toxicity and increase of adjuvanticity.     

Effect of Some Variables on theIn VivoDetermination of Scorpion and Viper Venom Toxicities

An adequate assessment of scorpion and snake venom LD50 is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD50 variation of Tunisian scorpion (Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57BI/6) of mice used, the route of venom injection, the venom-milking procedures (manually or electrically) and the venom batches have been studied over a 7-year period (1990-1996). Aag venom is 3-4 times more toxic than Bot venom. However for both venoms, the LD50 determined in C57BI/6 mice, in small body weight animal or by intraperitoneal route were respectively significantly lower than those determined in Swiss mice, in high body weight or by subcutaneous route. Significant LD50 variations (25-50%) were also seen from one electrically prepared batch to another. A good correlation (r = 0.982) was observed between the concentrations of the crude venom toxic fraction determined by ELISA and LD50 values when assessed in vivo. The LD50 variation of Tunisian viper (Cerastes cerastes: Cc and Vipera lebetina: VI) venoms with the strain (Swiss or BALB/c), sex and body weight of mice used, the season and the year of venom milking were also investigated over a 3-year period (1990-1992). No significant LD50 variations were observed with the mouse strain, the sex or the season of venom milking. However, LD50 varies significantly with the year of the venom collection and the body weight of mice used. Furthermore, SDS-PAGE analysis shows annual variation for VI venom composition where no such variations were observed for Cc venom. These results stress the need either for the standardization of the venom LD50 evaluation or of the venom quality used for the development of an efficient antivenom.     

急危重症眼镜蛇伤大鼠多项凝血指标动态变化

目的动态观察蛇伤后大鼠多项凝血指标、病理及皮下出血情况的改变,为蛇毒毒理及抗蛇毒制剂的研究提供实验数据。方法将SD大鼠注入2～4 LD50眼镜蛇毒造模,于注毒后不同时段(20～140 min)分别进行凝血功能、肝脏和肾脏病理等检测,处死大鼠后观察注毒部位皮下出血情况。结果大鼠注入4 LD50蛇毒20 min,凝血酶原时间(PT)、部分凝血酶原时间(APTT)延长;40 min时,纤维蛋白原(Fbg)明显下降;40 min后凝血功能逐渐回复至0 min水平。肾脏、肝脏组织见血管扩张、充血明显。注毒部位皮下出血范围于20～80min时最明显。2 LD50剂量组凝血功能的变化与4 LD50组相似,但其Fbg下降程度小于4 LD50组,皮下出血面积较4 LD50组小。结论眼镜蛇毒引起凝血功能改变,且其变化程度与剂量及注入时间密切相关。     

The dynamics of cytokine d nitric oxide secretion in mice injected with Tityus serrulatus scorpion venom

AIMS: The effects of Tityus serrulatus venom (TSV) were analysed with respect to the susceptibility of four isogenic mouse, the symptoms following injection of venom and the inflammatory mediators in an experimental model of severe envenomation induced in mice. METHODS: The susceptibility was analysed by lethal dose (LD50) determination, including the symptoms observed during envenomating and glucose levels. The detection of cytokines in serum from mice were analysed using enzyme-linked immunosorbent assay, and nitric oxide (NO) was analysed using nitrite determination. RESULTS: The estimated LD50 values were in micrograms per 100 microliters, and the susceptibility of mice to TSV varies with: (a) mouse strain and route of injection (A/J < BALB/c < C57Bl/6 = DBA); (b) mouse strain and sex (A/J female and male < BALB/c female and male); and (c) body weight (all groups of A/J < BALB/c groups). Among the mouse strains studied, BALB/c mice presented moderate sensibility to TSV, with changes in specific signs and serum levels of glucose, several cytokines and NO, when injected intraperitoneally (i.p.) with 1 LD50 of venom. Sweating, salivation and tremor were the specific signs that preceded death. The maximum levels of glucose in sera from mice injected i.p. with 1 LD50 of TSV were observed 60-90 min post-injection. Significant differences were observed in the time-course of cytokine levels, and the venom induced marked elevations of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10 and interferon gamma (IFN-gamma). The maximum levels of IL-1alpha and IL-1beta were observed 2 h post-injection. The more pronounced levels of IL-6 were observed 4 h post-injection. There was an early increase in IFN-gamma followed by an even higher level after 4 h. IL-10 levels peaked between 6 and 8 h, and this cytokine probably modulates the secretion of IFN-gamma. Tumor necrosis factor release was not detected in BALB/c mice injected with TSV. NO levels attained maximal release after 2 h, following venom injection, while a second peak for NO was at 6 h. CONCLUSIONS: These findings indicate that the susceptibility to the systemic effects of the venom varies among mice of different haplotypes, and that the cytokines such as IL-1, IL-6, IFN-gamma and NO are strongly involved in the pathogenesis caused by this venom and are correlated with the severity of envenomation.     

