Isolation of minax toxins from the venom of the scorpion Buthus minax and their metabolic effects

Two neurotoxins, minax toxins 1 and 2, were isolated from venom of the scorpion Buthus minax from the Sudan. Molecular weights of 7000 and 6800 and 66 and 62 amino acids were found for minax toxins 1 and 2, respectively. Both toxins contain four disulfide bonds, 1 mol each of phenylalanine, histidine, and tryptophan, no free sulfhydryl groups, and no methionine. Both minax toxins 1 and 2 are basic polypeptides with isoelectric points of 8.2 and 9.0, respectively. There is a significant increase in the calcium content of rat hearts envenomated with minax toxins 1 and 2 or crude venom. This confirms earlier electron microscopic findings of calcium deposits in the heart following scorpion envenomation. There is a concomitant decrease in the calcium and phosphorus content of rat serum following envenomation. It seems that neither scorpion toxins nor scorpion venoms affect the mineral metabolism of the bone. The present investigation indicates that scorpion toxins have not only a neurotoxic action but also broader biological effects such as mineral metabolism.     

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.     

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.     

Time-course of lipid peroxidation in different organs of mice treated with Echis pyramidum snake venom

This study examined the effect of Echis pyramidum (EP) venom on time-course of lipid peroxidation in different vital organs of mice. Adult male Swiss albino mice were injected with EP venom (2 mg/kg, i.p.); control mice received vehicle alone (normal saline). Mice were killed at 1, 3, 6, 12, and 24 h post-envenomation. The liver, lung, kidney, heart, and brain (cerebrum and cerebellum) were collected for the estimation of malondialdehyde (MDA), an index of lipid peroxidation. The results of this study showed that a single injection of EP venom caused a significant lipid peroxidation in all the organs studied. The onset of lipid peroxidation was as early as 1 h and persisted for several hours, suggesting an important role of oxidative stress in the cytotoxicity of EP venom.     

Effect of age on body distribution of Tityustoxin from Tityus serrulatus scorpion venom in rats

Previous research from our Laboratory has shown a greater susceptibility of young animals, when compared to adults, to envenomation by tityustoxin (TsTX), one of the main toxins from Tityus serrulatus scorpion venom. Our hypothesis is that a differential body distribution of TsTX among adult and young animals could account for the worse prognosis of scorpion envenomation in infants. Thus, TsTX labeled with technetium-99m was injected (6 microg, subcutaneous) in adult (150-160 day-old) and young (21-22 day-old) male rats. Groups of animals were sacrificed at different times after TsTX injection (0.08, 1.0, 3.0, 6.0, 12.0 and 24.0 hours) under Urethane anesthesia (140 mg/100 g, i.p.). The brain, heart, lungs, liver, kidneys, spleen and thyroid were excised and blood collected. Young rats presented a shorter latency toxin concentration peak in all studied organs except for the liver and the kidney, when compared to adults. The ratio between the area under the curve of the toxin concentration in each organ and that in blood (Kp) indicates higher accumulation in the organs of young animals mainly for brain, liver and heart. These observations suggest a faster toxin distribution in the organs of young rats. The higher uptake of TsTX in the brain is suggestive of a greater permeability for the toxin along the blood-brain barrier of young rats. In conclusion, the higher uptake in heart, together with data from the brain, may help to elucidate the clinical manifestations frequently observed in children under scorpion envenomation.     

Scorpion venom increases mRNA expression of lung cytokines

Previous studies have demonstrated that scorpion toxins increase the serum levels of IL-1, IL-6, INF-gamma, and GM-CSF in patients with severe shock and pulmonary edema. Moreover, it has been shown that experimental models of scorpion envenomation presented an increase in serum levels of IL-1, IL-6, IFN-gamma and nitric oxide. Thus, it is possible that the cytokine release may contribute to the onset and maintenance of the pulmonary edema induced by scorpion venom. This study was designed to investigate whether inflammatory and non-inflammatory cytokines, contribute to the pulmonary injury induced by infusion of Tityus serrulatus scorpion toxin in rats. We show that scorpion venom not only increases the expression of mRNA pulmonary inflammatory cytokines but also non-inflammatory cytokines as well. Moreover, the expression of IL-1alpha, IL-1beta and IL-6 mRNA was shown to be higher among the remaining detectable cytokines. The findings of this study provide additional insight towards the understanding of the pathophysiology of the pulmonary edema induced by scorpion venom. The increased level of pulmonary cytokines observed during the pulmonary edema may be responsible for the exacerbation and maintenance of the inflammatory response to scorpion venom in the lungs.     

