Cytoprotective effects of creosote bush (Larrea tridentata) and Southern live oak (Quercus virginiana) extracts against toxicity induced by venom of the black-tailed rattlesnake (Crotalus ornatus).

The venom of Crotalus ornatus (vCo) poses a threat to human health, as it contains a mixture of toxins that can cause cytotoxic, necrotic, and hemolytic effects. The present study assessed methanolic and acetone extracts from leaves and flowers of Larrea tridentata, as well as the bark of Quercus virginiana as potential suppressors of the toxic effects of vCo in vitro. The content of total phenols, flavonoids, and tannins of the plant extracts were quantified for the suppression of vCo cytotoxicity in two cell culture models, human lymphocytes and porcine aortic endothelial (PAE) cells. Extracts from Q. virginiana displayed a greater concentration of total phenols, flavonoids, and tannins. Co-incubation of lymphocytes and PAE cells with fixed concentrations of vCo and plant extracts resulted in decreased vCo-induced cytotoxicity. A 24-hour co-incubation of lymphocytes with vCo (2.36 ± 0.17 µg/mL) and 0.5 µg/mL of methanolic leaf extract from L. tridentata (LLM) significantly suppressed the venom-induced cytotoxicity by 37.33 ± 8.33%. Similarly, the LLM extract (4 µg/mL) caused a significant decrease in vCo cytotoxicity after 24 hours in PAE cells. In contrast, while the acetone extract of Q. virginiana bark (QA) suppressed cytotoxicity by 29.20 ± 3.51% (p < 0.001) in lymphocytes, it failed to protect PAE cells against vCo after 24 hours. In PAE cells, a shorter 4-hour co-incubation showed significant suppression of cytotoxicity with both extracts. Our results collectively suggest that LLM and QA possess cytoprotective properties against the in vitro toxic effects of vCo, and thus establish extracts from these plants as potential therapeutic interventions against Crotalus envenomation.

Functional Mining of the Crotalus Spp. Venom Protease Repertoire Reveals Potential for Chronic Wound Therapeutics.

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

Chlorpromazine and dimethyl sulfoxide modulate the catalytic activity of the plasma membrane Ca2+-ATPase from human erythrocyte.

The plasma membrane Ca2+-ATPase (PMCA) removes Ca2+ from the cytosol into the extracellular space. Its catalytic activity can be stimulated by calmodulin (CaM) or by limited proteolysis. We evaluated the effect of chlorpromazine (CPZ) and dimethyl sulfoxide (DMSO) over the hydrolytic activity of PMCA. Activity was monitored in three different forms: native, CaM-activated and proteolyzed by trypsin. CPZ appears to inhibit PMCA without directly interfering with the C-terminal site, since it is affected by CaM and proteolysis. Although the treatment of PMCA with trypsin and CaM produces an activation, it also produces an enzymatic form that is more sensitive to inhibition by CPZ. The same case was observed in the DMSO inhibition experiments. In the absence of CPZ, DMSO produces a progressive loss of activity, but in the presence of CPZ the profile of activity against DMSO changes and produces a recovery of activity, indicating a possible partition of CPZ by the solvent. Increasing Ca2+ concentrations indicated that CPZ interacts with PMCA rather than with CaM. This observation is supported by docking analysis that suggests that the CPZ-PMCA interaction is non-competitive. We propose that CPZ interacts with the state of lower affinity for Ca2 +.

Development of aqueous two-phase systems-based approaches for the selective recovery of metalloproteases and phospholipases A2 toxins from Crotalus molossus nigrescens venom.

