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Microcystin-LR (MC-LR) is a potent cyanotoxin that can reach several organs. However subacute exposure to sublethal doses of MC-LR has not yet well been studied. Herein, we evaluated the outcomes of subacute and sublethal MC-LR exposure on lungs. Male BALB/c mice were exposed to MC-LR by gavage (30 µg/kg) for 20 consecutive days, whereas CTRL mice received filtered water. Respiratory mechanics was not altered in MC-LR group, but histopathology disclosed increased collagen deposition, immunological cell infiltration, and higher percentage of collapsed alveoli. Mitochondrial function was extensively affected in MC-LR animals. Additionally, a direct in vitro titration of MC-LR revealed impaired mitochondrial function. In conclusion, MC-LR presented an intense deleterious effect on lung mitochondrial function and histology. Furthermore, MC-LR seems to exert an oligomycin-like effect in lung mitochondria. This study opens new perspectives for the understanding of the putative pulmonary initial mechanisms of damage resulting from oral MC-LR intoxication.

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AIM: We aimed to evaluate whether the streptozotocin-induced diabetic model can generate lung functional, histological and biochemical impairments and whether moderate exercise can prevent these changes. METHODS: Wistar rats were assigned to control (CTRL), exercise (EXE), diabetic (D) and diabetic with exercise (D+EXE) groups. We used the n5-STZ model of diabetes mellitus triggered by a single injection of streptozotocin (STZ, 120 mg/kg b.w., i.p.) in newborn rats on their 5th day of life. EXE and D+EXE rats were trained by running on a motorized treadmill, 5 days a week for 9 weeks. Blood glucose, body weight, food intake, exercise capacity, lung mechanics, morphology, and antioxidant enzymatic activity were analysed. RESULTS: On the 14th week of life, diabetic rats exhibited a significant impairment in post-prandial glycaemia, glucose tolerance, body weight, food intake, lung function (tissue viscance, elastance, Newtonian resistance and hysteresis), morphological parameters, redox balance and exercise capacity. Physical training completely prevented the diabetes-induced alterations, except for those on fasting blood glucose, which nevertheless remained stable. CONCLUSIONS: Mild diabetes in n5-STZ-treated rats jeopardized pulmonary mechanics, morphology and redox balance, which confirms the occurrence of diabetes-induced pneumopathy. Moreover, moderate exercise completely prevented all diabetes-induced respiratory alterations.

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Direct analysis of isolated mitochondria enables a better understanding of lung dysfunction. Despite well-defined mitochondrial isolation protocols applicable to other tissues, such as the brain, kidney, heart, and liver, a robust and reproductive protocol has not yet been advanced for the lung. We describe a protocol for the isolation of mitochondria from lung tissue aiming for functional analyses of mitochondrial O2 consumption, transmembrane potential, reactive oxygen species (ROS) formation, ATP production, and swelling. We compared our protocol to that used for heart mitochondrial function that is well-established in the literature, and achieved similar results.

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Mechanical ventilation (MV) is essential for the treatment of critical patients since it may provide a desired gas exchange. However, MV itself can trigger ventilator-associated lung injury in patients. We hypothesized that the mechanisms of lung injury through redox imbalance might also be associated with pulmonary inflammatory status, which has not been so far described. We tested it by delivering different tidal volumes to normal lungs undergoing MV. Healthy Wistar rats were divided into spontaneously breathing animals (control group, CG), and rats were submitted to MV (controlled ventilation mode) with tidal volumes of 4 mL/kg (MVG4), 8 mL/kg (MVG8), or 12 mL/kg (MVG12), zero end-expiratory pressure (ZEEP), and normoxia (FiO2 = 21%) for 1 hour. After ventilation and euthanasia, arterial blood, bronchoalveolar lavage fluid (BALF), and lungs were collected for subsequent analysis. MVG12 presented lower PaCO2 and bicarbonate content in the arterial blood than CG, MVG4, and MVG8. Neutrophil influx in BALF and MPO activity in lung tissue homogenate were significantly higher in MVG12 than in CG. The levels of CCL5, TNF-α, IL-1, and IL-6 in lung tissue homogenate were higher in MVG12 than in CG and MVG4. In the lung parenchyma, the lipid peroxidation was more important in MVG12 than in CG, MVG4, and MVG8, while there was more protein oxidation in MVG12 than in CG and MVG4. The stereological analysis confirmed the histological pulmonary changes in MVG12. The association of controlled mode ventilation and high tidal volume, without PEEP and normoxia, impaired pulmonary histoarchitecture and triggered redox imbalance and lung inflammation in healthy adult rats.