In vivo and in vitro protection against lethal activity of Buthus occitanus tunetanus venom with a recombinant protein

We report the use of recombinant scorpion toxin in the form of fusion protein as antigen for mice immunisation. The aim is to produce protective antisera against lethal activity of the venom from Tunisian scorpion Buthus occitanus tunetanus, responsible for several annually reported human cases of scorpion stings. The gene encoding Bot III (the most toxic alpha toxin of Buthus occitanus tunetanus) was fused to the sequence encoding synthetic ZZ domains of staphylococcal protein A. The construct ZZ-Bot III was expressed in the periplasm of E. coli as a fusion protein and purified by affinity chromatography. The recombinant fusion protein was characterized and used as antigen to generate antibodies in mice. The antibodies against the recombinant protein neutralize the toxic venom (10 LD50/ml) and also confer protection for immunized mice against antigenically related mammal toxins.     

Effect of bacterial lipopolysaccharide on the content of lipid peroxidation products in lungs and other organs of mice

The influence of lipopolysaccharide fromEscherichia coli (LPS, 17 mg/kg body weight) on the lipid peroxidation process in organs of mice was studied. The content of conjugated dienes (CD), lipid peroxides (LP), malondialdehyde (MDA) (all three lipid peroxidation by-products), peroxidase (PO) activity and wet-to-dry weight ratio in lungs, heart, spleen, kidneys and liver were determined 1.5 h after intravenous injection of LPS. Animals observed at this time-point had reduced activity and decreased body temperature by about 2°C, however, all analysed organs did not reveal any changes of wet-to-dry weight ratio comparing to organs from mice injected with sterile, pyrogen free 0,9% NaCl. Only extracts from heart and lungs showed significant increase in the tissue level of at least two lipid peroxidation products. The heart content of CD, MDA, and LP was about 1.5-, 1.3-, and 2.4-fold higher than in control group. In lungs CD and MDA increased 3.3- and 1.3-times but in spleen only content of LP was elevated. In these organs the suppression of PO activity was also observed. Liver and kidneys did not reveal any convincing enhancement of lipid peroxidation process and alterations of PO activity. Since free radical reactions are involved in lipid peroxidation process and inactivation of PO these results suggest that heart, lungs and spleen are the organs mostly exposed to oxidative stress during the first 1.5 h after single injection of LPS in mice.Abbreviations CD conjugated dienes - LP lipid peroxides - LPS lipopolysaccharide - MDA malondialdehyde - PMNL polymorphonuclear leukocytes - PO peroxidase - TBA thiobarbituric acid     

Analysis of the subproteomes of proteinases and heparin‐binding toxins of eight Bothrops venoms

Viperid snakes show the most complex snake‐venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2‐DE, and their subproteomes of proteinases were explored by 2‐D immunostaining and 2‐D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti‐coagulant and anti‐inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high‐affinity for heparin as composed of mainly serine proteinases and C‐type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin‐binding toxins. Only the non‐bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin‐bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.     

