Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution

The processes that drive the evolution of snake venom variability, particularly the role of diet, have been a topic of intense recent research interest. Here, we test whether extensive variation in venom composition in the medically important viper genus Echis is associated with shifts in diet. Examination of stomach and hindgut contents revealed extreme variation between the major clades of Echis in the proportion of arthropod prey consumed. The toxicity (median lethal dose, LD₅₀) of representative Echis venoms to a natural scorpion prey species was found to be strongly associated with the degree of arthropod feeding. Mapping the results onto a novel Echis phylogeny generated from nuclear and mitochondrial sequence data revealed two independent instances of coevolution of venom toxicity and diet. Unlike venom LD₅₀, the speed with which venoms incapacitated and killed scorpions was not associated with the degree of arthropod feeding. The prey-specific venom toxicity of arthropod-feeding Echis may thus be adaptive primarily by reducing venom expenditure. Overall, our results provide strong evidence that variation in snake venom composition results from adaptive evolution driven by natural selection for different diets, and underscores the need for a multi-faceted, integrative approach to the study of the causes of venom evolution.     

Possible recovery from an acute envenomation in male rats with LD50 of Echis coloratus crude venom: I-A seven days hematological follow-up study

The effect of an acute LD₅₀ dose of Echis coloratus crude venom in male albino rats was tested on blood parameters: white blood cells (WBCs), red blood cells (RBCs), platelets count, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), also serum glucose, total protein, triglycerides with alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) enzyme activities. The effect of the LD₅₀ dose was monitored over a period of seven days, with time intervals of 1, 3, 6, 12, 24, 72 h. All of the tested parameters show fluctuations with time and with tendency to regain normal control level after 12 h. At 12–24 h it seems to be crucial for the process of physiological recovery, in spite of the irreversible damage and tissue distraction. The process of physiological adaptation and recovery from the lethal destructive venom effect seems to stabilize after one week, leaving the animal alive with several biochemical altered metabolisms and disturbed physiological profile.     

Histological alterations and biochemical changes in the liver of sheep following Echis coloratus envenomation

Snake envenoming is a major problem in Al-Jouf Province of Saudi Arabia where most of these envenoming are caused by Echis coloratus which is the highest risk to human and animals in this Province. Little, if any, has been carried out on the histological alterations and biochemical changes in the liver of sheep following snake envenomation. Healthy adult male Ovis orientalis sheep were subjected to E. coloratus envenomation in an attempt to evaluate the histological alterations and biochemical changes in the liver. E. coloratus venom elevated glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride and total bilirubin while cholesterol was reduced. The histological alterations were mainly pyknosis, karyorrhexis, cytoplasmic vacuolation, necrosis, fatty changes and hepatocytes atrophy. Sinusoidal dilatation, Kupffer cell activation, amyloidosis, portal vein thrombosis, partial glycogen depletion and hepatic architecture distortion were also detected. The findings revealed that E. coloratus venom produced biochemical changes and histological alterations in the liver of the envenomated sheep that might affect the functions of this organ severely.     

Observations on the chemosensory responses of the midget faded rattlesnake (<Emphasis Type="Italic">Crotalus oreganus concolor</Emphasis>): discrimination of envenomated prey in a type II venom species

Rattlesnakes use prey chemical cues for ambush site selection and for relocating envenomated (E) prey following a predatory strike. The ability to discriminate between E and non-envenomated (NE) prey cues has been widely studied in rattlesnake species that produce type I venoms, which show high levels of snake venom metalloproteinase (SVMP) activity and low lethal toxicity [lethal dose which kills 50% of test animals (LD₅₀) >1.0 µg/g]. However, E vs. NE prey discrimination studies have not been conducted on rattlesnake species that produce a type II venom that consists of low SVMP activity and high lethal toxicity (LD₅₀ <1.0 µg/g). In the current study, long-term captive Crotalus oreganus concolor, which produce a type II venom, were tested for their ability to discriminate between chemical cues of natural (Sceloporus undulatus and Peromyscus maniculatus) and non-natural (Hemidactylus frenatus and Mus musculus) prey cues, as well as for their ability to discriminate between E and NE mouse carcasses, when prey envenomation occurred by a conspecific. Snakes showed significant levels of tongue flicking towards the chemical extracts of P. maniculatus and M. musculus, suggesting that C. oreganus concolor exhibit both innate and experience-based plasticity in response to prey chemical cues. In addition, C. oreganus concolor were able to discriminate between E and NE prey sources, when envenomation occurred by a conspecific, indicating that a type II venomous species can also discriminate between E and NE chemical cues.     

