Evaluation of Oxidative Stress Markers in Human Conjunctival Epithelial Cells Exposed to Diesel Exhaust Particles (DEP)

PURPOSE. The aim of this study was to evaluate oxidative stress markers in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP). METHODS. Reactive oxygen (ROS) and nitrogen (RNS) species production; hydrogen peroxide (H 2 O 2 ) levels; protein oxidation; antioxidant enzymes activities (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx], glutathione S-transferase [GST], and glutathione reductase [GR]); total reactive antioxidant potential (TRAP); reduced (GSH) and oxidized glutathione (GSSG) were evaluated. Transmission electron microscopy was performed to evaluate DEP uptake. RESULTS. Diesel exhaust particles were entrapped by membrane protrusions developed by IOBA-NHC. Cells exposed to DEP 50 and 100 lg/mL showed a significant increase in ROS, RNS, H 2 O 2 levels, SOD, GPx, and GST compared with the control group. A significant decay in GR was observed in both groups, meanwhile CAT levels remained unchanged. The group exposed to DEP 100 lg/mL displayed a significant increase in protein oxidation. In both groups, TRAP was significantly reduced as well as the GSH/GSSG ratio. CONCLUSIONS. The decrease in nonenzymatic antioxidants and the compensatory increase of SOD, GPX, and GST activities are consequence of the increase in ROS and RNS production due to DEP exposure and its accumulation inside the cells. The decay in GR activity leads to the decrease in GSH/GSSG recycling. These results suggest that oxidative stress could play an important role in the development of DEP effects on human conjunctival epithelial cells. Keywords: conjunctiva, oxidative stress, antioxidants, environmental pollution, diesel exhaust particles T he evidence on airborne particulate matter (PM) and its public health impact is consistent in showing adverse health effects at exposures that are currently experienced by urban populations in both developed and developing countries. 1 Ambient particles are known as both initiators and enhancers of the clinical manifestations of both allergic and nonallergic diseases in industrialized countries. Diesel exhaust particles (DEP) are one of main components of urban air pollution as they are the most common combustion-derived particles presented in traffic emissions. 7 The toxic mechanism developed in the respiratory mucosa, including epithelial and immune cells, after DEP exposure has been well-studied. The role of oxidative stress in particulate air pollution effects on ocular surface and, in particular, the involvement in the mechanism of damage after DEP exposure has not yet been reported. The aim of this study was to evaluate oxidative stress markers in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP). In this work, we evaluate oxidative stress markers in human conjunctival epithelial cells incubated with DEP at different concentrations analyzing the changes in pro-oxidant/antioxidant balance, the antioxidant enzymes activity and the levels of nonenzymatic antioxidants. Transmission electron microscopy analysis was also performed in order to evaluate if the particles were incorporated by cells and to determine morphologic changes associated with DEP incubation. MATERIALS AND METHODS Cell Culture The normal human conjunctival epithelium cell line (IOBA-NHC) was provided by Yolanda Diebold, PhD (University Institute of Applied Ophthalmobiology, University of Valladolid, Valladolid, Spain). Diesel Exhaust Particles Diesel exhaust particles from diesel motor combustion were provided by Paulo H. Saldiva, PhD (Laboratório de Poluição Atmosférica Experimental, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil). Diesel exhaust particles are composed of a carbonaceous core with adsorbed organic compounds, including polycyclic aromatic hydrocarbons, and trace of inorganic compounds. IOBA-NHC Incubation With DEP Cell monolayers of IOBA-NHC (80%-100% confluent) were incubated with DEP 10, 50, and 100 lg/mL (DEP 10, DEP 50 and DEP 100, respectively) or with DMEM-F12 (control group) for 24 hours. Cell monolayers were washed twice with PBS 1X (pH ¼ 7.40). Cells were removed with 0.25% trypsin-EDTA to perform the assays

Diesel exhaust particles (DEP) induce an early redox imbalance followed by an IL-6 mediated inflammatory response on human conjunctival epithelial cells

