Cross neutralization of Hypnale hypnale (hump-nosed pit viper) venom by polyvalent and monovalent Malayan pit viper antivenoms in vitro and in a rodent model

Hypnale hypnale (hump-nosed pit viper) is a medically important venomous snake in Sri Lanka and Southwestern India. Bite of this snake may result in hemostatic dysfunction, acute kidney injury and death. Clinical studies indicated that the locally available polyvalent antivenoms produced in India are not effective against hump-nosed pit viper envenoming. Hence, there is an urgent need to search for effective antivenom. In this paper, we examined the ability of Calloselasma rhodostoma (Malayan pit viper) monovalent antivenom and the Hemato polyvalent antivenom (both produced by Thai Red Cross Society, TRCS) to neutralize the lethality and toxic effects of H. hypnale venom, as C. rhodostoma is considered a sister taxon of H. hypnale. In vitro neutralization studies showed that the Hemato polyvalent antivenom effectively neutralized the lethality of H. hypnale venom (1.52 mg venom/mL antivenom) as well as the hemorrhagic, procoagulant and necrotic activities of the venom. The monovalent C. rhodostoma antivenom could also neutralize the lethality and toxic activities of the venom, but the potency was lower. The Hemato polyvalent antivenom also effectively protected mice from the lethal and local effects of H. hypnale venom in an in vivo rodent model of envenoming. Furthermore, the polyvalent antivenom could also effectively neutralize the venom of Daboia russelii (2.50 mg venom/mL antivenom), another common cause of snake bites in Sri Lanka and South India. These findings suggested that the Hemato polyvalent antivenom may be beneficial in the antivenom treatment of H. hypnale envenoming.     

Oxidative stress‐induced methemoglobinemia is the silent killer during snakebite: a novel and strategic neutralization by melatonin

Oxidative stress‐induced methemoglobinemia remained an untouched area in venom pharmacology till date. This study for the first time explored the potential of animal venoms to oxidize hemoglobin to methemoglobin. In in vitro whole‐blood assay, methemoglobin forming ability of venoms varied as Naja naja > Ophiophagus hannah > Echis carinatus > Daboia russellii > Apis mellifera > Mesobuthus tamulus > Hippasa partita. Being highly potential, N. naja venom was further studied to observe methemoglobin formation in RBCs and in combinations with PMNs and PBMCs, where maximum effect was observed in RBCs + PMNs combination. Naja naja venom/externally added methemoglobin‐induced methemoglobin formation was in parallel with ROS generation in whole blood/RBCs/RBCs + PMNs/RBCs + PBMCs. In in vivo studies, the lethal dose (1 mg/kg body weight, i.p.) of N. naja venom readily induced methemoglobin formation, ROS generation, expression of inflammatory markers, and hypoxia‐inducible factor‐3α. Although the mice administered with three effective doses of antivenom recorded zero mortality; the methemoglobin and ROS levels remained high. However, one effective dose of antivenom when administered along with melatonin (1:50; venom/melatonin, w/w), not only offered 100% survival of experimental mice, but also significantly reduced methemoglobin level, and oxidative stress markers including hypoxia‐inducible factor‐3α. This study provides strong drive that, complementing melatonin would not only reduce the antivenom load, but for sure greatly increase the success rate of antivenom therapy and drastically minimize the global incidence of snakebite deaths. However, further detailed investigations are needed before translating the combined therapy towards the bed side.     

A new monospecific ovine Fab fragment antivenom for treatment of envenoming by the Sri Lankan Russell's viper (Daboia Russelii Russelii): a preliminary dose-finding and pharmacokinetic study.

Russell's viper is the most important cause of life-threatening snake bite and acute renal failure in Sri Lanka. Only equine polyspecific antivenoms imported from India are available. They have not proved effective clinically or in clearing venom antigenemia and they frequently cause reactions. In an attempt to reduce mortality and morbidity, a new monospecific ovine Fab fragment antivenom (PolongaTab; Therapeutic Antibodies, Inc., London, United Kingdom) was raised against Sri Lankan Russell's viper venom. In a preliminary dose-finding study in 35 patients, an initial dose of 3-4 g restored blood coagulability permanently and stopped systemic bleeding, even in severely envenomed patients. Venom antigenemia disappeared within 1 hr of antivenom treatment but recurred, probably as a result of continued absorption of venom from the site of the bite, after the rapid clearance of therapeutic antibody. Twelve patients (34%) experienced early reactions that were usually mild and always responded to epinephrine.     