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C60 fullerene (C60) is a nano-pollutant that can damage the respiratory system. Eugenol exhibits significant anti-inflammatory and antioxidant properties. We aimed to investigate the time course of C60 emulsion-induced pulmonary and spermatic harms, as well as the effect of eugenol on C60 emulsion toxicity. The first group of mice (protocol 1) received intratracheally C60 emulsion (1.0 mg/kg BW) or vehicle and were tested at 12, 24, 72 and 96 h (F groups) thereafter. The second group of mice (protocol 2) received intratracheally C60 emulsion or vehicle, 1 h later were gavaged with eugenol (150 mg/kg) or vehicle, and experiments were done 24 h after instillation. Lung mechanics, morphology, redox markers, cytokines and epididymal spermatozoa were analyzed. Protocol 1: Tissue damping (G) and elastance (H) were significantly higher in F24 than in others groups, except for H in F72. Morphological and inflammatory parameters were worst at 24 h and subsequently declined until 96 h, whereas redox and spermatic parameters worsened over the whole period. Eugenol eliminated the increase in G, H, cellularity, and cytokines, attenuated oxidative stress induced by C60 exposure, but had no effect on sperm. Hence, exposure to C60 emulsion deteriorated lung morphofunctional, redox and inflammatory characteristics and increased the risk of infertility. Furthermore, eugenol avoided those changes, but did not prevent sperm damage.

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C60 fullerene (C60) nanoparticles, a nanomaterial widely used in technology, can offer risks to humans, overcome biological barriers, and deposit onto the lungs. However, data on its putative pulmonary burden are scanty. Recently, the C60 interaction with mitochondria has been described in vitro and in vivo. We hypothesized that C60 impairs lung mechanics and mitochondrial function. Thirty-five male BALB/c mice were randomly divided into two groups intratracheally instilled with vehicle (0.9% NaCl + 1% Tween 80, CTRL) or C60 (1.0 mg/kg, FUL). Twenty-four hours after exposure, 15 FUL and 8 CTRL mice were anesthetized, paralyzed, and mechanically ventilated for the determination of lung mechanics. After euthanasia, the lungs were removed en bloc at end-expiration for histological processing. Lung tissue elastance and viscance were augmented in FUL group. Increased inflammatory cell number, alveolar collapse, septal thickening, and pulmonary edema were detected. In other six FUL and six CTRL mice, mitochondria expressed reduction in state 1 respiration [FUL = 3.0 ± 1.14 vs. CTRL = 4.46 ± 0.9 (SEM) nmol O2/min/mg protein, p = 0.0210], ATP production (FUL = 122.6 ± 18 vs. CTRL = 154.5 ± 14 µmol/100 µg protein, p = 0.0340), and higher oxygen consumption in state 4 [FUL = 12.56 ± 0.9 vs. CTRL = 8.26 ± 0.6], generation of reactive oxygen species (FUL 733.1 ± 169.32 vs. CTRL = 486.39 ± 73.1 nmol/100 µg protein, p = 0.0313) and reason ROS/ATP [FUL = 8.73 ± 2.3 vs. CTRL = 2.99 ± 0.3]. In conclusion, exposure to fullerene C60 impaired pulmonary mechanics and mitochondrial function, increased ROS concentration, and decrease ATP production.

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Silicosis is an occupational lung disease caused by inhalation of silica particles. It is characterized by intense lung inflammation, with progressive and irreversible fibrosis, leading to impaired lung function. Purinergic signaling modulates silica-induced lung inflammation and fibrosis through P2X7 receptor. In the present study, we investigate the role of P2Y12, the G-protein-coupled subfamily prototype of P2 receptor class in silicosis. To that end, BALB/c mice received an intratracheal injection of PBS or silica particles (20 mg), without or with P2Y12 receptor blockade by clopidogrel (20 mg/kg body weight by gavage every 48 h) - groups CTRL, SIL, and SIL + Clopi, respectively. After 14 days, lung mechanics were determined by the end-inflation occlusion method. Lung histology was analyzed, and lung parenchyma production of nitric oxide and cytokines (IL-1ß, IL-6, TNF-α, and TGF-ß) were determined. Silica injection reduced animal survival and increased all lung mechanical parameters in relation to CTRL, followed by diffuse lung parenchyma inflammation, increased neutrophil infiltration, collagen deposition and increased pro-inflammatory and pro-fibrogenic cytokine secretion, as well as increased nitrite production. Clopidogrel treatment prevented silica-induced changes in lung function, and significantly reduced lung inflammation, fibrosis, as well as cytokine and nitrite production. These data suggest that inhibition of P2Y12 signaling improves silica-induced lung inflammation, preventing lung functional changes and mortality. Our results corroborate previous observations of silica-induced lung changes and expand the understanding of purinergic signaling in this process.