马氏蝎毒毒素的分离纯化及其对红细胞膜作用的初步研究

本文用山东产马氏蝎(Buthus martensii kashi)粗毒为材料,经SephadexG-50和Sp-Sephadex C-25二次柱层析,分离纯化获得三个毒峰部分,毒性比粗毒分别提高40—100倍。 纯度鉴定表明三个毒峰的聚丙烯酰胺凝胶电泳和等电聚焦电泳均为一条带,等电点分别为8.7,9.1,9.1,分子量用SDS-不连续聚丙烯酰胺凝胶电泳测定分别为6,600,5,000和8,500。对纯化蝎毒毒素的氨基酸组分也作了分析。 蝎毒毒素对人红细胞膜作用的初步探索结果表明:它使人红细胞膜的Na.K-ATP酶活性和膜脂流动性有所降低。     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

Ethanol-Induced Cell Death by Lipid Peroxidation in PC12 Cells

Free radical generation is hypothesized to be the cause of alcohol-induced tissue injury. Using fluorescent cis-parinaric acid and TBARS, lipid peroxidation was shown to be increased in the presence of trace amounts of free ferrous ion in PC12 cells. This increase in lipid peroxidation was enhanced by ethanol in a dose dependent manner and also correlated with loss of cell viability, as measured by increased release of lactate dehydrogenase (LDH). Resveratrol, a potent antioxidant, had a protective effect against lipid peroxidation and cell death. These findings strongly suggest that ethanol-induced tissue injury and cell death is a free radical mediated process, and may be important in alcohol-related premature aging and other degenerative diseases.     

Effect of Chronic N-Acetyl Cysteine Administration on Oxidative Status in the Presence and Absence of Induced Oxidative Stress in Rat Striatum

Antioxidants have possible therapeutic value in neurodegenerative disorders, although they may have pro-oxidant effects under certain conditions. Glutathione (GSH) is a key free radical scavenger. N-acetylcysteine (NAC) bolsters GSH and intracellular cysteine and also has effective free radical scavenger properties. The effects of chronic NAC administration (50 mg/kg/day, 500 mg/kg/day, 1500 mg/kg/day × 21 days) on cellular markers of oxidative status was studied in striatum of healthy male Sprague-Dawley rats as well as in animals with apparent striatal oxidative stress following chronic haloperidol treatment (1.5 mg/kg/day × 3 weeks). In non-haloperidol treated animals, NAC 50 and 500 mg/kg did not affect oxidative status, although NAC 1,500 mg/kg significantly increased striatal superoxide levels, decreased lipid peroxidation and increased consumption of reduced glutathione (GSH). Haloperidol alone evoked a significant increase in superoxide and lipid peroxidation. All NAC doses blocked haloperidol induced increases in superoxide levels, while NAC 500 mg/kg and 1,500 mg/kg prevented haloperidol-associated lipid peroxidation levels and also increased the GSSG/GSH ratio. NAC may protect against conditions of striatal oxidative stress, although possible pro-oxidative actions at high doses in otherwise healthy individuals, e.g. to offset worsening of neurodegenerative illness, should be viewed with caution.     

Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes

We elucidated the protective effect of quercetin, a polyphenolic flavonoid, on lipid peroxidation, endogenous antioxidant status and DNA damage during nicotine-induced toxicity in cultured rat peripheral blood lymphocytes as compared to N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine (3 mM) with and without quercetin and NAC (1 mM) in RPMI-1640 medium for 1 h. In preliminary experiments to fix the effective dose of quercetin, different doses of quercetin (25, 50, 75, 100 and 200 microM) were administered to lymphocytes with nicotine, and lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides) were analysed. A 75 microM dose of quercetin was found to be effective as evidenced by decreased lipid peroxidation. To evaluate the protective potential of quercetin against genotoxic effects of nicotine we used comet and micronucleus assays, which are valid parameters to assess genetic damage. In addition, biochemical changes including lipid peroxidation and antioxidant status were assessed. There were significant increases in the levels of lipid peroxidation, comet parameters and micronuclei frequencies, followed by decrease in the endogenous antioxidant status, in nicotine-treated lymphocytes, which were brought back to near normal by quercetin or NAC treatment. The protective effect of quercetin against nicotine toxicity was comparable to that of NAC. These findings suggest that quercetin can be as effective as NAC in protecting rat peripheral lymphocytes against nicotine-induced cellular and DNA damage.     

