{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T20:00:58Z","timestamp":1790280058195,"version":"4.1.0"},"reference-count":34,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T00:00:00Z","timestamp":1771804800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100010325","name":"Secretar\u00eda General de Ciencia y T\u00e9cnica, Universidad Nacional del Nordeste, Argentina","doi-asserted-by":"publisher","award":["PI 21F014"],"award-info":[{"award-number":["PI 21F014"]}],"id":[{"id":"10.13039\/501100010325","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Argentina","award":["ACT210097"],"award-info":[{"award-number":["ACT210097"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Phospholipases A2 (PLA2s) are key mediators of the cytotoxic and inflammatory activities of snake venoms. While PLA2 isoforms from Bothrops diporus venom have been characterized and shown to possess antimetastatic and antiangiogenic properties, their impact on mitochondrial bioenergetics and immune modulation has not yet been investigated. In this study, we examined the bioenergetic and immunomodulatory effects of B. diporus PLA2s using integrated biochemical, metabolic, and multiplex cytokine analyses. In MDA-MB-231 breast cancer cells, pooled PLA2s induced a dose-dependent decrease in MTT-reducing activity, increased mitochondrial ROS, caused \u0394\u03c8m hyperpolarization, and decreased NADH autofluorescence, collectively indicating sustained mitochondrial stress. Real-time impedance measurements further revealed a marked inhibition of cell proliferation. In human PBMCs, pooled PLA2s elicited a dynamic cytokine program, inducing early cytotoxic (Granzyme B) and chemotactic (CCL2, CCL3, CCL4) mediators, followed by late pro-inflammatory and regulatory factors such as IL-6, TNF-\u03b2, IL-10 and IL-15. Analysis of a single purified PLA2 isoform (Fraction 6) confirmed activation of the canonical IL-6\/TNF-\u03b1\/IL-1\u03b2 axis but uniquely induced IL-10 and CCL20, revealing isoform-specific immunomodulatory properties. Altogether, these findings provide the first integrated characterization of mitochondrial and immune perturbations induced by B. diporus PLA2s, expanding their recognized biological scope and underscoring their potential as molecular templates for novel pharmacological strategies targeting mitochondrial vulnerabilities or modulating the tumor immune microenvironment.<\/jats:p>","DOI":"10.3390\/toxins18020114","type":"journal-article","created":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T08:58:21Z","timestamp":1771837101000},"page":"114","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["New Insights into the Bioenergetic and Immunomodulatory Properties of Phospholipases A2 from Bothrops diporus Venom"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-2495-868X","authenticated-orcid":false,"given":"Daniela J.","family":"Sasovsky","sequence":"first","affiliation":[{"name":"Grupo de Investigaciones Biol\u00f3gicas y Moleculares (GIByM), Instituto de Qu\u00edmica B\u00e1sica y Aplicada del Nordeste Argentino (IQUIBA NEA), Universidad Nacional del Nordeste, Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Corrientes W3400, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6837-5739","authenticated-orcid":false,"given":"Ana K.","family":"Oliveira","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA 22903, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dilza Trevisan","family":"Silva","sequence":"additional","affiliation":[{"name":"School of Health Sciences, Universidade Positivo, Curitiba 81280-330, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7916-6721","authenticated-orcid":false,"given":"Gonzalo A.","family":"Ojeda","sequence":"additional","affiliation":[{"name":"Laboratorio de Productos Naturales, Instituto de Qu\u00edmica B\u00e1sica y Aplicada del Nordeste Argentino (IQUIBA NEA), Universidad Nacional del Nordeste, Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Corrientes W3400, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-8861-806X","authenticated-orcid":false,"given":"Cristopher","family":"Almarza","sequence":"additional","affiliation":[{"name":"Network for Snake Venom Research and Drug Discovery, Santiago 8320000, Chile"},{"name":"Laboratorio de Plasticidad Metab\u00f3lica y Bioenerg\u00e9tica, Programa de Farmacolog\u00eda Molecular, N\u00facleo Interdisciplinario de Farmacolog\u00eda e Inmunolog\u00eda, Instituto de Ciencias Biom\u00e9dicas, Facultad de Medicina, Universidad de Chile, Santiago 8320000, Chile"},{"name":"Interuniversity Center for Healthy Aging (CIES), Consortium of Universities of the State of Chile (CUECH), Santiago 8320000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2419-6469","authenticated-orcid":false,"given":"Bruno","family":"Lomonte","sequence":"additional","affiliation":[{"name":"Instituto Clodomiro Picado, Facultad de Microbiolog\u00eda, Universidad de Costa Rica, San Jos\u00e9 11501, Costa Rica"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jay W.","family":"Fox","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA 22903, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"F\u00e9lix