Protective effects of melatonin against oxidative damage induced by Egyptian cobra (Naja haje) crude venom in rats

Naja haje envenomation is one of the leading causes of death due to snakebite. Antiserum therapy sometimes fails to provide enough protection against venom toxicity. In this study, we investigated the protective effects of melatonin against N. haje venom in rats. The animals were injected with venom (0.25 mg/kg) and/or melatonin (10 mg/kg) and compared with vehicle-treated rats. There was oxidative/nitrosative damage and apoptosis in the liver, heart, and kidneys of venom-injected rats. Melatonin counteracted the increased lipoperoxidation and nitric oxide, prevented decreased glutathione peroxidase and reductase activity, reduced the glutathione disulfide/glutathione (GSSG/GSH) ratio, and maintained the GSH pool. Furthermore, melatonin administration was associated with a reduction of apoptosis, which was increased in venom-injected rats. Overall, these results suggest that melatonin mitigates oxidative/nitrosative stress in venom-induced cardio-hepato-renal injury in rats. Our results suggest that melatonin treatment may ameliorate some of the effects of N. haje envenomation.     

Melatonin prevents increased asymmetric dimethylarginine in young rats with bile duct ligation

Abstract: Identifying and treating kidney injury in cirrhosis is important. Bile duct ligation (BDL) is a commonly used cholestatic liver disease model. We hypothesized that asymmetric dimethylarginine (ADMA) is involved in BDL‐induced oxidative stress and kidney injury, which can be prevented by melatonin. We also intended to elucidate whether increased ADMA is due to increased protein arginine methyltransferase‐1 (PRMT1, ADMA‐synthesizing enzyme) and/or decreased dimethylarginine dimethylaminohydrolase (DDAH, ADMA‐metabolizing enzyme). Three groups of young rats were studied, sham (N = 7), untreated BDL rats (N = 9), and melatonin‐treated BDL rats (N = 6, BDL + M). Melatonin‐treated BDL rats received daily melatonin 1 mg/kg/day via intraperitoneal injection. One‐third of the young BDL rats died compared with none in the BDL + M group. All surviving rats were killed 14 days after surgery. BDL rats had higher plasma aspartate aminotransferase, alanine aminotransferase, direct and total bilirubin, and ammonia levels than shams. They also had kidney injury characterized by increased tubulointerstitial injury scores and plasma creatinine and symmetric dimethylarginine levels, which melatonin prevented. Plasma ADMA levels were elevated in BDL rats, combined with increased hepatic PRMT1 and decreased renal DDAH activity. In addition, melatonin increased hepatic DDAH2 expression, increased DDAH activity and concomitantly decreased ADMA contents in both the liver and kidney. In conclusion, melatonin therapy decreased mortality and prevented kidney injury induced by BDL via reduction of ADMA (by increasing DDAH activity) and oxidative stress.     

Glutathione‐dependent induction of local and systemic defense against oxidative stress by exogenous melatonin in cucumber (Cucumis sativus L.)

Melatonin is involved in defending against oxidative stress caused by various environmental stresses in plants. In this study, the roles of exogenous melatonin in regulating local and systemic defense against photooxidative stress in cucumber (Cucumis sativus) and the involvement of redox signaling were examined. Foliar or rhizospheric treatment with melatonin enhanced tolerance to photooxidative stress in both melatonin‐treated leaves and untreated systemic leaves. Increased melatonin levels are capable of increasing glutathione (reduced glutathione [GSH]) redox status. Application of H₂O₂ and GSH also induced tolerance to photooxidative stress, while inhibition of H₂O₂ accumulation and GSH synthesis compromised melatonin‐induced local and systemic tolerance to photooxidative stress. H₂O₂ treatment increased the GSH/oxidized glutathione (GSSG) ratio, while inhibition of H₂O₂ accumulation prevented a melatonin‐induced increase in the GSH/GSSG ratio. Additionally, inhibition of GSH synthesis blocked H₂O₂‐induced photooxidative stress tolerance, whereas scavenging or inhibition of H₂O₂ production attenuated but did not abolish GSH‐induced tolerance to photooxidative stress. These results strongly suggest that exogenous melatonin is capable of inducing both local and systemic defense against photooxidative stress and melatonin‐enhanced GSH/GSSG ratio in a H₂O₂‐dependent manner is critical in the induction of tolerance.     