The role of C3 as an opsonin in the early stages of infection.

In order to investigate the role of C3 in host defense in vivo, normal AKR/J mice, genetically deficient in C5, were depleted of serum C3 by the injection of purified cobra venom factor (CoVF). Concurrent with their C3 depletion, their serum opsonizing activity decreased to a level less than 20% of normal. When these mice were challenged with an intraperitoneal injection of pneumococci 2 hr after the CoVF treatment, the LD50 was from 30 to 80 times lower than the LD50 in saline-treated control animals. When the CoVF was given only 6 hr after the pneumococcal challenge, the LD50 was the same as in the control mice. If the pneumococci were first preopsonized in vitro and then injected into CoVF-treated animals, the LD50 was the same as that in control animals. These experiments demonstrate that C3 plays a significant role in vivo in the host's defense against infection and that a major part of that role is through its action as an opsonin. Furthermore, these experiments demonstrate that the role of C3 is most significant during the early stages of bacterial invasion.     

Toxicological and immunological aspects of scorpion venom (Tytius pachyurus): neutralizing capacity of antivenoms produced in Latin America

The toxicity and immunochemical properties of Tityus pachyurus Pocock scorpion venom was characterized, as well as the neutralization capacity against it by three anti-scorpion antivenoms (Alacramyn, Instituto Bioclón, México; Suero antiescorpiónico, Instituto Butantán, Sao Paulo, Brasil; and Suero antiescorpiónico, Centro de Biotecnología, Universidad Central de Venezuela, Caracas, Venezuela). The venom yield, obtained by manual milking, 680+/-20 microg venom, a 50% lethal dose in mice was 4.8 microg/kg (90 microg for an 18-20 g mouse). The most common symptoms of venom poisoning in mice were sialorrhea, respiratory distress, profuse sweating, ataxia, behavior alterations (restlessness, somnolence) and hyperglycemia at 3 and 24 hours after subcutaneous venom injection (0.5 LD50). The neutralizing capacity of Bioclón (México City) and Butantán (Sao Paulo) antivenoms (for a 50% effective dose) was 330 and 292 microg venom/ml antivenom, respectively. The Biotecnología (Caracas) antivenom did not neutralize the lethal effect of venom. By electrophoresis (SDS-PAGE) was demonstrated that the venom contains proteins from less than 14 kd to 97 kd. The Western blots indicated immunological reactivity of the three antivenoms with most of venom components, including proteins of low molecular mass (<14 kd). The results allow to conclude that T. pachyurus venom is neutralized efficiently by anti-scorpion antivenoms produced in México and Brasil.     

乙醇对中华眼镜蛇毒毒性的影响

目的为了探索乙醇对眼镜蛇毒毒性的影响。方法将眼镜蛇毒不同浓度致死量经不同浓度乙醇体外处理后,分别于小白鼠皮下注射、口服,将致死量蛇毒皮下注射后的小白鼠立即于局部注射乙醇,观察蛇毒毒性情况。结果小白鼠经皮下注射致死量眼镜蛇毒后,在局部注射50％（或异蛇米酒）、75％乙醇0．1～0．2ml有一定的保护作用;口服100倍皮下注射致死量眼镜蛇毒未发现有毒性表现,口服经50％乙醇处理后的眼镜蛇毒（100倍皮下注射致死量）未增加小鼠死亡率。结论眼镜蛇毒体外经过乙醇处理后毒性有所下降。口服少量的眼镜蛇毒是安全的。眼镜蛇毒与乙醇混合后口服未见蛇毒毒性增加。     

A mammal toxin derived from the venom of a chactoid scorpion

1. It has been shown that the low toxicity to mammals (LD50 of about 200 mg per kg mice body weight) of the chactoid scorpion venom Scorpio maurus palmatus (Scorpionidae) is due to a single low molecular weight basic protein. 2. This compound was purified by the aid of gel filtration and ion exchange column chromatography, possessed about 80% of the mice lethality of the crude venom with an increase of about 60 fold in its specific toxicity. 3. It is composed of 32 amino acids (mol. wt = 3478) and devoid of isoleucine, leucine, phenylalanine, histidine and tryptophan. 4. The unique amino acid composition of the present toxin is compared to those of the well known buthoid scorpion venom mammal toxins and some other toxins derived from the same venom. 5. It is the first chemically characterized chactoid toxin.     

Amino-acid sequence of the minor neurotoxin from Acalyptophis peronii venom

Sea snakes, Acalyptophis peronii, were captured in the Gulf of Thailand and their venom was isolated. A. peronii venom contains two neurotoxins called major and minor toxin. The complete amino-acid sequence of the minor toxin was identified and compared to that of the major toxin. The only difference between the major and the minor toxins is in the 43rd residue. The major toxin at this position contains glutamine, while the minor toxin contains glutamic acid. The LD50 of the minor toxin is 0.170 microgram/g in mice when injected intravenously. The toxicity is slightly lower than that of the major toxins, which has an LD50 of 0.125 microgram/g.     

