Influência da variabilidade geográfica sobre o potencial neurotóxico e reatividade cruzada com antiveneno comercial de venenos de subespécies de Crotalus durissus (Viperidae: Crotalinae) distribuídas no Brasil

Abstract

Envenomation by Crotalus snakes are known for causing significant neurotoxic and myotoxic effects, primarily attributed to crotoxin, a phospholipase A2 (PLA2) toxin that constitutes 70–90% of these venoms. Recent studies have demonstrated that geographic variability affects the PLA2 compositional profile of C. d. terrificus and C. d. collilineatus venoms, revealing a greater complexity of isoforms in specimens from the central-west and southeastern regions of Brazil. In this study, we assessed how geographic variability affects the neuromuscular activity of venoms from Crotalus durissus cascavella (CDV), C. d. collilineatus (CDC), C. d. ruruima (CDR), and C. d. terrificus (CDT), including the cross-reactivity of these venoms with commercial anti-Crotalus antivenom (AV) and PLA2 inhibition by varespladib (VPL). PLA2 activity was measured through spectrophotometry using 3-octanoyloxy benzoic acid as an artificial substrate. The mouse phrenic nerve-diaphragm preparation was set up in an Ugo Basile SRL myographic system under a tension of 1 g. Twitches were induced through supramaximal stimuli (0.1 Hz, 0.2 ms, ~6 V) and recorded with a DY1 force displacement transducer connected to a DataCapsule-Evo and LabScribe4 software. Results are expressed as mean ± SD (n=3–4). The CDR venom exhibited the highest PLA2 activity (75.5±2 ΔA425nm/min, n=3), while the CDV venom demonstrated greater sensitivity to VPL (0.3 mM), resulting in a ~97% inhibition of its PLA2 activity (p<0.05 compared to venom alone, n=3). CDC venom (10–100 μg/ml) induced significant neuromuscular facilitation, followed by a complete blockade within a 120 min incubation period. In contrast, CDV venom (10–100 μg/ml) exhibited greater potency at the neuromuscular junction, fully blocking twitch responses at all concentrations within 80–110 min. CDR venom resulted in a complete blockade only at the highest concentration of 100 μg/ml, which occurred within approximately 110 minutes. AV (antivenom:venom ratio 1:1.5 v/w) demonstrated partial efficacy against the venoms of both CDT and CDV (30 g/ml), avoiding the occurrence of 90% blockade for each. However, it exhibited limited cross-reactivity when tested against CDC and CDR (30 g/ml) venoms. In contrast, VPL at a concentration of 0.3 mM effectively inhibited the 90% blockade induced by both CDT and CDV (30 µg/ml) venoms, as well as avoiding the 50% blockade for CDC (30 µg/ml). The immunoreactivity with antivenom revealed the major enzymatic groups in these venoms, with C. d. cascavella and C. d. ruruima venoms being positive for crotamina. In conclusion, the venoms of these subspecies exhibit significant variability in their neuromuscular effects observed. Inhibition of PLA2 activity by VPL resulted in a marked reduction of the neuromuscular blockade induced by these venoms. Furthermore, VPL demonstrated greater efficacy than AV in mitigating the neuromuscular actions associated with CDC, CDT, and CDV venoms.