Melatonin inhibits nuclear factor kappa B activation and oxidative stress and protects against thioacetamide induced liver damage in rats

BACKGROUND/AIMS: Free radical-mediated oxidative stress has been implicated in the pathogenesis of acute liver injury. The aim of our study was to investigate whether melatonin, a potent free radical scavenger could prevent fulminant hepatic failure in rats. METHODS: Liver damage was induced by two consecutive injections of thioacetamide (TAA, 300 mg/kg/i.p.) at 24 h intervals. Treatment with melatonin (3 mg/kg/daily, i.p) was initiated 24 h prior to TAA. RESULTS: Twenty-four h after the second TAA injection, serum liver enzymes and blood ammonia were lower in rats treated with TAA+melatonin compared to TAA (P<0.001). Liver histology was significantly improved and the mortality in the melatonin-treated rats was decreased (P<0.001). The increased nuclear binding of nuclear factor kappa B in the livers of the TAA-treated rats, was inhibited by melatonin. The hepatic levels of thiobarbituric acid reactive substances, protein carbonyls and inducible nitric oxide synthase were lower in the TAA+melatonin-treated group (P<0.01), indicating decreased oxidative stress and inflammation. CONCLUSIONS: In a rat model of TAA-induced fulminant hepatic failure, melatonin improves survival and reduces liver damage and oxidative stress. The results suggest a causative role of oxidative stress in TAA-induced hepatic damage and suggest that melatonin may be utilized to reduce liver injury associated with oxidative stress.     

ELISA confirmation of acute and past envenoming by the monocellate Thai cobra (Naja kaouthia)

The monocellate Thai cobra (Naja kaouthia) is a major cause of snake bite mortality and morbidity throughout Thailand, but neither the local nor the systemic effects of its venom are diagnostic. Species diagnosis is important because only monospecific antivenoms are available for treatment in Thailand. We tested the ability of the ELISA technique to detect venom antigen in the sera of 58 acute snake bite cases including 4 fatalities, and venom antibody in 51 patients bitten between 1 month and 19 years previously. N. kaouthia venom antigen was found in 8 of 33 patients with only local envenoming and in 14 of 20 with local plus systemic (neurotoxic) envenoming, but the mean venom concentration was 33 times greater in the latter group. The serum of 1 fatal case contained banded krait (Bungarus fasciatus) but no cobra venom antigen. N. kaouthia venom antibody was present in sera of patients bitten between 1 month and 7 years previously. Antibody was found in 6 of 8 patients who had had local envenoming alone but in only 19 of 41 who had had systemic envenoming treated by antivenom. The titer of antibody declined with an approximate half time of 2-3 years. One patient had a significant titer of B. fasciatus venom antibody. This study confirms the value of ELISA-immunodiagnosis and the predominance of N. kaouthia as a cause of neurotoxic envenoming in the Bang Phli area. However, the attribution of 1 fatal case to B. fasciatus bite suggests that patients with neurotoxic signs should be given B. fasciatus antivenom if they fail to respond to cobra antivenom.     

Haemostatic disturbances in patients bitten by Russell''s viper (Vipera russelli siamensis) in Burma