A.","family":"Urra","sequence":"additional","affiliation":[{"name":"Network for Snake Venom Research and Drug Discovery, Santiago 8320000, Chile"},{"name":"Laboratorio de Plasticidad Metab\u00f3lica y Bioenerg\u00e9tica, Programa de Farmacolog\u00eda Molecular, N\u00facleo Interdisciplinario de Farmacolog\u00eda e Inmunolog\u00eda, Instituto de Ciencias Biom\u00e9dicas, Facultad de Medicina, Universidad de Chile, Santiago 8320000, Chile"},{"name":"Interuniversity Center for Healthy Aging (CIES), Consortium of Universities of the State of Chile (CUECH), Santiago 8320000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1875-7664","authenticated-orcid":false,"given":"Soledad","family":"Bustillo","sequence":"additional","affiliation":[{"name":"Grupo de Investigaciones Biol\u00f3gicas y Moleculares (GIByM), Instituto de Qu\u00edmica B\u00e1sica y Aplicada del Nordeste Argentino (IQUIBA NEA), Universidad Nacional del Nordeste, Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Corrientes W3400, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"13057","DOI":"10.1016\/S0021-9258(17)36794-7","article-title":"Diversity of group types, regulation, and function of phospholipase A2","volume":"269","author":"Dennis","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.biochi.2012.09.007","article-title":"Emerging roles of secreted phospholipase A2 enzymes: An update","volume":"95","author":"Murakami","year":"2013","journal-title":"Biochimie"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.toxicon.2007.08.007","article-title":"Systemic and local myotoxicity induced by snake venom group II phospholipases A2: Comparison between crotoxin, crotoxin B and a Lys49 PLA2 homologue","volume":"51","author":"Gutierrez","year":"2008","journal-title":"Toxicon"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1016\/j.toxicon.2003.11.002","article-title":"Excitement ahead: Structure, function and mechanism of snake venom phospholipase A2 enzymes","volume":"42","author":"Kini","year":"2003","journal-title":"Toxicon"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"203639","DOI":"10.1155\/2014\/203639","article-title":"Antitumoral activity of snake venom proteins: New trends in cancer therapy","volume":"2014","author":"Calderon","year":"2014","journal-title":"Biomed. Res. Int."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1000181","DOI":"10.4172\/2161-0495.1000181","article-title":"Snake venom cytotoxins, phospholipase A2s, and metalloproteinases: Mechanisms of action and biomedical applications","volume":"4","author":"Gasanov","year":"2014","journal-title":"J. Clin. Toxicol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1042\/BST20200110","article-title":"Cytotoxicity of snake venom enzymatic toxins: Phospholipase A2 and L-amino acid oxidase","volume":"48","author":"Hiu","year":"2020","journal-title":"Biochem. Soc. Trans."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1042\/BJ20050890","article-title":"The basal proton conductance of mitochondria depends on adenine nucleotide translocase content","volume":"392","author":"Brand","year":"2005","journal-title":"Biochem. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1038\/nrd2803","article-title":"Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?","volume":"8","author":"Trachootham","year":"2009","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4745","DOI":"10.7314\/APJCP.2014.15.12.4745","article-title":"Roles of oxidative stress in the development and progression of breast cancer","volume":"15","author":"Nourazarian","year":"2014","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.canlet.2016.03.042","article-title":"Reactive oxygen species (ROS) and cancer: Role of antioxidative nutraceuticals","volume":"387","author":"Prasad","year":"2017","journal-title":"Cancer Lett."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1016\/j.arr.2012.10.004","article-title":"Oxidative stress and cancer: An overview","volume":"12","author":"Sosa","year":"2013","journal-title":"Ageing Res. Rev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2570","DOI":"10.1002\/jcp.25349","article-title":"Mitochondria and mitochondrial ROS in cancer: Novel targets for anticancer therapy","volume":"231","author":"Yang","year":"2016","journal-title":"J. Cell. Physiol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Resiere, D., Mehdaoui, H., and Neviere, R. (2022). Inflammation and oxidative stress in snakebite envenomation: A brief descriptive review and clinical implications. Toxins, 14.","DOI":"10.3390\/toxins14110802"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Urra, F.A., Vivas-Ruiz, D.E., Sanchez, E.F., and Araya-Maturana, R. (2022). An emergent role for mitochondrial bioenergetics in the action of snake venom toxins on cancer cells. Front. Oncol., 12.","DOI":"10.3389\/fonc.2022.938749"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Zambelli, V.O., Picolo, G., Fernandes, C.A.H., Fontes, M.R.M., and Cury, Y. (2017). Secreted phospholipases A2 from animal venoms in pain and analgesia. Toxins, 9.","DOI":"10.3390\/toxins9120406"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/s40409-018-0170-y","article-title":"Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom","volume":"24","author":"Cedro","year":"2018","journal-title":"J. Venom. Anim. Toxins Incl. Trop. