Cytotoxic Effects Caused by Functionalized Carbon Nanotube in Murine Macrophages

Background/Aims: The development of new nanomaterials has been growing in recent decades to bring benefits in several areas, especially carbon-based nanoparticles, which have unique physical-chemical properties and allow to take on several applications. Consequently, the use of new nanomaterials without previous toxicological studies raises concern about possible harmful health effects. The aim of this study was to investigate the cytotoxic profile of a new multi-walled carbon nanotube (MWCNT) functionalized with tetraethylenepentamine called OCNT-TEPA using in vitro assays in murine macrophage cells linage J774 A.1. Methods: OCNT-TEPA was characterized by transmission electron microscopy (TEM) and high resolution TEM (HR-TEM), scanning electron microscopy (SEM), zeta potential and dynamic light scattering (DLS), and its cytotoxic effects were evaluated at 24 and 48 hours by cell viability assays (MTT and NR), morphology and cell recovery (optic microscopy and clonogenic assay), formation of reactive oxygen (ROS) and nitric oxide (NO) species, inflammatory profile (IL-6 and TNF cytokines), mitochondrial membrane potential analysis (MMP), activation of the caspase 3 pathway and cell death (flow cytometry). Results: The data showed a significant decrease in cell viability, increased production of ROS and NO, alteration of mitochondrial membrane potential, increased levels of inflammatory cytokines, alteration of cell morphology, activation of the Caspase 3 pathway and consequently cell death, in the highest concentrations of OCNT-TEPA tested in the periods of 24 and 48 hours. Conclusion: The analyses showed that OCNT-TEPA has a dose-dependent cytotoxic profile, which may be harmful to murine macrophages (J774 A.1) and may represent a health risk

Carbon Black CB-EDA Nanoparticles in Macrophages: Changes in the Oxidative Stress Pathway and in Apoptosis Signaling

The influence of black carbon nanoparticles on J774.A1 murine cells was investigated with the objective of exploring the cytotoxicity of black carbon functionalized with ethylenediamine CB-EDA. The results showed that CB-EDA has a cytotoxic profile for J774.A1 macrophages in a time- and dose-dependent manner. When phagocytosed by the macrophage, CB-EDA triggers a mechanism that leads to apoptosis. In this process, there is an increase in oxidative stress pathways due to the activation of nitric oxide and then ROS. This causes an imbalance in redox function and a disruption of membrane integrity that occurs due to high levels of LDH, in addition to favoring the release of the pro-inflammatory cytokines IL-6, IL-12, and tumor necrosis factor (TNF) in an attempt to modulate the cell. However, these stimuli are not sufficient to repair the cell and the level of mitochondrial integrity is affected, causing a decrease in cell viability. This mechanism may be correlated with the activation of the caspasse-3 pathway, which, when compromised, cleaves and induces cells death via apoptosis, either through early or late apoptosis. In view of this, the potential for cell damage was investigated by analyzing the oxidative and inflammatory profile in the macrophage lineage J774.A1 and identifying potential mechanisms and metabolic pathways connected to these processes when cells were exposed to NP CB-EDA for both 24 h and 48 h.The authors would like to thank Márcia Regina Cominetti (Departamento de Gerontologia, Universidade Federal de São Carlos, São Carlos, SP, Brazil) for availability of equipment. M.A. was supported by the Margarita Salas postdoctoral contract MGS/2021/21 (UP2021-021) financed by the European Union—Next Generation EU. The authors would also like to express their gratitude for the financial aid from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2013/07296-2), the Financiadora de Estudos e Projetos—FINEP, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES (Financial Code 001) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq

HGPRT and PNP: Recombinant Enzymes from <i>Schistosoma mansoni</i> and Their Role in Immunotherapy during Experimental Murine Schistosomiasis