Patients who are severely envenomed by Russell's viper develop DIC which is frequently associated with spontaneous bleeding and incoagulable blood. These haemostatic disturbances may be responsible for death or organ/tissue damage both through haemorrhage and microvascular occlusion by fibrin thrombi. The most striking laboratory features of the coagulopathy developing after Russell's viper bite in the 42 patients studied were depletion of fibrinogen (mean 0.09 g/l, range 0-0.6), factor V (6.5 u/dl, range 0-17), factor X (35 u/dl, range 1-85), factor XIIIa (57 u/dl, range 15-82), plasminogen (61 u/dl, range 10-92), antiplasmin (36 u/dl, range 14-62). Protein C (49 u/dl, range 15-100) and platelets (104 x 10(9)/l, range 25-197). Intense fibrinolytic activity was detected in all cases with marked elevation of FDPs (1614 micrograms/ml, range 350-3000), a large proportion of which were cross-linked (1058 micrograms/ml, range 38-3000). The monospecific Burmese antivenom appeared to be very effective in neutralizing the venom procoagulants and in restoring blood coagulability. Moreover, the unexpectedly normal level of AT III provides a theoretical basis for the use of heparin to enhance the inactivation of those serine proteases present before antivenom administration.     

Melatonin enhances the occurrence of autophagy induced by oxidative stress in Arabidopsis seedlings

The beneficial effect that melatonin has against mitochondrial dysfunctioning seems to be linked to mitophagy. Roles for melatonin have been demonstrated in promoting health and preventing disease, as well as activating the process of autophagy in general. However, no reports have been made about how the application of melatonin regulates that process when plants are exposed to oxidative stress. We investigated the influence of different concentrations of melatonin (0.0, 0.5, 5.0, 10.0, or 50.0 μm ) on autophagy under methyl viologen (MV)‐induced oxidative stress. Arabidopsis seedlings that were pretreated with 5 or 10 μm melatonin underwent relatively strong induction of autophagy, as evidenced by the number of monodansylcadaverine (MDC)‐stained autophagosomes in root samples. Pretreatment with 10 μm melatonin also alleviated MV‐induced photo‐oxidation damage and significantly reduced the accumulation of oxidized proteins. Those responses might have been due to the strong upregulation of genes that involved in AtATG8‐PE conjugation pathway, which enhanced the capacity for autophagy. Histochemical staining revealed that both and H₂O₂ were highly accumulated upon MV exposure, although the response did not differ significantly between control and melatonin‐pretreated seedlings. By contrast, exogenous melatonin upregulated the expression of two genes for H₂O₂‐scavenging enzymes, that is, AtAPX1 and AtCATs. The activation of autophagy by melatonin without an alteration in ROS production may be part of a survival mechanism that is enhanced by melatonin after cellular damage. Therefore, it represents a second level of defense to remove damaged proteins when antioxidant activities are compromised.     

Melatonin alleviates cadmium‐induced liver injury by inhibiting the TXNIP‐NLRP3 inflammasome

Cadmium (Cd) is a persistent environmental and occupational contaminant that accumulates in the liver and induces oxidative stress and inflammation. Melatonin possesses potent hepatoprotective properties against the development and progression of acute and chronic liver injury. Nevertheless, the molecular mechanism underlying the protective effects of melatonin against Cd‐induced hepatotoxicity remains obscure. In this study, we aimed to investigate the effects of melatonin on Cd‐induced liver inflammation and hepatocyte death. Male C57BL/6 mice were intraperitoneally injected with melatonin (10 mg/kg) once a day for 3 days before exposure to CdCl₂ (2.0 mg/kg). We found that Cd induced hepatocellular damage and inflammatory infiltration as well as increased serum ALT/AST enzymes. In addition, we showed that Cd triggered an inflammatory cell death, which is mediated by the NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome. Moreover, melatonin treatment significantly alleviated Cd‐induced liver injury by decreasing serum ALT/AST levels, suppressing pro‐inflammatory cytokine production, inhibiting NLRP3 inflammasome activation, ameliorating oxidative stress, and attenuating hepatocyte death. Most importantly, melatonin markedly abrogated Cd‐induced TXNIP overexpression and decreased the interaction between TXNIP and NLRP3 in vivo and in vitro. However, treatment with siRNA targeting TXNIP blocked the protective effects of melatonin in Cd‐treated primary hepatocytes. Collectively, our results suggest that melatonin confers protection against Cd‐induced liver inflammation and hepatocyte death via inhibition of the TXNIP‐NLRP3 inflammasome pathway.     

