Deborah R. Tasat

Lung oxidative metabolism after exposure to ambient particles

The aim of this work was to study the time course of the oxidative metabolism in mice lung after exposure to ambient particles (ROFA). Swiss mice were intranasally instilled with a ROFA suspension (0.20 mg/kg). Animals were sacrificed 1 or 3 h after the exposure. Eighty percentage of increased oxygen consumption was observed in tissue cubes after 1 h of exposure. This observation was accompanied by an increased NADPH oxidase activity (40%) and mitochondrial oxygen consumption in state 3 (19%). NO production by lung homogenates was found to be increased by 43% after 3 h of exposure. Phospholipid oxidation in lung homogenates showed a 29% increase after 1 h of exposure, while a 30% increase in the carbonyl content was found only after 3 h of exposure. Our data show the relative importance of different sources of reactive oxygen species (NADPH oxidase activity and mitochondrial respiration) to the increased tissue oxygen consumption, oxidative damage and antioxidant status observed in an acute model of ROFA particles exposure.

Time course of systemic oxidative stress and inflammatory response induced by an acute exposure to Residual Oil Fly Ash

It is suggested that systemic oxidative stress and inflammation play a central role in the onset and progression of cardiovascular diseases associated with the exposure to particulate matter (PM). The aim of this work was to evaluate the time changes of systemic markers of oxidative stress and inflammation, after an acute exposure to Residual Oil Fly Ash (ROFA). Female Swiss mice were intranasally instilled with a ROFA suspension (1.0mg/kg body weight) or saline solution, and plasma levels of oxidative damage markers [thiobarbituric acid reactive substances (TBARSs) and protein carbonyls], antioxidant status [reduced (GSH) and oxidized (GSSG) glutathione, ascorbic acid levels, and superoxide dismutase (SOD) activity], cytokines levels, and intravascular leukocyte activation were evaluated after 1, 3 or 5h of exposure. Oxidative damage to lipids and decreased GSH/GSSG ratio were observed in ROFA-exposed mice as early as 1h. Afterwards, increased protein oxidation, decreased ascorbic acid content and SOD activity were found in this group at 3h. The onset of an adaptive response was observed at 5h after the ROFA exposure, as indicated by decreased TBARS plasma content and increased SOD activity. The observed increase in oxidative damage to plasma macromolecules, together with systemic antioxidants depletion, may be a consequence of a systemic inflammatory response triggered by the ROFA exposure, since increased TNF-α and IL-6 plasma levels and polymorphonuclear leukocytes activation was found at every evaluated time point. These findings contribute to the understanding of the increase in cardiovascular morbidity and mortality, in association with environmental PM inhalation.

Diesel Exhaust Particles Selectively Induce Both Proinflammatory Cytokines and Mucin Production in Cornea and Conjunctiva Human Cell Lines

PURPOSE To evaluate the effect of diesel exhaust particles (DEP) on the viability, proliferation, apoptosis, secretion of cytokines (IL-6, IL-8, and TNF-α), and mucin gene transcription (MUC1, MUC5AC, and MUC16) in human epithelial cells of the cornea (HCLE) and conjunctiva (IOBA-NHC). METHODS HCLE and IOBA-NHC cells were incubated with DEP (10-500 μg/mL) for 24 hours. Cell proliferation was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptotic cells were measured by an annexin V-FITC and propidium iodide kit for flow cytometry. Proinflammatory cytokines were determined by an ELISA kit. Mucin gene transcription was quantified by real-time PCR. RESULTS DEP significantly decreased the viability, proliferation, and secretion of IL-8, but increased the secretion of IL-6 on both HCLE and IOBA-NHC cell lines in a dose-dependent manner. Neither cornea nor conjunctiva cells incubated with DEP released TNF-α. DEP induced a significant increase in the percentage of apoptotic cells in IOBA-NHC, whereas no changes were observed in HCLE. Finally, DEP significantly decreased the transcription levels of MUC1 and MUC16 in HCLE, but increased the transcription levels of MUC1, MUC5AC, and MUC16 in IOBA-NHC. CONCLUSIONS These findings suggest that human corneal and conjunctival epithelial cells incubated with DEP showed cytotoxicity and an inflammatory response mediated by IL-6, not by TNF-α or IL-8. Also, the decrease in mucin expression in the cornea cells might leave exposed areas in the cornea for contact with DEP. Finally, the increase in mucin expression in the conjunctiva cells might be involved at least in the clearance of DEP to protect the ocular epithelium.