In vitro alpha-amylase inhibition and in vivo antioxidant potential of Amaranthus spinosus in alloxan-induced oxidative stress in diabetic rats

Amaranthus spinosus Linn. (Amaranthaceae), commonly known as “Mulluharivesoppu” in Kannada, is used in the Indian traditional system of medicine for the treatment of diabetes. The present study deals with the scientific evaluation of alpha amylase and the antioxidant potential of methanol extract of A. spinosus (MEAS). The aim of this study was to investigate in vitro alpha-amylase enzyme inhibition by CNPG3 (2-chloro-4-nitrophenol α-d-maltotrioside) and in vivo antioxidant potential of malondialdehyde (MDA), glutathione (GSH), catalase (CAT) and total thiols (TT) in alloxan-induced diabetic rats of a methanolic extract of A. spinosus. Blood sugar was also determined in MEAS-treated alloxan-induced diabetic rats. MEAS showed significant inhibition of alpha-amylase activity and IC₅₀ 46.02 μg/ml. Oral administration of MEAS (200 and 400 mg/kg) for 15 days showed significant reduction in the elevated blood glucose, MDA and restores GSH, CAT and TT levels as compared with a diabetic control. The present study provides evidence that the methanolic extract of A. spinosus has potent alpha amylase, anti-diabetic and antioxidant activities.     

Effects of Echis pyramidum Snake Venom on Hepatic and Renal Antioxidant Enzymes and Lipid Peroxidation in Rats

The effects of Echis pyramidum venom (EPV) (0.25, 0.50, and 1.00 mg/kg) on activities of superoxide dismutase (SOD) and catalase (CAT) and levels of thiobarbituric acid reactive substances (TBARS) and total thiols (T‐SH) in liver and kidneys of rats were investigated. EPV significantly and dose dependently decreased the activities of SOD and CAT in livers. Although the kidney SOD and CAT activities were not affected by low and medium doses of EPV, the high dose significantly reduced the activities of these enzymes. Liver and kidney TBARS levels were not affected by the low and medium doses of EPV, whereas the high dose significantly increased the TBARS after 6 h postdosing. There was a significant depletion of T‐SH in liver and kidneys of rats exposed to a high dose of EPV. The acute phase oxidative stress due to an EPV injection points toward the importance of an early antioxidant therapy for the management of snake bites.     

Protective effects of Parinari curatellifolia flavonoids against acetaminophen-induced hepatic necrosis in rats

In the present study, we investigated the hepatoprotective potential of Parinari curatellifolia Planch (Chrysobalanaceae) in experimental rats in order to ascertain the validity of folkloric claims of its effectiveness in the treatment of hepatic-related disorders. Flavonoid extract of P. curatellifolia seed, PCF (10-, 20- or 30 mg/kg body weight) or silymarin (25 mg/kg), dissolved in corn oil, was administered by gavage to experimental animals once daily for 14 consecutive days before liver damage was chemically induced through the administration of acetaminophen (2 g/kg p.o.) on the 14th day. Hepatoprotection was assessed by analyzing liver homogenate and serum for markers of hepatotoxicity – alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT) and lactate dehydrogenase (LDH) activities as well as prothrombin time (PT). Evaluation of biochemical indices of oxidative stress – level of lipid peroxides (LPO), activities of superoxide dismutase (SOD) and catalase, along with histological assessment of hepatic tissue sections were also carried out. Results revealed that all doses of PCF significantly (P < 0.001) and dose dependently prevented acetaminophen-induced increase in serum activities of hepatic enzymes (ALT, AST, GGT, LDH) and PT. Furthermore, PCF (10- and 20 mg/kg) significantly (P < 0.001) reduced lipid peroxidation in liver tissue and restored the activities of the antioxidant enzymes SOD and catalase toward normal levels. Histopathology of the liver tissue showed that PCF mitigated the toxicant-induced hepatocellular necrosis, reduced inflammatory cell infiltration and enhanced hepatocyte regeneration. The results indicated that P. curatellifolia flavonoids demonstrated remarkable hepatoprotective activity in acute liver injury caused by acetaminophen.     