Immunological properties of Hypnale hypnale (hump-nosed pit viper) venom: antibody production with diagnostic and therapeutic potentials

Envenomation by hump-nosed pit viper (Hypnale hypnale, Hh) in Sri Lanka has caused significant morbidity and mortality, attributed to 35% of total venomous snakebites. In Southwestern India (Kerala), H. hypnale was increasingly identified as a dangerous and common source of envenomation, second to the Russell's viper but ahead of the cobra bites. Unfortunately, there is still no specific antivenom to date. This study aims to investigate the immunological properties of the venom and to assess the feasibility of specific Hh antivenom production as well as the development of a diagnostic assay. Hh venom elicited satisfactory titers of anti-Hh IgG in rabbits after 3rd immunization. The anti-Hh IgG, isolated with caprylic acid precipitation method, was effective in neutralizing the venom lethality (potency=48 LD(50) per ml IgG) as well as its procoagulant, hemorrhagic and necrotic effects, indicating the possibility to produce the specific antivenom using the common immunization regime. Cross-reactivity studies using indirect ELISA showed that anti-Hh IgG cross-reacted extensively with several Asiatic crotalid venoms, particularly that of Calloselasma rhodostoma (73.6%), presumably due to the presence of venom antigens common to both snakes. Levels of immunological cross-reactivity were vastly reduced with double-sandwich ELISA. Further work demonstrated that the assay was able to distinguish and quantify venoms of H. hypnale, Daboia russelii and Echis carinatus sinhaleyus (three common local viperid) used to spike human sera at various concentrations. The assay hence may be a useful investigating tool for diagnosing biting species and studying the time course profile of venom concentrations in blood.     

Melatonin reduces cardiac remodeling and improves survival in rats with isoproterenol‐induced heart failure

Melatonin was previously shown to reduce blood pressure and left ventricular (LV) remodeling in several models of experimental heart damage. This study investigated whether melatonin prevents LV remodeling and improves survival in isoproterenol‐induced heart failure. In the first experiment, four groups of 3‐month‐old male Wistar rats (12 per group) were treated for 2 wk as follows: controls, rats treated with melatonin (10 mg/kg/day) (M), rats treated with isoproterenol (5 mg/kg/day intraperitoneally the second week) (Iso), and rats treated with melatonin (2 wk) and isoproterenol (the second week) in corresponding doses (IsoM). In the second experiment, 30 rats were treated with isoproterenol and 30 rats with isoproterenol plus melatonin for a period of 28 days and their mortality was investigated. Isoproterenol‐induced heart failure with hypertrophy of the left and right ventricles (LV, RV), lowered systolic blood pressure (SBP) and elevated pulmonary congestion. Fibrotic rebuilding was accompanied by alterations of tubulin level in the LV and oxidative stress development. Melatonin failed to reduce the weight of the LV or RV; however, it curtailed the weight of the lungs and attenuated the decline in SBP. Moreover, melatonin decreased the level of oxidative stress and of insoluble and total collagen and partly prevented the beta‐tubulin alteration in the LV. Most importantly, melatonin reduced mortality and prolonged the average survival time. In conclusion, melatonin exerts cardioprotective effects and improves outcome in a model of isoproterenol‐induced heart damage. The antiremodeling effect of melatonin may be of potential benefit in patients with heart failure.     

Melatonin prevents experimental liver cirrhosis induced by thioacetamide in rats

Abstract:  Liver cirrhosis is a critical stage of chronic liver diseases that can produce liver failure, portal hypertension and hepatocarcinoma. Sustained oxidative stress plays a key role in cell damage and fibrosis induced during liver cirrhosis. We evaluated the effect of oxidative stress regulation by melatonin on the development of parenchymal destruction and stellate cell activation in experimental liver cirrhosis. Melatonin was administered to rats with liver cirrhosis induced by thioacetamide (TAA) for 1 or 3 months. Liver injury was assessed by serological analysis, as well as hematoxylin-eosin staining and the in situ apoptosis detection assay in liver sections. Oxidative stress was evaluated by lipoperoxide and reduced glutathione levels, and by the measurement of catalase and superoxide dismutase activities in liver and serum respectively. The activation of stellate cells was evaluated by α -smooth muscle actin expression in liver sections. Our results showed that TAA induced oxidative stress with extensive tissue damage and enhanced α -smooth muscle actin expression in liver. Melatonin prevented the oxidative stress-related changes associated with TAA toxicity. In conclusion, the study showed that melatonin prevents the tissue damage and fibrosis associated with TAA-induced liver cirrhosis in rats.     