Schistosomiasis is a parasitic infection caused by trematode worms (also called blood flukes) of the genus Schistosoma sp., which affects over 230 million people worldwide, causing 200,000 deaths annually. There is no vaccine or new drugs available, which represents a worrying aspect, since there is loss of sensitivity of the parasite to the medication recommended by the World Health Organization, Praziquantel. The present study evaluated the effects of the recombinant enzymes of S. mansoni Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT), Purine Nucleoside Phosphorylase (PNP) and the MIX of both enzymes in the immunotherapy of schistosomiasis in murine model. These enzymes are part of the purine salvage pathway, the only metabolic pathway present in the parasite for this purpose, being essential for the synthesis of DNA and RNA. Female mice of Swiss and BALB/c strains were infected with cercariae and treated, intraperitoneally, with three doses of 100 µg of enzymes. After the immunotherapy, the eggs and adult worms were counted in the feces; the number of eosinophils from the fluid in the peritoneal cavity and peripheral blood was observed; and the quantification of the cytokine IL-4 and the production of antibodies IgE was analyzed. The evaluation of the number of granulomas and collagen deposition via histological slides of the liver was performed. The results demonstrate that immunotherapy with the enzyme HGPRT seems to stimulate the production of IL-4 and promoted a significant reduction of granulomas in the liver in treated animals. The treatment with the enzyme PNP and the MIX was able to reduce the number of worms in the liver and in the mesenteric vessels of the intestine, to reduce the number of eggs in the feces and to negatively modulate the number of eosinophils. Therefore, immunotherapy with the recombinant enzymes of S. mansoni HGPRT and PNP might contribute to the control and reduction of the pathophysiological aspects of schistosomiasis, helping to decrease the morbidity associated with the infection in murine model

Bactericidal activity of Ag4V2O7/β-AgVO3 heterostructures against antibiotic-resistant Klebsiella pneumoniae

Although Ag-based materials are efficient against antibiotic-resistant bacteria, their high toxicity to living organisms represents a major challenge for obtaining useful products. In this work, we report the bactericidal activity of Ag4V2O7/β-AgVO3 heterostructures, which proved to be effective against Klebsiella pneumoniae (ATCC 1706, a standard strain; A54970, a multidrug-resistant carbapenemase (KPC)-producing strain; A34057, a multidrug-resistant strain capable of producing extended spectrum beta-lactamases (ESBL); and a community-isolated strain, A58240) at minimum inhibitory concentrations (MIC) as low as 62.5 μg/mL. This activity is higher than that reported for the individual silver vanadates (Ag4V2O7 or β-AgVO3) owing to the synergistic interactions between both semiconductors. However, the most efficient heterostructure was found to be toxic to mouse 3 T3 fibroblasts and to L. sativa and C. sativus seeds, as indicated by MTT ((4,5 - dimethylthiazol -2yl) 2,5 -diphenylbromide), neutral red assays and germination index measurements. The antimicrobial, phytotoxic and cytotoxic activities were all associated with an efficient generation of reactive oxygen species (ROS) in the heterostructure, especially radical dotOH and radical dotO2− radicals. The ROS production by Ag4V2O7/β-AgVO3 heterostructures was measured through photodegradation studies with Rhodamine B. While the bactericidal activity of the heterostructures is promising, especially when compared to Ag-based materials, their use in practical applications will require encapsulation either to avoid leaching or to mitigate their toxicity to humans, animals and plants.J.A. acknowledges the Universitat Jaume I (project UJI-B2019-30) and the Ministerio de Ciencia, Innovación y Universidades (Spain) (project PGC2018094417-B-I00) for financially supporting this research. M.A. was supported by the Margarita Salas postdoctoral contract MGS/2021/21 (UP2021-021) financed by the European Union-NextGenerationEU. F.F.A. and J.M.A.R. were sponsored by PETROBRAS (Project No. 2017/00010-7). The authors would also like to thank the financial aid from the Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (2013/07296-2, 2016/13423-5, 2018/22214-6), the Financiadora de Estudos e Projetos – FINEP, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Financial Code 001) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Grant no.141964/2018-9)