Reduction of ultraviolet light-induced oxidative stress by amino acid-based iron chelators

The generation of free radicals by ultraviolet (UV) light accelerates skin aging, which is known as photoaging. Cutaneous iron catalyzes the generation of free radicals. We designed novel antioxidants that suppressed the iron-catalyzed free radical generation and the ensuing UV-induced damage by mimicking the binding site of iron sequestering proteins. These antioxidants, N-(2-hydroxybenzyl)amino acids, were prepared by condensation of amino acids such as glycine and L-serine with salicylaldehyde and followed by catalytic reduction. The compounds formed a 2:1 complex to iron ion. These amino acid derivatives inhibited the iron-induced hydroxyl radical generation (the Fenton reaction). The compounds also suppressed UV-induced lipid peroxidation in murine dermal fibroblast homogenates. In addition, N-(2-hydroxybenzyl)-L-serine showed protective activity against UV-induced cytotoxicity in murine dermal fibroblasts. Desferrioxamine, a strong iron sequestering compound, was effective in inhibiting the Fenton reaction and the lipid peroxidation, but it was ineffective in protecting against UV-induced cytotoxicity. The results suggest that UV-induced oxidative stress can be reduced by these amino acid derivatives.     

Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects of L-carnitine

Excitotoxicity and disrupted energy metabolism are major events leading to nerve cell death in neurodegenerative disorders. These cooperative pathways share one common aspect: triggering of oxidative stress by free radical formation. In this work, we evaluated the effects of the antioxidant and energy precursor, levocarnitine ( l -CAR), on the oxidative damage and the behavioral, morphological, and neurochemical alterations produced in nerve tissue by the excitotoxin and free radical precursor, quinolinic acid (2,3-pyrindin dicarboxylic acid; QUIN), and the mitochondrial toxin, 3-nitropropionic acid (3-NP). Oxidative damage was assessed by the estimation of reactive oxygen species formation, lipid peroxidation, and mitochondrial dysfunction in synaptosomal fractions. Behavioral, morphological, and neurochemical alterations were evaluated as markers of neurotoxicity in animals systemically administered with l -CAR, chronically injected with 3-NP and/or intrastriatally infused with QUIN. At micromolar concentrations, l -CAR reduced the three markers of oxidative stress stimulated by both toxins alone or in combination. l -CAR also prevented the rotation behavior evoked by QUIN and the hypokinetic pattern induced by 3-NP in rats. Morphological alterations produced by both toxins (increased striatal glial fibrillary acidic protein-immunoreactivity for QUIN and enhanced neuronal damage in different brain regions for 3-NP) were reduced by l -CAR. In addition, l -CAR prevented the synergistic action of 3-NP and QUIN to increase motor asymmetry and depleted striatal GABA levels. Our results suggest that the protective properties of l -CAR in the neurotoxic models tested are mostly mediated by its characteristics as an antioxidant agent.     

Prenatal exposure to the contaminant perfluorooctane sulfonate elevates lipid peroxidation during mouse fetal development but not in the pregnant dam

Abstract

Perfluorooctane sulfonate (PFOS), a member of the perfluorinated chemical family, has been convincingly demonstrated to affect lipid metabolism in animals and humans and readily crosses the placenta to exert its effects on the developing fetuses. While its exact mechanism is still not clear, PFOS exposure has long been suggested to exert its toxicity via oxidative stress and/or altered gene expression. Levels of PFOS and malondialdehyde in various organs and cell cultures have been widely determined as general indicators of non-specific lipid peroxidation after PFOS exposure. In this study, the oxidation of precise polyunsaturated fatty acids and their metabolites, derived from enzymatic and non-enzymatic pathways was determined following PFOS exposure in both adult and maternal/fetal mice. CD-1 mice were exposed to 3 mg/kg body weight/day of PFOS in corn oil by oral gavage until late gestation (GD17). We demonstrated that lipid peroxidation was particularly and exclusively affected in fetuses exposed to PFOS, but this was not the case in the maternal mice, where limited effects were observed in the enzymatic oxidation pathway. In this study, we demonstrated that PFOS-induced lipid peroxidation might have a greater impact in free radical generation in fetuses than in dams and could be responsible for affecting fetal development. In addition, antioxidant enzymes, such as superoxide dismutase and catalase, appeared to maintain oxidative stress homeostasis partially in adult mice exposed to PFOS. Taken together, our results might elucidate the mechanism of how PFOS induces oxidative stress in vivo.     

Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes

Abstract

We elucidated the protective effect of quercetin, a polyphenolic flavonoid, on lipid peroxidation, endogenous antioxidant status and DNA damage during nicotine-induced toxicity in cultured rat peripheral blood lymphocytes as compared to N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine (3 mM) with and without quercetin and NAC (1 mM) in RPMI-1640 medium for 1 h. In preliminary experiments to fix the effective dose of quercetin, different doses of quercetin (25, 50, 75, 100 and 200 μM) were administered to lymphocytes with nicotine, and lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides) were analysed. A 75 μM dose of quercetin was found to be effective as evidenced by decreased lipid peroxidation. To evaluate the protective potential of quercetin against genotoxic effects of nicotine we used comet and micronucleus assays, which are valid parameters to assess genetic damage. In addition, biochemical changes including lipid peroxidation and antioxidant status were assessed. There were significant increases in the levels of lipid peroxidation, comet parameters and micronuclei frequencies, followed by decrease in the endogenous antioxidant status, in nicotine-treated lymphocytes, which were brought back to near normal by quercetin or NAC treatment. The protective effect of quercetin against nicotine toxicity was comparable to that of NAC. These findings suggest that quercetin can be as effective as NAC in protecting rat peripheral lymphocytes against nicotine-induced cellular and DNA damage.     

Promethazine inhibits the formation of aldehydic products of lipid peroxidation but not covalent binding resulting from the exposure of rat liver fractions to CCl4.

Promethazine is known to have protective activity in relation to CCl4-induced liver necrosis. This hepatoprotective property has been investigated with regard to the free radical scavenging and antioxidant properties of promethazine using isolated hepatocytes and microsomal suspensions. CCl4 is activated in both systems to free radical metabolites that bind covalently to lipid and protein, and initiate lipid peroxidation. A large number of carbonyl products is produced during CCl4-induced lipid peroxidation; promethazine strongly inhibits the production of all classes of carbonyl compounds in both microsomal suspensions and isolated hepatocytes. In contrast, promethazine is a very weak inhibitor of the covalent binding of metabolites of CCl4. We conclude that promethazine acts by scavenging the trichloromethylperoxyl radical and lipid peroxyl radicals, and is a weak scavenger of the trichloromethyl radical. These data, when considered together with the hepatoprotective effects of promethazine, suggest that lipid peroxidation is of relatively more importance than covalent binding in the pathogenesis of CCl4-induced liver necrosis.     

Protective effect of N‐acetylcysteine against ischemia/reperfusion injury in rat urinary bladders

Ischemia/reperfusion (I/R) injury represents an important cause of bladder contractile dysfunction. One of the major causes leading to this dysfunction is thought to be reactive oxygen species formation. In this study, we investigated the potential benefit of N‐acetylcysteine (NAC), a potent antioxidant that neutralizes free radicals, in a rat model of urinary bladder injury. NAC treatment rescues the reduction of contractile response to I/R injury in a dose‐dependent manner. In addition, all levels of reactive oxygen species, lipid peroxidation, and NADPH‐stimulated superoxide production in the I/R operation + NAC (I/R + NAC) group also decreased compared with a marked increase in the I/R operation + saline (I/R + S) group. Moreover, an in situ fluorohistological approach also showed that NAC reduces the generation of intracellular superoxides enlarged by I/R injury. Together, our findings suggest that NAC has a protective effect against the I/R‐induced bladder contractile dysfunction via radical scavenging property. Copyright © 2013 John Wiley & Sons, Ltd.