Snake venoms are rich sources of proteins with potential biotechnological and pharmaceutical applications. Among them, metalloproteases (MPs) and phospholipases A2 (PLA2) are the most abundant. Their isolation involves a multistep chromatographic approach, which has proven to be effective, however implies high operating costs and long processing times. In this study, a cost-effective and simple method based on aqueous two-phase systems (ATPS) was developed to recover MPs and PLA2 from Crotalus molossus nigrescens venom. A system with PEG 400 g mol-1, volume ratio (VR) 1, tie line length (TLL) 25% w/w and pH 7 showed the best performance for PLA2 recovery. In systems with PEG 400 g mol-1, VR 1, TLL 15% w/w, pH 7 and 1 and 3% w/w of NaCl, selective recovery of MP subtype P-III was achieved; whereas, in a system with PEG 400 g mol-1, VR 1, TLL 25% w/w and pH 8.5, MP subtypes P-I and P-III were recovered. Due to their low costs, ethanol-salt systems were also evaluated, however, failed to differentially partition PLA2 and MPs. The use of ATPS could contribute to the simplification and cost reduction of protein isolation processes from snake venoms and other toxin fluids, as well as potentially aid their biochemical, proteomic and biological analyses.

Practical context of enzymatic treatment for wound healing: A secreted protease approach (Review).

Skin wounds have been extensively studied as their healing represents a critical step towards achieving homeostasis following a traumatic event. Dependent on the severity of the damage, wounds are categorized as either acute or chronic. To date, chronic wounds have the highest economic impact as long term increases wound care costs. Chronic wounds affect 6.5 million patients in the United States with an annual estimated expense of $25 billion for the health care system. Among wound treatment categories, active wound care represents the fastest-growing category due to its specific actions and lower costs. Within this category, proteases from various sources have been used as successful agents in debridement wound care. The wound healing process is predominantly mediated by matrix metalloproteinases (MMPs) that, when dysregulated, result in defective wound healing. Therapeutic activity has been described for animal secretions including fish epithelial mucus, maggot secretory products and snake venom, which contain secreted proteases (SPs). No further alternatives for use, sources or types of proteases used for wound healing have been found in the literature to date. Through the present review, the context of enzymatic wound care alternatives will be discussed. In addition, substrate homology of SPs and human MMPs will be compared and contrasted. The purpose of these discussions is to identify and propose the stages of wound healing in which SPs may be used as therapeutic agents to improve the wound healing process.

Acanthocytosis and brain damage in area postrema and choroid plexus: Description of novel signs of Loxosceles apachea envenomation in rats.

Loxocelism is a neglected medical problem that depends on its severity, can cause a cutaneous or viscero-cutaneous syndrome. This syndrome is characterized by hemostatic effects and necrosis, and the severity of the loxoscelism depends on the amount of venom injected, the zone of inoculation, and the species. In the Chihuahuan desert, the most abundant species is L. apachea. Its venom and biological effects are understudied, including neurological effects. Thus, our aim is to explore the effect of this regional species of medical interest in the United States-Mexico border community, using rat blood and central nervous system (CNS), particularly, two brain structures involved in brain homeostasis, Area postrema (AP) and Choroid plexus (PC). L. apachea specimens were collected and venom was obtained. Different venom concentrations (0, 0.178 and 0.87 µg/g) were inoculated into Sprague-Dawley rats (intraperitoneal injection). Subsequently, blood was extracted and stained with Wright staining; coronal sections of AP were obtained and stained with Hematoxylin-Eosin (HE) staining and laminin γ immunolabelling, the same was done with CP sections. Blood, AP and CP were observed under the microscope and abnormalities in erythrocytes and fluctuation in leukocyte types were described and quantified in blood. Capillaries were also quantified in AP and damage was described in CP. L. apachea venom produced a segmented neutrophil increment (neutrophilia), lymphocyte diminishment (leukopenia) and erythrocytes presented membrane abnormalities (acanthocytosis). Extravasated erythrocytes were observed in HE stained sections from both, AP and CP, which suggest that near to this section a hemorrhage is present; through immunohistofluorescence, a diminishment of laminin γ was observed in AP endothelial cells and in CP ependymal cells when these structures were exposed to L. apachea venom. In conclusion, L. apachea venom produced leukopenia, netrophilia and acanthocytosis in rat peripheral blood, and also generated hemorrhages on AP and CP through degradation of laminin γ.

Rattlesnake Crotalus molossus nigrescens venom induces oxidative stress on human erythrocytes.