Immunoreactivity and neutralization study of Chinese Bungarus multicinctus antivenin and lab-prepared anti-bungarotoxin antisera towards purified bungarotoxins and snake venoms

Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of B. multicinctus envenomation is attributed to the lethal components of α-, β-, γ- and κ- bungarotoxins contained in the venom. Although anti-B. multicinctus sera were produced in Shanghai, Taiwan and Vietnam, the most widely clinic used product was term as B. multicinctus antivenin and manufactured by Shanghai Serum Bio-technology Co. Ltd. In the present investigation, high purity α-, β- and γ-bungarotoxins were separately isolated from B. multicinctus crude venom. Rabbit anti- α-, β- and γ-bungarotoxin antisera were prepared by common methods, respectively. LD₅₀ values of α-, β- and γ-bungarotoxins were systematically determined via three administration pathways (intraperitoneal, intramuscular and intravenous injections) in Kunming mice. LD₅₀ values of β-bungarotoxin were closely related with injection routines but those of both α- and γ-bungarotoxins were not dependent on the injection routines. Commercial B. multicinctus antivenin showed strong immunoreaction with high molecular weight fractions of the B. multicinctus but weakly recognized low molecular weight fractions like α- and γ-bungarotoxins. Although B. multicinctus antivenin showed immunoreaction with high molecular weight fractions of Bungarus fasciatus, Naja atra, Ophiophagus hannah venoms but the antivenin only demonstrated animal protection efficacy against O. hannah venom. These results indicated that the high molecular weight fractions of the O. hannah played an important role in venom lethality but those of B. fasciatus and N. atra did not have such a role.     

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

An adequate assessment of scorpion and snake venom LD₅₀is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD₅₀variation of Tunisian scorpion (Androctonus australis garzonii: Aag andButhus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57Bl/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 LD₅₀determined in C57Bl/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 LD₅₀variations (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 LD₅₀values when assessedin vivo.The LD₅₀variation of Tunisian viper (Cerastes cerastes: Cc andVipera 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 LD₅₀variations were observed with the mouse strain, the sex or the season of venom milking. However, LD₅₀varies 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 LD₅₀evaluation or the venom quality used for the development of an efficient antivenom.     

Antioxidant,antimicrobial, toxicity and analgesic properties of ethanol extract of Solena amplexicaulis root

Background

This study was subjected to investigate different pharmacological properties of ethanol extract of Solena amplexicaulis root.

Results

The extract contains flavonoid, alkaloid, saponin and steroid compounds. The extract exhibited excellent antioxidant activity in DPPH radical scavenging activity. The extract also showed potent activity in brine shrimp lethality bioassay. The LC₅₀ value was found to 44.677 μg/ml. The extract showed better anti-bacterial activity against gram-negative bacteria. In antifungal assay, the maximum 79.31% of anti-mycotic activity was observed against Aspergillus ochraceus while minimum 44.2% against Rhizopus oryzae. MIC value ranged between 1500–3000 μg/ml. The extract was found moderately toxic with a 24-hr LD₅₀ value of 81.47 mg/kg in Swiss albino mice. The degree of inhibition by the ethanolic extract of the root was found less than that of standard analgesic drug diclofenac sodium. The extract also showed moderate anti-inflammatory and antinociceptive activity and anti-diabetic property. Reducing power of the extract was comparable with standard ascorbic acid. Moderate in vitro thrombolytic activity, lipid peroxidation inhibition property, metal chelating ability and stress-protective activity was also observed.

Conclusion

Ethanol extract of Solena amplexicaulis root can be valuable for treatment of different diseases.     

Effect of Ottoman Viper (Montivipera xanthina (Gray, 1849)) Venom on Various Cancer Cells and on Microorganisms

Cytotoxic and antimicrobial effects of Montivipera xanthina venom against LNCaP, MCF-7, HT-29, Saos-2, Hep3B, Vero cells and antimicrobial activity against selected bacterial and fungal species: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, E. coli O157H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus epidermidis ATCC 12228, S. typhimirium CCM 5445, Proteus vulgaris ATCC 6957 and Candida albicans ATCC 10239 were studied for evaluating the potential medical benefit of this snake venom. Cytotoxicity of venom was determined using MTT assay. Snake venom cytotoxicity was expressed as the venom dose that killed 50 % of the cells (IC₅₀). The antimicrobial activity of venom was studied by minimal inhibitory concentration (MIC) and disc diffusion assay. MIC was determined using broth dilution method. The estimated IC₅₀ values of venom varied from 3.8 to 12.7 or from 1.9 to 7.2 μg/ml after treatment with crude venom for 24 or 48 h for LNCaP, MCF-7, HT-29 and Saos-2 cells. There was no observable cytotoxic effect on Hep3B and Vero cells. Venom exhibited the most potent activity against C. albicans (MIC, 7.8 μg/ml and minimal fungicidal concentration, 62.5 μg/ml) and S. aureus (MIC, 31.25 μg/ml). This study is the first report showing the potential of M. xanthina venom as an alternative therapeutic approach due to its cytotoxic and antimicrobial effects.     