Low doses of urban air particles from Buenos Aires promote oxidative stress and apoptosis in mice lungs

Air pollution consists of a wide range of gaseous and particulate pollutants. Exposure to particulate matter (PM) can cause oxidative stress within the lung, which in turn can negatively impact health. The mechanisms by which PM causes oxidative stress include the release of trace metals or organic components from the particle. Previously, we have characterized urban air particles from downtown Buenos Aires (UAP-BA) and, by using in vivo animal studies, found that they are able to generate lung inflammation. Purpose: We studied lung responses to low doses of UAP-BA (15 µg), with special emphasis on oxidative balance. Methods: We assessed cell viability, total cell number (TCN) and cell differential (CD) on bronchoalveolar lavages (BAL), oxidative metabolism in lung homogenates by tertbutylhydroperoxide-initiated chemiluminescence (CL), thiobarbituric reactive substances (TBARS), total reactive antioxidant potential (TRAP), reduced glutathione (GSH), and apoptosis in lung sections. Results: We found that low UAP-BA exposure increases TCN, modifies CD, and decreases cell viability in the BAL. In lung homogenates, TBARS and CL rose while TRAP and GSH showed no alteration when compared to controls. Occurrence of apoptosis evaluated by TUNEL assay was markedly augmented in UAP-BA exposed animals. Conclusions: Our data further implicate oxidative stress as a possible inducer of apoptosis in lungs from animals exposed to low concentrations of this urban environmental contaminant.

Reactive oxygen species produced by NADPH oxidase and mitochondrial dysfunction in lung after an acute exposure to Residual Oil Fly Ashes

Reactive O2 species production triggered by particulate matter (PM) exposure is able to initiate oxidative damage mechanisms, which are postulated as responsible for increased morbidity along with the aggravation of respiratory diseases. The aim of this work was to quantitatively analyse the major sources of reactive O2 species involved in lung O2 metabolism after an acute exposure to Residual Oil Fly Ashes (ROFAs). Mice were intranasally instilled with a ROFA suspension (1.0mg/kg body weight), and lung samples were analysed 1h after instillation. Tissue O2 consumption and NADPH oxidase (Nox) activity were evaluated in tissue homogenates. Mitochondrial respiration, respiratory chain complexes activity, H2O2 and ATP production rates, mitochondrial membrane potential and oxidative damage markers were assessed in isolated mitochondria. ROFA exposure was found to be associated with 61% increased tissue O2 consumption, a 30% increase in Nox activity, a 33% increased state 3 mitochondrial O2 consumption and a mitochondrial complex II activity increased by 25%. During mitochondrial active respiration, mitochondrial depolarization and a 53% decreased ATP production rate were observed. Neither changes in H2O2 production rate, nor oxidative damage in isolated mitochondria were observed after the instillation. After an acute ROFA exposure, increased tissue O2 consumption may account for an augmented Nox activity, causing an increased O2(-) production. The mitochondrial function modifications found may prevent oxidative damage within the organelle. These findings provide new insights to the understanding of the mechanisms involving reactive O2 species production in the lung triggered by ROFA exposure.

Simvastatin pretreatment prevents ambient particle-induced lung injury in mice

Air particulate pollution negatively affects the health of the population exposed, being the lung the main target organ. Simvastatin (SV) is widely used for the prevention and risk reduction of coronary disease. Its pleiotropic effects may provide benefit for lung diseases. Here, we investigated the preventive effect of simvastatin pretreatment on acute intranasal exposure to ROFA (Residual Oil Fly Ash), and UAP (Urban Air Particle from Buenos Aires). Male BALB/c mice were randomized in two groups to receive either saline (control, C) solution or SV (1 mg/kg bw /day; ip) for 14 days. After SV treatment, ROFA or UAP (1 mg/kg bw) or saline were intranasally delivered for 24 hours generating 4 subgroups for the ROFA experiment (C, SV, ROFA and SV+ROFA) and 3 subgroups for the UAP experiment (C, SV, UAP and SV+UAP). Biomarkers of lung injury were examined in BAL cells evaluating total cell number (TCN), cell differential (CD) and superoxide anion generation (O2-), in lung homogenates assessing superoxide dismutase activity (SOD) and tumor necrosis factor α (TNFα); and in blood samples determining interleukin 6 (IL-6) production. ROFA and UAP produced an acute pulmonary injury, characterized by an increase in BAL, TCN and neutrophilic inflammatory influx, a rise in O2- generation, and production of the proinflammatory TNFα cytokine. SV pretreatment had no significant effect per se on any of these biomarkers but prevented the pulmonary cytotoxicity and inflammation induced by ROFA and UAP. Our results encourage further studies to determine the preventive effects on lung injury induced by air pollutants.