BACKGROUND: Globally, snake envenomation is a well-known cause of death and morbidity. In many cases of snakebite, myonecrosis, dermonecrosis, hemorrhage and neurotoxicity are present. Some of these symptoms may be provoked by the envenomation itself, but others are secondary effects of the produced oxidative stress that enhances the damage produced by the venom toxins. The only oxidative stress effect known in blood is the change in oxidation number of Fe (from ferrous to ferric) in hemoglobin, generating methemoglobin but not in other macromolecules. Currently, the effects of the overproduction of methemoglobin derived from snake venom are not extensively recorded. Therefore, the present study aims to describe the oxidative stress induced by Crotalus molossus nigrescens venom using erythrocytes. METHODS: Human erythrocytes were washed and incubated with different Crotalus molossus nigrescens venom concentrations (0-640 µg/mL). After 24 h, the hemolytic activity was measured followed by attenuated total reflectance-Fourier transform infrared spectroscopy, non-denaturing PAGE, conjugated diene and thiobarbituric acid reactive substances determination. RESULTS: Low concentrations of venom (<10 µg/mL) generates oxyhemoglobin release by hemolysis, whereas higher concentrations produced a hemoglobin shift of valence, producing methemoglobin (>40 µg/mL). This substance is not degraded by proteases present in the venom. By infrared spectroscopy, starting in 80 µg/mL, we observed changes in bands that are associated with protein damage (1660 and 1540 cm-1) and lipid peroxidation (2960, 2920 and 1740 cm-1). Lipid peroxidation was confirmed by conjugated diene and thiobarbituric acid reactive substance determination, in which differences were observed between the control and erythrocytes treated with venom. CONCLUSIONS: Crotalus molossus nigrescens venom provokes hemolysis and oxidative stress, which induces methemoglobin formation, loss of protein structure and lipid peroxidation.

In vitro hemotoxic, α-neurotoxic and vasculotoxic effects of the Mexican black-tailed rattlesnake (Crotalus molossus nigrescens) venom.

The Mexican black-tailed rattlesnake Crotalus molossus nigrescens is distributed in the Mexican plateau. Its venom is known to cause hemolysis and presents fibrinogen coagulase, collagenase and fibrinolytic activities. These activities may be associated with hemostatic alterations, such as platelet aggregation, hemolysis and fibrinolysis, often described in ophidic accidents. However, the mechanisms of action of the C. m. nigrescens venom remain unclear. In this study we investigated the in vitro hemotoxic, neurotoxic, and vasculotoxic effects of the venom. We found that this venom produces two types of hemolytic responses, Oxyhemoglobin release and Methemoglobin formation. As a result of the cytotoxicity to endothelial cells produces morphological biphasic toxicity. The first step in this process is characterized by morphological changes, as well as the loss of cellular adhesion and reduction in thickness. The second phase is characterized by massive cellular aggregation and death. It also induced laminin, type IV collagen, perlecan and nidogen degradation. However, the venom did not modulate the muscular fetal and neuronal nicotinic acetylcholine receptors activity. Thus, we concluded that the C. m. nigrescens venom produced hemolysis and hemorrhages via degradation of the basement membrane components and endothelial cell cytotoxicity, but not by neurotoxicity at the receptor level in nicotinic acetylcholine receptors.

Capillary damage in the area postrema by venom of the northern black-tailed rattlesnake (Crotalus molossus molossus).

The Northern black-tailed rattlesnake (Crotalus molossus molossus) venom is mainly hemotoxic, hemorrhagic, and neurotoxic. Its effects in the central nervous system are unknown and only poorly described for all Viperidae species in general. This is why we are interested in describe the damage induced by C. m. molossus venom in rat brain, particularly in the area postrema capillaries. Four C. m. molossus venom doses were tested (0.02, 0.05, 0.10 and 0.20mg/kg) injected intramuscularly at the lower limb, incubated by 24 hours and the brains were harvested. Area postrema coronal sections were stained with Haematoxylin and Eosin, and examined to observe the venom effect in quantity of capillaries and porphology. Starting from the 0.10mg/kg treatment we observed lysed extravasated erythrocytes and also capillary breakdown, as a consequence of hemorrhages appearance. The number of capillaries decreased significantly in response to the venom dose increment. Hemorrhages could be caused by the metalloproteinase activity on the basal membrane and the apoptosis generated by L-amino acid oxidases. Hemolysis could be caused by phospholipase A2 hemotoxic effect. We conclude that C. m. molossus crude venom produces hemolysis, capillary breakdown, hemorrhages, and the reduction in number of capillaries in the area postrema.

