Search

The lethality neutralization assay in mice is the gold standard for the evaluation of the preclinical efficacy and specification fulfillment of snake antivenoms. However, owing to the animal suffering involved, this assay is a candidate to be replaced by in vitro alternatives or, at least, improved by the reduction of the number of animals used per experiment, the introduction of analgesia, and the refinement of the test. Since these tests are usually run for 24 or 48 h, one possibility to refine it is to shorten the endpoint observation time of the assay and so limiting the duration of suffering. To assess the effect of this modification of the standard procedure on the analytical properties of the assay, we compared the median lethal dose (LD50) and median effective dose (ED50) values, estimated through observation times of 6, 24 and 48 h. We used African and Latin American snake venoms and several batches of two polyspecific antivenoms. A significant correlation was found between LD50 and ED50 values estimated at the three observation times. Although some LD50 and ED50 values were significantly different at these time points, results of 6 h were robust enough to be used in the characterization of new antivenoms, the verification of specification compliance, and the parallel comparison of formulations. Our observations support the modification of the standard procedures used for assessing neutralizing ability of antivenoms by carrying out the observations at 6 h instead of 24 or 48 h, with the consequent reduction in the suffering inflicted upon mice during these assays. However, the shortening of the observation time in the lethality tests must be validated for each venom and antivenom before its introduction in the routine procedures., Graphical abstract Image 1, Highlights • The lethality neutralization assay in mice is the gold standard for the preclinical evaluation of snake antivenoms. • This assay is a candidate to be improved by the 3Rs philosophy (i.e., replacement, reduction and refinement). • The effect of the reduction of the endpoint observation time on the analytical properties of the test was assessed. • A significant correlation was found between LD50 and ED50 values estimated at 6 h and 24 or 48 h. • A 6-h observation time limits the duration of the suffering without reducing the analytical quality of the test.

Lethality and cytotoxicity assays of snake venoms and their neutralization by antivenom require many mice for the experiments. Recent developments have prompted researchers to seek alternative strategies that minimize the use of mice in line with Russel and Burch's 3Rs philosophy (Replacement, Reduction, and Refinement). Artemia salina is an animal model widely used for toxicity screening. However, its use in snake venom toxinology is limited by a lack of data. The present study compared the toxicity of venoms from Bitis arietans, Naja ashei, and Naja subfulva using mice and Artemia salina. In the Artemia salina test at 24 h and the dermonecrotic test in mice, the toxicity of the venoms was in the order Naja ashei ~ Naja subfulva > Bitis arietans. In the lethality test in mice, the toxicity of the venoms was in the order Naja subfulva > Naja ashei > Bitis arietans. These findings suggest that the toxicity of the venoms in Artemia salina and the dermonecrotic bioassay in mice have a similar trend but differ from the lethality test in mice. Therefore, it may be relevant to further explore the Artemia salina bioassay as a potential surrogate test of dermonecrosis in mice. Studies with more venoms may be needed to establish the correlation between the Artemia salina bioassay and the dermonecrotic assay in mice., Graphical abstract Image 1, Highlights • Bitis arietans, Naja ashei, and Naja subfulva snake venom-induced toxicities were compared in Artemia salina and in mice. • Venom-induced toxicity in Artemia salina has a similar trend to necrosis in mice but not lethality. • The Artemia salina bioassay may be further explored along the 3Rs (Reduction, Replacement, and Refinement) concept.

Snake venom may vary in composition and toxicity across the geographic distribution of a species. In the case of the three species of the Neotropical rattlesnakes Crotalus simus, C. culminatus and C. tzabcan recent research has revealed that their venoms can contain a neurotoxic component (crotoxin homologs), but is not always the case. In the present work, we detected and quantified crotoxin homologs in venom samples from three species distributed across Mexico, to describe variation at the individual and subspecific levels, using slot blot and ELISA immunoassays. We found that all C. simus individuals analyzed had substantial percentages of crotoxin homologs in their venoms (7.6–44.3%). In contrast, C. culminatus lacked them completely and six of ten individuals of the species C. tzabcan had low percentages (3.0–7.7%). We also found a direct relationship between the lethality of a venom and the percentage of crotoxin homologs it contained, indicating that the quantity of this component influences venom lethality in the rattlesnake C. simus., Highlights • Monoclonal antibodies were produced that specifically recognized crotoxin homologs in venoms of Crotalus species. • Crotoxin homologs were quantified in three species of Crotalus: C. simus, C. culminatus and C. tzabcan. • All specimens of C. simus contained crotoxin homologs at different levels, while C. culminatus venoms lacked them completely. • In C. tzabcan, some venoms possess and other lack crotoxin homologs.

Several snake species possess, in their circulating blood, endogenous PLA2 inhibitors (sbPLIs) with the primary function of natural protection against toxic enzymes from homologous and heterologous venoms. Among the three structural classes of sbPLIs – named α, β, and γ − the β class (sbβPLIs) is the least known with only four identified sequences, so far. The last class of inhibitors encompass molecules with leucine rich repeats (LRRs) motifs containing repeating amino acid segments. In the present study, we identified and characterized putative sbβPLIs from the liver and venom glands of six Latin American pit vipers belonging to Bothrops and Crotalus genera. The inhibitor from Crotalus durissus terrificus snakes (CdtsbβPLI) was chosen as a reference for the construction of the first in silico structural model for this class of inhibitors, using molecular modeling and molecular dynamics simulations. Detailed analyses of the electrostatic surface of the CdtsbβPLI model and protein-protein docking with crotoxin B from homologous venoms predict the interacting surface between these proteins., Highlights • Transcripts of phospholipases A2 inhibitors from the β-class (sbβPLIs) were identified in Latin American pit vipers. • Structural features of sbβPLIs were compared and discussed, including their characteristic leucine-rich repeats (LRRs). • One sbβPLI (CdtsbβPLI) was chosen for the in silico construction of the first structural model of a sbβPLI. • A possible mechanism of interaction between sbβPLIs and basic snake venom PLA2s was suggested. • Docking predictions between CdtsbβPLI and crotoxin B highlighted the amino acids residues at the interaction surfaces.