Comparative histological, histochemical and ultrastructural studies of the nephron of selected snakes from the Egyptian area

The present study was aimed to compare and contrast the histochemical, histological and ultrastructural variations (microanatomical differences) in the nephrons of selected snake species, Eryx jaculus (Boidae), Psammophis sibilans (Colubridae), Naja haje (Elapidae) and Echis pyramidum (Viperidae) from Egypt. The structural studies were carried out by conventional light and electron microscopy. The nephron, the renal unit of snakes, consists of renal corpuscle, proximal tubule, intermediate segment, distal tubule and collecting tubule. The renal corpuscles have large capillaries with clear and dark fenestrated endothelial cells. The proximal tubule showed long microvilli, cytoplasmic vacuoles, developed endoplasmic reticulum and abundant mitochondria. The intermediate segment was lined by ciliated cells. The lining cells of the distal tubules showed few microvilli, abundant dense mitochondria and clear vesicles of mucous appeared in the terminal portion. The collecting tubules consisted of mucous cells. In summary, the ultra-structure studies of nephrons revealed several interspecies similarities and also some intra-species differences in species of snakes.     

The hepatoprotective effects of Hypericum perforatum L. on hepatic ischemia/reperfusion injury in rats

Little is known about the effective role of Hypericum perforatum on hepatic ischemia–reperfusion (I/R) injury in rats. Hence, albino rats were subjected to 45 min of hepatic ischemia followed by 60 min of reperfusion period. Hypericum perforatum extract (HPE) at the dose of 50 mg/kg body weight (HPE₅₀) was intraperitonally injected as a single dose, 15 min prior to ischemia. Rats were sacrificed at the end of reperfusion period and then, biochemical investigations were made in serum and liver tissue. Liver tissue homogenates were used for the measurement of malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPx) levels. At the same time alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were assayed in serum samples and compared statistically. While the ALT, AST, LDH activities and MDA levels were significantly increased, CAT and GPx activities significantly decreased in only I/R-induced control rats compared to normal control rats (p < 0.05). Treatment with HPE₅₀ significantly decreased the ALT, AST, LDH activities and MDA levels, and markedly increased activities of CAT and GPx in tissue homogenates compared to I/R-induced rats without treatment–control group (p < 0.05). In oxidative stress generated by hepatic ischemia–reperfusion, H. perforatum L. as an antioxidant agent contributes an alteration in the delicate balance between the scavenging capacity of antioxidant defence systems and free radicals in favour of the antioxidant defence systems in the body.     

Bee venom ameliorates cardiac dysfunction in diabetic hyperlipidemic rats

High levels of blood glucose and lipids are well-known risk factors for heart diseases. Bee venom is a natural product that has a potent hypoglycemic, hypolipidemic, anti-inflammatory, and antioxidant effects. The current study aimed to determine the bee venom effects on cardiac dysfunction compared to combined therapy of metformin and atorvastatin in diabetic hyperlipidemic rats. The median lethal dose of bee venom was estimated, and then 50 adult male albino rats were categorized into five groups. One group was fed a standard diet and served as a negative control, while the other groups were given nicotinamide and streptozotocin injections to induce type 2 diabetes. After confirming diabetes, the rats were fed a high-fat diet for four weeks. The four groups were divided as follows: one group served as a positive control, whereas the other three groups were treated with bee venom (0.5 mg/kg), bee venom (1.23 mg/kg), and combined therapy of metformin (60 mg/kg) and atorvastatin (10 mg/kg), respectively, for four weeks. Upon termination of the experiment, blood samples and heart tissue were obtained. Administration of bee venom using both doses (0.5 and 1.23 mg/kg) and combined therapy of metformin and atorvastatin revealed a significant decrease in the concentrations of glucose, total cholesterol, triacylglycerol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, troponin I, creatine kinase, and lactate dehydrogenase activities. Moreover, a significant decrease had been detedcted in malondialdehyde, nuclear factor-kappa-β levels, and relative mRNA expression of vascular cell adhesion molecule-1 and galectin-3 in heart tissue compared to the positive control (P < 0.0001). Furthermore, there was a significant increase in bodyweight levels of insulin, high-density lipoprotein cholesterol, and total antioxidant capacity in heart tissue compared to the positive control (P < 0.0001). The results indicate that bee venom can ameliorate cardiac dysfunction through attenuating oxidative stress and downregulating the NF-κβ signaling pathway.     