Rattlesnake Crotalus molossus nigrescens venom induces oxidative stress on human erythrocytes

Abstract Background Globally, snake envenomation is a well-known cause of death and morbidity. In many cases of snakebite, myonecrosis, dermonecrosis, hemorrhage and neurotoxicity are present. Some of these symptoms may be provoked by the envenomation itself, but others are secondary effects of the produced oxidative stress that enhances the damage produced by the venom toxins. The only oxidative stress effect known in blood is the change in oxidation number of Fe (from ferrous to ferric) in hemoglobin, generating methemoglobin but not in other macromolecules. Currently, the effects of the overproduction of methemoglobin derived from snake venom are not extensively recorded. Therefore, the present study aims to describe the oxidative stress induced by Crotalus molossus nigrescens venom using erythrocytes. Methods Human erythrocytes were washed and incubated with different Crotalus molossus nigrescens venom concentrations (0640 g/mL). After 24 h, the hemolytic activity was measured followed by attenuated total reflectance-Fourier transform infrared spectroscopy, non-denaturing PAGE, conjugated diene and thiobarbituric acid reactive substances determination. Results Low concentrations of venom ( 10 g/mL) generates oxyhemoglobin release by hemolysis, whereas higher concentrations produced a hemoglobin shift of valence, producing methemoglobin (>40 g/mL). This substance is not degraded by proteases present in the venom. By infrared spectroscopy, starting in 80 g/mL, we observed changes in bands that are associated with protein damage (1660 and 1540 cm1) and lipid peroxidation (2960, 2920 and 1740 cm1). Lipid peroxidation was confirmed by conjugated diene and thiobarbituric acid reactive substance determination, in which differences were observed between the control and erythrocytes treated with venom. Conclusions Crotalus molossus nigrescens venom provokes hemolysis and oxidative stress, which induces methemoglobin formation, loss of protein structure and lipid peroxidation.

Rattlesnake Crotalus molossus nigrescens venom induces oxidative stress on human erythrocytes

Background Globally, snake envenomation is a well-known cause of death and morbidity. In many cases of snakebite, myonecrosis, dermonecrosis, hemorrhage and neurotoxicity are present. Some of these symptoms may be provoked by the envenomation itself, but others are secondary effects of the produced oxidative stress that enhances the damage produced by the venom toxins. The only oxidative stress effect known in blood is the change in oxidation number of Fe (from ferrous to ferric) in hemoglobin, generating methemoglobin but not in other macromolecules. Currently, the effects of the overproduction of methemoglobin derived from snake venom are not extensively recorded. Therefore, the present study aims to describe the oxidative stress induced by Crotalus molossus nigrescens venom using erythrocytes. Methods Human erythrocytes were washed and incubated with different Crotalus molossus nigrescens venom concentrations (0-640 μg/mL). After 24 h, the hemolytic activity was measured followed by attenuated total reflectance-Fourier transform infrared spectroscopy, non-denaturing PAGE, conjugated diene and thiobarbituric acid reactive substances determination. Results Low concentrations of venom (<10 μg/mL) generates oxyhemoglobin release by hemolysis, whereas higher concentrations produced a hemoglobin shift of valence, producing methemoglobin (>40 μg/mL). This substance is not degraded by proteases present in the venom. By infrared spectroscopy, starting in 80 μg/mL, we observed changes in bands that are associated with protein damage (1660 and 1540 cm−1) and lipid peroxidation (2960, 2920 and 1740 cm−1). Lipid peroxidation was confirmed by conjugated diene and thiobarbituric acid reactive substance determination, in which differences were observed between the control and erythrocytes treated with venom. Conclusions Crotalus molossus nigrescens venom provokes hemolysis and oxidative stress, which induces methemoglobin formation, loss of protein structure and lipid peroxidation.(AU)