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OBJECTIVE: To evaluate the different components of the resistance of the respiratory system, respiratory muscle strength and to investigate the occurrence of expiratory flow limitation (EFL) in patients with morbid obesity (MO) when seated. METHODS: The sample was composed of MO (BMI≥40 kg/m2) and non-obese individuals (NO) with a BMI between 18 and 30 kg/m2. The protocol consisted of the anthropometric assessment and the following measures of respiratory function: spirometry, maximal inspiratory and expiratory pressures (MIP and MEP, respectively) and impulse oscillometry. The group comparison was performed using T-test for unpaired samples. The correlations were evaluated by the Pearson test with a significance level of 5%. RESULTS: Fifty MO (age 40±10.4 years, 1.64±0.09 m, 138.8±33.6 kg and 50.7±8.9 kg/m2), and 30 NO (age 37.6±11.5 years, 1.67±0.09 m, 65.2±10.3 kg and 23.2±22 kg/m2) were evaluated. The MO showed higher values of total, peripheral, airways, tissue and central resistance when compared to the NO. No patient showed EFL. The waist circumference was associated with spirometric variables, MIP, and MEP. The waist-to-hip ratio was correlated to respiratory mechanics and spirometric variables, MIP, and MEP. CONCLUSION: Morbidly obese patients with no obstructive spirometric pattern show increased total, airway, peripheral, and tissue respiratory system resistance when compared to nonobese. These individuals, however, do not present with expiratory flow limitation and reduced respiratory muscles strength.

OBJETIVO: avaliar os diferentes componentes da resistência do sistema respiratório e a força muscular respiratória, bem como investigar a ocorrência de limitação de fluxo expiratório (LFE) de pacientes obesos mórbidos (OM) na posição sentada. MÉTODOS: a amostra foi composta de OM (IMC ≥ 40 kg/m2) e de indivíduos não obesos (NO) com IMC entre 18 e 30 kg/m2. O protocolo foi composto de: avaliação antropométrica e da função respiratória (espirometria, pressões inspiratória (PIM) e expiratória máximas (PEM) e oscilometria de impulso). Na comparação entre os grupos, foi utilizado o teste T para amostras não pareadas. As correlações foram avaliadas pelo teste de Pearson, e o nível de significância foi de 5%. RESULTADOS: Foram avaliados 50 OM (idade 40,0 ± 10,4 anos, 1,64 ± 0,09 m, 138,8 ± 33,6 kg e 50,7 ± 8,9 kg/m2), além de 30 NO (idade 37,6 ± 11,5 anos, 1,67 ± 0,09 m, 65,2 ± 10,3 kg e 23,2 ± 22 kg/m2). Os OM apresentaram maiores valores de resistência total, central, de vias aéreas, tecidual e periférica quando comparados aos NO. Nenhum paciente apresentou LFE. A circunferência abdominal se associou com variáveis espirométricas PIM e PEM. A relação cintura-quadril se correlacionou com variáveis de mecânica respiratória, além das espirométricas PIM e PEM. CONCLUSÕES: pacientes com obesidade mórbida e sem padrão espirométrico obstrutivo apresentam aumento nas resistências total, de vias aéreas, periférica e tecidual do sistema respiratório quando comparados a não obesos. Esses indivíduos, entretanto, não apresentam limitação de fluxo expiratório e redução da força muscular respiratória.

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INTRODUCTION: Application of lipopolysaccharide (LPS) is a widely employed model to mimic acute respiratory distress syndrome (ARDS). Available data regarding LPS-induced biomechanical changes on pulmonary epithelial cells are limited only to P. aeruginosa LPS. Considering that LPS from different bacteria could promote a specific mechanical response in epithelial cells, we aim to assess the effect of E. coli LPS, widely employed as a model of ARDS, in the biomechanics of alveolar epithelial cells. METHODS: Young's modulus (E) of alveolar epithelial cells (A549) was measured by atomic force microscopy every 5 min throughout 60 min of experiment after treatment with LPS from E. coli (100 µg/mL). The percentage of cells presenting actin stress fibers (F-actin staining) was also evaluated. Control cells were treated with culture medium and the values obtained were compared with LPS-treated cells for each time-point. RESULTS: Application of LPS induced significant increase in E after 20 min (77%) till 60 min (104%) in comparison to controls. Increase in lung epithelial cell stiffness induced by LPS was associated with a higher number of cells presenting cytoskeletal remodeling. CONCLUSIONS: The observed effects of E. coli LPS on alveolar epithelial cells suggest that this widely-used LPS is able to promote a quick formation of actin stress fibers and stiffening cells, thereby facilitating the disruption of the pulmonary epithelial barrier.

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