Investigations of anticholinestrase and antioxidant potentials of methanolic extract,subsequent fractions,crude saponins and flavonoids isolated from Isodon rugosus

Background

Based on the ethnomedicinal uses and the effective outcomes of natural products in various diseases, this study was designed to evaluate Isodon rugosus as possible remedy in oxidative stress, alzheimer’s and other neurodegenerative diseases. Acetylecholinestrase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of crude methanolic extract (Ir.Cr), resultant fractions (n-hexane (Ir.Hex), chloroform (Ir.Cf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq)), flavonoids (Ir.Flv) and crude saponins (Ir.Sp) of I. rugosus were investigated using Ellman’s spectrophotometric method. Antioxidant potential of I. rugosus was determined using DPPH, H₂O₂ and ABTS free radicals scavenging assays. Total phenolic and flavonoids contents of plant extracts were determined and expressed in mg GAE/g dry weight and mg RTE/g of dry sample respectively.

Results

Among different fractions Ir.Flv and Ir.Cf exhibited highest inhibitory activity against AChE (87.44 ± 0.51, 83.73 ± 0.64%) and BChE (82.53 ± 0.71, 88.55 ± 0.77%) enzymes at 1 mg/ml with IC₅₀ values of 45, 50 for AChE and 40, 70 μg/ml for BChE respectively. Activity of these fractions were comparable to galanthamine causing 96.00 ± 0.30 and 88.61 ± 0.43% inhibition of AChE and BChE at 1 mg/ml concentration with IC₅₀ values of 20 and 47 μg/ml respectively. In antioxidant assays, Ir.Flv, Ir.Cf, and Ir.EtAc demonstrated highest radicals scavenging activities in DPPH and H₂O₂ assays which were comparable to ascorbic acid. Ir.Flv was found most potent with IC₅₀ of 19 and 24 μg/ml against DPPH and H₂O₂ radicals respectively. Whereas antioxidant activates of plant samples against ABTS free radicals was moderate. Ir.Cf, Ir.EtAc and Ir.Cr showed high phenolic and flavonoid contents and concentrations of these compounds in different fractions correlated well to their antioxidant and anticholinestrase activities.

Conclusion

It may be inferred from the current investigations that the Ir.Sp, Ir.Flv and various fractions of I. rugosus are good sources of anticholinesterase and antioxidant compounds. Different fractions can be subjected to activity guided isolation of bioactive compounds effective in neurological disorders.     

Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom

Background

Multifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms.

Methods

The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.

Results

Bl-LAAO is a 60 kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H₂O₂. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC₅₀ of 0.07 μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H₂O₂ in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.

Conclusion

Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H₂O₂ which kill the cells.

General significance

These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.     

Effects of Melothriamaderaspatana leaf extract on antioxidant status in sham-operated and uninephrectomized DOCA-salt hypertensive rats

The present study was designed to investigate the antihypertensive and antioxidant effect of Melothria maderaspatana leaf extract (MME) on sham-operated and DOCA-salt (deoxycorticosterone acetate) induced hypertensive rats. Administration of DOCA-salt significantly increased the systolic (from 127 to 212 mm Hg) and diastolic (from 91 to 174 mm Hg) blood pressure compared to sham-operated control rats, while treatment with MME significantly reduced the systolic (from 212 to 135 mm Hg) and diastolic (from 174 to 96 mm Hg) blood pressure compared to hypertensive control. In DOCA-salt rats, the plasma and tissue concentration of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxide (LOOH) significantly increased and administration of MME significantly reduced these parameters towards the levels in sham-operated control. In hypertensive rats, activities of the enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and levels of non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione (GSH) decreased significantly in the plasma and tissues. Administration of MME returned the enzymatic and non-enzymatic antioxidants towards sham-operated control. MME shows both antihypertensive and antioxidant properties in DOCA-salt hypertensive rats and, among the three different doses tested, 200 mg/kg caused the maximum effect.     

Involvement of von Willebrand factor and botrocetin in the thrombocytopenia induced by Bothrops jararaca snake venom

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na₂-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf^-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na₂-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf^-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.