Melatonin promotes seed germination under high salinity by regulating antioxidant systems,ABA and GA4 interaction in cucumber (Cucumis sativus L.)

Although previous studies have found that melatonin can promote seed germination, the mechanisms involved in perceiving and signaling melatonin remain poorly understood. In this study, it was found that melatonin was synthesized during cucumber seed germination with a peak in melatonin levels occurring 14 hr into germination. This is indicative of a correlation between melatonin synthesis and seed germination. Meanwhile, seeds pretreated with exogenous melatonin (1 μm ) showed enhanced germination rates under 150 mm NaCl stress compared to water‐pretreated seeds under salinity stress. There are two apparent mechanisms by which melatonin alleviated salinity‐induced inhibition of seed germination. Exogenous melatonin decreased oxidative damage induced by NaCl stress by enhancing gene expression of antioxidants. Under NaCl stress, compared to untreated control, the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly increased by approximately 1.3–5.0‐fold, with a concomitant 1.4–2.0‐fold increase of CsCu‐ZnSOD, CsFe‐ZnSOD, CsCAT, and CsPOD in melatonin‐pretreated seeds. Melatonin also alleviated salinity stress by affecting abscisic acid (ABA) and gibberellin acid (GA) biosynthesis and catabolism during seed germination. Compared to NaCl treatment, melatonin significantly up‐regulated ABA catabolism genes (e.g., CsCYP707A1 and CsCYP707A2, 3.5 and 105‐fold higher than NaCl treatment at 16 hr, respectively) and down‐regulated ABA biosynthesis genes (e.g., CsNECD2, 0.29‐fold of CK2 at 16 hr), resulting in a rapid decrease of ABA content during the early stage of germination. At the same time, melatonin positively up‐regulated GA biosynthesis genes (e.g., GA20ox and GA3ox, 2.3 and 3.9‐fold higher than NaCl treatment at 0 and 12 hr, respectively), contributing to a significant increase of GA (especially GA₄) content. In this study, we provide new evidence suggesting that melatonin alleviates the inhibitory effects of NaCl stress on germination mainly by regulating the biosynthesis and catabolism of ABA and GA₄.     

Mechanisms of isoproterenol‐induced cardiac mitochondrial damage: protective actions of melatonin

Mitochondrial dysfunction due to oxidative damage is the key feature of several diseases. We have earlier reported mitochondrial damage resulting from the generation of oxidative stress as a major pathophysiological effect of isoproterenol (ISO)‐induced myocardial ischemia in rats. That melatonin is an antioxidant that ameliorates oxidative stress in experimental animals as well as in humans is well established. We previously demonstrated that melatonin provides cardioprotection against ISO‐induced myocardial injury as a result of its antioxidant properties. The mechanism of ISO‐induced cardiac mitochondrial damage and protection by melatonin, however, remains to be elucidated in vitro. In this study, we provide evidence that ISO causes dysfunction of isolated goat heart mitochondria. Incubation of cardiac mitochondria with increasing concentrations of ISO decreased mitochondrial succinate dehydrogenase (SDH) activity, which plays a pivotal role in mitochondrial bioenergetics, as well as altered the activities of other key enzymes of the Kreb's cycle and the respiratory chain. Co‐incubation of ISO‐challenged mitochondria with melatonin prevented the alterations in enzyme activity. That these changes in mitochondrial energy metabolism were due to the perpetration of oxidative stress by ISO was evident from the increased levels of lipid peroxidation and decreased reduced glutathione/oxidized glutathione ratio. ISO‐induced oxidative stress also altered mitochondrial redox potential and brought about changes in the activity of the antioxidant enzymes manganese superoxide dismutase and glutathione peroxidase, eventually leading to alterations in total ATPase activity and membrane potential. Melatonin ameliorated these changes likely through its antioxidant abilities suggesting a possible mechanism of cardioprotection by this indole against ISO‐induced myocardial injury.     

Coagulopathy after snake bite byBothrops neuwiedi: Case report and results of in vitro experiments

Summary Coagulation studies were performed in a patient who had been bitten by a snake of the speciesBothrops neuwiedi. The patient presented with hemorrhagic necrosis at the envenomization site and considerable bleeding from venous puncture sites. He developed a severe defibrination syndrome with a clottable fibrinogen level of approximately 0.1 g/l. Fibrinogen was not measurable by clotting time assay. Fibrin degradation products were greatly elevated. Treatment with antivenom caused an anaphylactic reaction within ten minutes and serum sickness after three days. In vitro experiments revealed thatB. neuwiedi venom directly activates Factors II and X, but does not activate Factor XIII. In vivo consumption of Factor XIII afterB. neuwiedi envenomization is ascribed to the action of Factor IIa. At low venom concentrations clotting is initiated by activation of prothrombin by the venom either directly or via Factor X activation. Treatment with heparin might be beneficial in coagulopathy secondary to snake bite by reducing circulating active thrombin. The venom contains thrombinlike proteases which cause slow clotting of fibrinogen, and plasmin-like components causing further proteolysis of fibrinogen and fibrin. Antivenom has no effect on the proteolytic action of the snake venom. The in vivo effects of antivenom are presumably caused by acceleration of the elimination of venom components from the circulation. Intravenous administration of antivenom caused normalization of blood coagulation parameters within 48 h.     

Melatonin and its metabolites as copper chelating agents and their role in inhibiting oxidative stress: a physicochemical analysis

The copper sequestering ability of melatonin and its metabolites cyclic 3‐hydroxymelatonin (3OHM), N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK), and N¹‐acetyl‐5‐methoxykynuramine (AMK) was investigated within the frame of the Density Functional Theory. It was demonstrated that these compounds are capable of chelating copper ions, yielding stable complexes. The most likely chelation sites were identified. Two different mechanisms were modeled, the direct‐chelation mechanism (DCM) and the coupled‐deprotonation‐chelation mechanism (CDCM). It is proposed that, under physiological conditions, CDCM would be the main chelation route for Cu(II). It was found that melatonin and its metabolites fully inhibited the oxidative stress induced by Cu(II)‐ascorbate mixtures, via Cu(II) chelation. In the same way, melatonin, AFMK, and 3OHM also prevented the first step of the Haber–Weiss reaction, consequently turning off the ˙OH production via the Fenton reaction. Therefore, it is proposed that, in addition to the previously reported free radical scavenging cascade, melatonin is also involved in a concurrent ‘chelating cascade’, thereby contributing to a reduction in oxidative stress. 3OHM was identified as the most efficient of the studied compounds for that purpose, supporting the important role of this metabolite in the beneficial effects of melatonin against oxidative stress.     

Effects of melatonin on thymic and oxidative stress dysfunctions during Trypanosoma cruzi infection

Although the exact etiology of Chagas disease is not completely elucidated, thymic atrophy and oxidative stress are believed to be important contributors to the pathogenesis during acute Trypanosoma cruzi (T. cruzi ) infection. We hypothesized that exogenous melatonin, administered by gavage (5 mg/kg, p.o., gavage) to young (5 weeks old) and middle‐aged (18 months old) male Wistar rats, would modulate thymic oxidative damage and reverse the age‐related thymus regression during T. cruzi acute infection. Increased levels of superoxide anion (O₂^?) were detected in the thymus of infected animals, and treatment with melatonin reverted this response. We found reduced TBARS levels as well as a significant increase in superoxide dismutase (SOD ) activity in the thymus of all middle‐aged melatonin‐treated animals, infected or not with T. cruzi . Furthermore, melatonin increased the thymic expression of SOD 1 and SOD 2 in middle‐aged control animals. Nox2 expression was not affected by melatonin treatment in young or middle‐aged animals. Melatonin reverted the age‐related thymic regression as revealed by the increase in thymus weight, total number of thymocytes, and reduction in age‐related accumulation of double‐negative thymocytes. This is the first report to directly examine the effects of melatonin treatment on the thymic antioxidant/oxidant status and thymic changes during T. cruzi infection. Our results revealed new antioxidant features that turn melatonin a potentially useful compound for the treatment of Chagas disease, a condition in which an excessive oxidative damage occurs.