Defibrinogenation by batroxobin and acylated batroxobin in rats

The thrombin-like snake venom enzyme, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about 1 hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effect of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.     

Mice primed with swainsonine are protected against doxorubicin-induced lethality.

The anthracycline, doxorubicin is a potent cancer chemotherapeutic agent whose therapeutic usefulness is limited by both a dose- and time-dependent cardiomyopathy. We tested the ability of an immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) to protect C57BL/6 mice against lethality within 70 days following a single bolus intraperitoneal injection of LD50/14 doxorubicin. Also, we sought the potential mechanisms responsible for this protection. This extended 70-day study in mice, which may be considered equivalent to a period of 4 to 5 years in humans, has clinical implication for delayed cardiotoxic sequela of therapy with high dose doxorubicin. Mice were pretreated with swainsonine or its diluent buffer, phosphate buffered saline for ten consecutive days prior to a single bolus intraperitoneal injection of a LD50/14 doxorubicin. We have previously defined this swainsonine pretreatment regimen as one of the two optimal conditions for swainsonine rescue of mice from death induced by LD50/14 doxorubicin. The survival and well being of groups of mice pretreated with swainsonine and phosphate buffered saline prior to LD50/14 doxorubicin, sham-treated and untreated were monitored daily for up to 70 days. The bone marrow cellularity of the mice were quantified, and in vitro progenitor cell assays were used to determine the effects of these treatment regimens on bone marrow competence following doxorubicin treatment. The effects of these treatment regimens on heart morphology and hematologic toxicities were also determined. This swainsonine pretreatment regimen significantly abrogated doxorubicin-induced lethality and prolonged survival of mice by facilitating restoration of bone marrow cellularity, accelerating restoration of blood hematocrit and total leukocyte levels, enhancing the proliferation and differentiation of bone marrow pluripotent stem cells along the different paths to progenitor lineages, and preserving the heart morphology. This study strongly suggests a potential role for swainsonine with doxorubicin in cancer chemotherapy.     

PLGA-奥沙利铂缓释微球急性毒性实验

目的了解PLGA-奥沙利铂缓释微球和奥沙利铂小鼠皮下给药的急性毒性。方法采用Bliss法分别测定PLGA-奥沙利铂缓释微球和奥沙利铂小鼠皮下给药对动物的半数致死量（LD50）。结果PLGA-奥沙利铂缓释微球小鼠皮下给药时的LD50为660.90 mg/kg,奥沙利铂小鼠皮下给药时的LD50为15.24 mg/kg,相差达43.37倍。结论PLGA-奥沙利铂缓释微球能够显著降低药物对动物的急性毒性。     

乙醇对五步蛇（Agkistrodonacutus）毒毒性的影响

目的为了探索乙醇对五步蛇毒毒性的影响。方法将五步蛇毒不同浓度致死量经不同浓度乙醇体外处理后,选择小白鼠分别给予皮下注射、口服,并将致死量蛇毒于小白鼠皮下注射后立即于局部注射乙醇,观察蛇毒毒性情况。结果使用乙醇体外处理后的五步蛇毒毒性明显下降,小白鼠经皮下注射致死量五步蛇毒后立即在局部注射50％异蛇米酒或75％乙醇0．1～0．2ml有一定的保护作用,口服20倍致死量五步蛇毒未发现有毒性表现。结论五步蛇毒体外经过乙醇处理后毒性明显下降。口服五步蛇毒是安全的。蛇毒进入皮下后立即用乙醇处理有一定的治疗效果。     

Effect of gamma irradiation on toxicity and immunogenicity of Androctonus australis hector venom

An investigation was made of the radiosensitivity of the toxic and immunological properties of Androctonus australis hector venom. This venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a 60Co source. The results showed that venom toxicity was abolished for the two radiation doses (1 and 2 kGy) with, respectively, 10 and 25 times its initial LD50 value. However, irradiated venoms were immunogenic, and the antibodies elicited by them were able to recognize the native venom by enzyme-linked immunosorbent assay. Antisera raised against these toxoids (1 and 2 kGy) had a higher neutralizing capacity and immunoreactivity against all components of native venom than did the antiserum produced against the native venom. The antiserum of rabbits immunized with 2-kGy-irradiated venom was more efficient than 1-kGy-irradiated toxoid antiserum. Indeed, in vivo protection assays showed that the mice immunized with 2-kGy-irradiated venom resisted lethal doses (i.p.) of A. australis hector venom.     