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.toxicon.2019.07.004","article-title":"Isolation of two basic phospholipases A2 from Bothrops diporus snake venom: Comparative characterization and synergism between Asp49 and Lys49 variants","volume":"168","author":"Bustillo","year":"2019","journal-title":"Toxicon"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"111217","DOI":"10.1016\/j.cbi.2024.111217","article-title":"Comparative in vitro and in silico analysis of the ability of basic Asp49 phospholipase A2 and Lys49-phospholipase A2-like myotoxins from Bothrops diporus venom to inhibit the metastatic potential of murine mammary tumor cells and endothelial cell tubulogenesis: Asp49 vs Lys49 phospholipases A2: Inhibition of metastasis and angiogenesis","volume":"402","author":"Sasovsky","year":"2024","journal-title":"Chem.-Biol. Interact."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1007\/s00441-025-04008-7","article-title":"Exploring the antiangiogenic effects of phospholipases A2 from Bothrops diporus venom","volume":"402","author":"Sasovsky","year":"2025","journal-title":"Cell Tissue Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"40485","DOI":"10.1074\/jbc.M607704200","article-title":"Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells","volume":"281","author":"Criddle","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1016\/j.toxicon.2003.11.006","article-title":"Inflammatory effects of snake venom myotoxic phospholipases A2","volume":"42","author":"Teixeira","year":"2003","journal-title":"Toxicon"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.toxicon.2012.09.006","article-title":"Phospholipases A2: Unveiling the secrets of a functionally versatile group of snake venom toxins","volume":"62","author":"Gutierrez","year":"2013","journal-title":"Toxicon"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Mackessy, S.P. (2021). Snake venom phospholipase A2 toxins. Handbook of Venoms and Toxins of Reptiles, CRC Press.","DOI":"10.1201\/9780429054204"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1006\/abbi.1993.1311","article-title":"Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): Subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction","volume":"303","author":"Berridge","year":"1993","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"990","DOI":"10.1016\/j.ijbiomac.2022.03.111","article-title":"Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49-PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae)","volume":"206","author":"Proleon","year":"2022","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1152\/physrev.2000.80.1.315","article-title":"Mitochondria and neuronal survival","volume":"80","author":"Nicholls","year":"2000","journal-title":"Physiol. Rev."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1016\/j.toxicon.2008.08.015","article-title":"Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions","volume":"52","author":"Amora","year":"2008","journal-title":"Toxicon"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1186\/s40409-018-0180-9","article-title":"Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells","volume":"24","author":"Benati","year":"2018","journal-title":"J. Venom. Anim. Toxins Incl. Trop. Dis."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/0014-4800(91)90002-F","article-title":"Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu","volume":"55","author":"Gutierrez","year":"1991","journal-title":"Exp. Mol. Pathol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1016\/j.cbpc.2011.06.003","article-title":"Isolation and functional characterization of proinflammatory acidic phospholipase A2 from Bothrops leucurus snake venom","volume":"154","author":"Nunes","year":"2011","journal-title":"Comp. Biochem. Physiol. C Toxicol. Pharmacol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1016\/j.toxicon.2005.06.017","article-title":"Activation of cellular functions in macrophages by venom secretory Asp49 and Lys49 phospholipases A2","volume":"46","author":"Zuliani","year":"2005","journal-title":"Toxicon"},{"key":"ref_33","first-page":"361","article-title":"MitoSOX-based flow cytometry for detecting mitochondrial ROS","volume":"2","author":"Kauffman","year":"2016","journal-title":"React. Oxyg. Species (Apex)"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/S0006-3495(99)77214-0","article-title":"Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives","volume":"76","author":"Scaduto","year":"1999","journal-title":"Biophys. J."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/18\/2\/114\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T09:10:27Z","timestamp":1771837827000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/18\/2\/114"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,23]]},"references-count":34,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["toxins18020114"],"URL":"https:\/\/doi.org\/10.3390\/toxins18020114","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,23]]}}}