The role of oxidative stress in red blood cell responses to Bothrops atrox venom

Bothrops atrox envenoming causes significant hematotoxicity, yet the contribution of oxidative stress in red blood cells (RBCs) damage remains incompletely defined. We investigated the oxidative mechanisms of B. atrox venom on RBCs using complementary in vitro and in vivo model, with attention to sex-specific responses. For this, Human RBCs were incubated with 0.1-1 μg venom for 1-24h. Hemolysis, methemoglobin, oxidative stress markers and antioxidant status were quantified. For in vivo analysis mice received intravenous dose of venom (½ LD₅₀) and plasma and RBCs oxidative parameters were measured after 24h. In vitro, venom induced dose- and time-dependent hemolysis reaching 708 % and 647 % over controls in RBCs from male and female donors, after 24h at 1 μg. Similarly, methemoglobin increased by 925 % (male) and 687 % (female). Oxidant parameters significantly increased and antioxidants decreased in RBCs from both sexes. Microscopy revealed echinocytes and spherocytes after venom exposure. In vivo, envenoming increased ROS in (+157.6 % in males, +89.2 % in females), NO (+84.8 %; +82.4 %), and ONOO^- (+93.1 %; +135.5 %) in RBCs compared to controls. Similar changes were also observed in mice plasma with marked decrease in antioxidant capacity mainly in males. In conclusion, B. atrox venom causes severe RBCs damage, with oxidative stress serving as one of the key contributing factors. The resulting changes lead to hemolysis and loss of membrane function. In additions, our results show that RBC oxidative damages are from endogenous and exogenous origin. Greater susceptibility in males point to sex-specific redox vulnerability. These findings support exploring antioxidant-based adjunctive therapies in B. atrox envenomings.