Age-related lung cell response to urban Buenos Aires air particle soluble fraction

Exposure to particulate matter (PM) may alter lung homeostasis inducing changes in fluid balance and host defense. Bioavailability of soluble PM compounds like polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and transition metals has been shown to play a key role in lung injury. We have previously characterized the size, shape, and chemical components of urban air particles from Buenos Aires (UAP-BA) and their biological impact on lungs. Herein, we evaluate the possible toxic effect of UAP-BA-soluble fraction (UAP-BAsf) on pulmonary cells obtained from young (1-2 months old) and aged (9-12 months old) Wistar rats using phagocytosis, oxidant-antioxidant generation, and apoptosis as endpoints. UAP-BA were collected in downtown BA and residual oil fly ash (ROFA), employed as a positive control, was collected from Boston Edison Co., Mystic Power Plant, Mystic, CT, USA. Both particle-soluble fractions (sf) were employed at concentrations ranging from 0 to 100 microg/mL. UAP-BAsf and ROFAsf even at the lowest dose assayed (10 microg/mL) showed in both lung cell populations the ability to stimulate phagocytosis and increase superoxide anion (O(2)(-)) generation. Both types of air particles caused a marked intracellular oxidant stress in aged pulmonary cells that may contribute to subsequent cell activation and production of proinflammatory mediators, leading to cell dysfunction. These data suggest that the impact of UAP-BAsf on phagocytosis, oxidant radical generation, and apoptosis is clearly dependent on the maturational state of the animal and might have different mechanisms of action.

Impact through time of different sized titanium dioxide particles on biochemical and histopathological parameters

Due to corrosion, a titanium implant surface can be a potential source for the release of micro (MPs) and nano-sized particles (NPs) into the biological environment. This work sought to evaluate the biokinetics of different sized titanium dioxide particles (TiO2 ) and their potential to cause cell damage. Wistar rats were intraperitoneally injected with 150 nm, 10 nm, or 5nm TiO2 particles. The presence of TiO2 particles was evaluated in histologic sections of the liver, lung, and kidney and in blood cells at 3 and 12 months. Ultrastructural analysis of liver and lung tissue was performed by TEM, deposit concentration in tissues was determined spectroscopically, and oxidative metabolism was assessed by determining oxidative membrane damage, generation of superoxide anion (O2(-)), and enzymatic and non-enzymatic antioxidants. TiO2 particles were observed inside mononuclear blood cells and in organ parenchyma at 3 and 12 months. TiO2 deposits were consistently larger in liver than in lung tissue. Alveolar macrophage O2(-) generation and average particle size correlated negatively (p < 0.05). NPs were more reactive and biopersistent in lung tissue than MPs. Antioxidant activity, particularly in the case of 5 nm particles, failed to compensate for membrane damage in liver cells; the damage was consistent with histological evidence of necrosis.

In vivo comparative biokinetics and biocompatibility of titanium and zirconium microparticles

Titanium and zirconium are biomaterials that present a layer of titanium dioxide (TiO(2)) or zirconium dioxide (ZrO(2)). As a result of corrosion, microparticles can be released into the bioenvironment, and their effect on tissues is seemingly associated with differences in the physicochemical properties of these metals. The aim of this study was to perform a long-term evaluation of the distribution, destination, and potential risk of TiO(2) and ZrO(2) microparticles that might result from the corrosion process. Wistar rats were i.p. injected with an equal dose of either TiO(2) or ZrO(2) suspension. The following end-points were evaluated at 3, 6, and 18 months: (a) the presence of particles in blood cells and liver and lung tissue, (b) Ti and Zr deposit quantitation, (c) oxidant-antioxidant balance in tissues, and (d) O(2)(-) generation in alveolar macrophages. Ti and Zr particles were detected in blood mononuclear cells and in organ parenchyma. At equal doses and times postadministration, Ti content in organs was consistently higher than Zr content. Ti elicited a significant increase in O(2)(-) generation in the lung compared to Zr. The consumption of antioxidant enzymes was greater in the Ti than in the Zr group. The present study shows that the biokinetics of TiO(2) and ZrO(2) depends on particle size, shape, and/or crystal structure.

Direct and indirect air particle cytotoxicity in human alveolar epithelial cells

Air particulate matter has been associated with adverse impact on the respiratory system leading to cytotoxic and proinflammatory effects. The biological mechanisms behind these associations may be initiated by inhaled small size particles, particle components (soluble fraction) and/or mediators released by particle-exposed cells (conditioned media). The effect of Urban Air Particles from Buenos Aires (UAP-BA) and Residual Oil Fly Ash (ROFA) a surrogate of ambient air pollution, their Soluble Fractions (SF) and Conditioned Media (CM) on A549 lung epithelial cells was examined. After 24 h exposure to TP (10 and 100 μg/ml), SF or CM, several biological parameters were assayed on cultured A549 cells. We tested cell viability by MTT, superoxide anion (O₂(-)) generation by NBT and proinflammatory cytokine (TNFα, IL-6 and IL-8) production by ELISA. UAP-BA particles or its SF (direct effect) did not modify cell viability and generation of O₂(-) for any of the doses tested. On the contrary, UAP-BA CM (indirect effect) reduced cell viability and increased both generation of O₂(-) and IL-8 production. Exposure to ROFA particles, SF or ROFA CM reduced proliferation and O₂(-) but, stimulated IL-8. It is worth to note that UAP-BA and ROFA depicted distinct effects on particle-exposed A549 cells implicating morphochemical dependence. These in vitro findings support the hypothesis that particle-induced lung inflammation and disease may involve lung-derived mediators.