Toxicity during early development of the mouse nervous system of a scorpion neurotoxin active on sodium channels

The lethal effects of scorpion envenomation is due to neurotoxins active on voltage-sensitive sodium channels. Dysfunctions of the peripheral and central nervous systems with neurological manifestations are commonly observed after scorpion stings, specially in young children. Since the neurotoxicity of venom fraction is greatly higher by intracerebroventricular than by subcutaneous injections, a direct effect of venom on CNS cannot be excluded specially in infants where the blood-brain barrier is not fully functional. We investigated the activity of a neurotoxin from the scorpion Androctonus australis hector (AahII) in newborn mice at 3, 7 and 14 days after birth and in adults. Young mice (P3, P7) were more sensitive to AahII injected subcutaneously than were adults, but were less sensitive to intracerebroventricular injection. The affinity of AahII for its receptor site on brain synaptosomes from P3 and P7 mice was slightly higher and the density of the binding sites was half that of adult mice. After subcutaneous injection of [125I]-AahII it was also observed that a small amount of radioactivity was found in brains of neonate mice but not in that of adults. This amount is however extremely lower than the value of the LD50 determined by intracerebroventricular injection. Results are consistent with a peripheral action of AahII and show that its toxic activity changes during the mouse nervous system development.     

Feeding behavior and venom toxicity of coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity

The feeding behavior and venom toxicity of the coral snake Micrurus nigrocinctus (Serpentes: Elapidae) on its natural prey in captivity were investigated. Coral snakes searched for their prey (the colubrid snake Geophis godmani) in the cages. Once their preys were located, coral snakes stroke them with a rapid forward movement, biting predominantly in the anterior region of the body. In order to assess the role of venom in prey restraint and ingestion, a group of coral snakes was 'milked' in order to drastically reduce the venom content in their glands. Significant differences were observed between snakes with venom, i.e., 'nonmilked' snakes, and 'milked' snakes regarding their behavior after the bite. The former remained hold to the prey until paralysis was achieved, whereas the latter, in the absence of paralysis, moved their head towards the head of the prey and bit the skull to achieve prey immobilization by mechanical means. There were no significant differences in the time of ingestion between these two groups of coral snakes. Susceptibility to the lethal effect of coral snake venom greatly differed in four colubrid species; G. godmani showed the highest susceptibility, followed by Geophis brachycephalus, whereas Ninia psephota and Ninia maculata were highly resistant to this venom. In addition, the blood serum of N. maculata, but not that of G. brachycephalus, prolonged the time of death of mice injected with 2 LD(50)s of M. nigrocinctus venom, when venom and blood serum were incubated before testing. Subcutaneous injection of coral snake venom in G. godmani induced neurotoxicity and myotoxicity, without causing hemorrhage and without affecting heart and lungs. It is concluded that (a) M. nigrocinctus venom plays a role in prey immobilization, (b) venom induces neurotoxic and myotoxic effects in colubrid snakes which comprise part of their natural prey, and (c) some colubrid snakes of the genus Ninia present a conspicuous resistance to the toxic action of M. nigrocinctus venom.     

Diabody mixture providing full protection against experimental scorpion envenoming with crude Androctonus australis venom

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.     

Purification and chemical characterization of the major neurotoxin from the venom of Pelamis plautrus.

A major toxin was isolated from the venom of the sea snake Pelamis platurus (yellow-bellied sea snake) by Sephadex G-50 and carboxymethylcellulose column chromatography. The LD50 of the pure toxin (Pelamis toxin a) was 0.044 mug/g in mice representing a tenfold increase in toxicity after purification. The toxin was homogeneous in acrylamide disc gel electrophoresis and eluted as a single peak after isoelectric focusing in a sucrose density gradient column. The isoelectric point was 9.69; thus it is a highly basic protein. The toxin contained 55 amino acid residues with four disulfide linkages. When all disulfide linkages were reduced and alkylated, the toxic action of the pure toxin disappeared leading to the conclusion that the disulfide bonds of the neurotoxin were essential for toxic action.     

Toxicity and Immunogenic Properties of Liposomal form of Vipera Libetina Venom

Abstract

The incorporation of Vipera libetina venom into liposomes obtained from pure egg phosphatidyl choline by the reverse phase evaporation method decreases its toxicity by 3-fold - in mice LD₅₀ for the native toxin is 2.22 mg/kg body weight and for the liposomal toxin 6.9 mg/kg. Subcutaneous injection of liposomal preparation into mice stimulates the development of cellular immunity and reproduces the reaction of the delayed-type hypersensitivity. It is. also, shown that after a single dose immunization of mice with liposomes containing 1xLD₅₀ dose of the venom, the.titer.of antibodies increases at the early postinfection period and is maintained on high level longer than after the injection of the native venom. Thus, liposom.es can be succesfully used for antiserum production and protective immunization against Vipera libetina venom.