The aim of this study was to evaluate the time course of oxidative stress markers and inflammatory mediators in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP) for 1, 3, and 24 h. Reactive oxygen species (ROS) production, lipid and protein oxidation, Nrf2 pathway activation, enzymatic antioxidants, glutathione (GSH) levels and synthesis, as well as cytokine release and cell proliferation were analyzed. Cells exposed to DEP showed an increase in ROS at all time points. The induction of NADPH oxidase-4 appeared later than mitochondrial superoxide anion production, when the cell also underwent a proinflammatory response mediated by IL-6. DEP exposure triggered the activation of Nrf2 in IOBA-NHC, as a strategy for increasing cellular antioxidant capacity. Antioxidant enzyme activities were significantly increased at early stages except for glutathione reductase (GR) that showed a significant decrease after a 3-h-incubation. GSH levels were found increased after 1 and 3 h of incubation with DEP, despite the increase in its consumption by the antioxidant enzymes as it works as a cofactor. GSH recycling and the de novo synthesis were responsible for the maintenance of its content at these time points, respectively. After 24 h, the decrease in GR and glutamate cysteine ligase as wells as the enhanced activity of glutathione peroxidase and glutathione S-transferase produced a depletion in the GSH pool. Lipid-peroxidation was found increased in cells exposed to DEP after 1-h-incubation, whereas protein oxidation was found increased in cells exposed to DEP after a 3-h-incubation that persisted after a longer exposure. Furthermore, DEP lead IOBA-NHC cells to hyperplasia after 1 and 3 h of incubation, but a decrease in cell proliferation was found after longer exposure. ROS production seems to be an earlier event triggered by DEP on IOBA-NHC, comparing to the proinflammatory response mediated by IL-6. Despite the fact that under short periods of exposure to DEP lipids and then proteins are targets of oxidative damage, the viability of the cells is not affected at early stages, since cell hyperplasia was detected as compensatory mechanism. Although after 24 h Nrf2 pathway is still enhanced, the epithelial cell capacity to maintain redox balance is exceeded. The antioxidant enzymes activation and the depleted GSH pool are not capable of counteracting the increased ROS production, leading to oxidative damage.Fil: Lasagni Vitar, Romina Mayra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; ArgentinaFil: Tau, Julia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Janezic, Natasha S.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; ArgentinaFil: Tesone, Agustina I.. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; ArgentinaFil: Hvozda Arana, Ailen Gala. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; ArgentinaFil: Reides, Claudia G.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Berra, Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferreira, Sandra M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; ArgentinaFil: Llesuy, Susana Francisca. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Urban air pollution induces redox imbalance and epithelium hyperplasia in mice cornea

The aim of the study was to evaluate the time course of the effects of urban air pollutants on the ocular surface, focusing on the morphological changes, the redox balance, and the inflammatory response of the cornea. 8-week-old mice were exposed to urban or filtered air (UA-group and FA-group, respectively) in exposure chambers for 1, 2, 4, and 12 weeks. After each time, the eyes were enucleated and the corneas were isolated for biochemical analysis. UA-group corneas exhibited a continuous increase in NADPH oxidase-4 levels throughout the exposure time, suggesting an increased production of reactive oxygen species (ROS). After 1 week, an early adaptive response to ROS was observed as an increase in antioxidant enzymes. After 4 weeks, the enzymatic antioxidants were decreased, meanwhile an increase of the glutathione was shown, as a later compensatory antioxidant response. However, redox imbalance took place, evidenced by the increased oxidized proteins, which persisted up to 12 weeks. At this time point, corneal epithelium hyperplasia was also observed. The inflammatory response was modulated by the increase in IL-10 levels after 1 week, which early regulates the release of TNF-α and IL-6. These results suggest that air pollution alters the ocular surface, supported by the observed cellular hyperplasia. The redox imbalance and the inflammatory response modulated by IL-10 play a key role in the response triggered by air pollutants on the cornea. Taking into account this time course study, the ocular surface should also be considered as a relevant target of urban air pollutants.Fil: Lasagni Vitar, Romina Mayra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Hvozda Arana, Ailen Gala. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Janezic, Natasha S.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Marchini, Timoteo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Tau, Julia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martinefski, Manuela Romina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tesone, Agustina Inés. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; ArgentinaFil: Racca, Lourdes. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; ArgentinaFil: Reides, Claudia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Tripodi, Valeria Paula. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Evelson, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Berra, Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; ArgentinaFil: Llesuy, Susana Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; ArgentinaFil: Ferreira, Sandra María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentin