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Macrophages count on two O 2 -consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O 2 • - ) and nitric oxide ( • NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O 2 • - and • NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO 2 ) normally ranges within 2-14 %, with a typical average pO 2 value for most tissues ca. 5 %. Given that O 2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O 2 • - and • NO production. Also, O 2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O 2 • - and • NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO 2 ; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O 2 ) which are far from the physiological pO 2 . Here, the role of O 2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO 2 and O 2 • - and • NO formation rates were measured, obtaining a K M of 26 and 42 μM O 2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO 2 , reaching a maximum at ≥ 10 % O 2 , but even at levels as low as 2 % O 2 , a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO 2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked by Bothrops snakes. The present study aimed to assess whether Bothrops jararacussu and Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways., Methods: Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t(½) values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model., Results: At the highest concentration tested (120 μg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 μg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 μg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum., Conclusion: Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system by B. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.