Oxidative stress induction by short time exposure to ozone on THP-1 cells.

Ozone is a major component of air pollution mainly formed by photochemical reactions of nitrogen oxides with volatile organic compounds and/or carbon monoxide. Numerous studies have shown the association between ozone exposure with pulmonary injuries. This pollutant is a strong oxidant exerting its biological action either by direct reaction with target molecules or by generating reactive oxygen species which result in its biological effects and its toxicity. In order to study the effects of an induced oxidative stress by ozone on THP-1 cell, a human macrophage-like cell line, we used an in vitro system which has been previously used to study the rapid responses to ozone exposure. Using this system, THP-1 cells were subjected to short time exposure (30 min) followed by different incubation times ranging from 4 to 24 h. Our results show that ozone exposure provokes an alteration of the cell membrane translating an induction of lipid peroxidation resulting in a 3.2-fold increase of thiobarbituric reactive substances (TBARS), an increase by 35% of heme oxygenase-1 (HO-1) expression, and significant modifications of the redox status evaluated by glutathione measurement and of antioxidant enzyme activities in THP-1 cells. Our in vitro model constitutes a very interesting tool for the measurement of ozone effect on rapid modifications induced by this pollutant as well as intracellular modifications due to an oxidative stress.

Modification of membrane markers on THP-1 cells after ozone exposure in the presence or absence of fMLP.

The impacts of ozone and N-formyl-methionyl-leucyl-phenylalanine (fMLP) on the detection of membrane markers on non-differentiated THP-1 cells were evaluated for in vitro exposures. Several markers, specific for monocytes and macrophages, were identified on the THP-1 cells, allowing their use as a model for alveolar macrophages. Ozone exposure modified not only the detection of membrane markers, especially CD13 and CD14, monocyte and macrophage markers, but also the detection of the specific receptor for fMLP, formyl peptide receptor (FPR). Activation by fMLP also reduced the detection of the CD antigens, and a combined exposure to ozone and fMLP amplified this decrease, probably due to an additive effect of these chemicals. Overall, these results suggest important membrane rearrangements for short-term treatments to ozone and/or fMLP.

Ozone-mediated cytotoxicity after short-term exposure and its relation to the production of cellular metabolites (NO, H2O2).

Alveolar macrophages secrete numerous mediators, playing an important role in host defence. Among these mediators, nitric oxide (NO) and hydrogen peroxide (H2O2) are both involved in bactericidal killing and trigger the release of other cellular metabolites. We have analyzed the effect of an atmosphere polluted with ozone (0.03-0.5 ppm v/v) on the monocytic cell line THP-1, as a model for alveolar macrophages, in vitro. NO and H2O2 were chosen to evaluate cell response to ozone. Cell injury was evaluated using lactate dehydrogenase (LDH) liberation into the medium. An exposure to 0.5 ppm ozone proved to be more toxic to the cells, than 0.1 or 0.03 ppm, evidenced by more LDH being liberated and cytotoxicity reaching values up to 64%. For all ozone concentrations, H2O2 production reached a peak value after 10-15 min of exposure, after which the concentration of extracellular H2O2 production diminished rapidly. The highest NO concentrations were measured with 0.5 ppm ozone, reaching a maximum value of 1460 nmol/L per 5 x 10(6) cells, which is 1.55 times higher than for nonexposed cells. Lower concentrations barely induced higher NO concentrations compared to nonexposed cells. The results indicate that ozone effects not only the viability of human monocytes but also the release of antibacterial and defense signaling molecules and suggest that ozone-mediated cytotoxicity may be related to the secretion of NO and H2O2.

A new approach to evaluate toxicity of gases on mobile cells in culture.

A novel technique is described that measures the degree of toxicity of short-term exposure to gaseous pollutants or other chemical compounds on cultured cells, in 30 min. This technique, based on the study of the mobility properties of activated macrophages, consists of an image analysis procedure incorporating a specific exposure chamber (EC). The EC, which is developed from commercial culture flasks (50 ml, 25 cm(2) of culture surface), was first used to maintain cells in culture conditions, overnight, prior to the assay. In order to measure toxicity, it was then connected to the gaseous pollutant or chemical source. After exposing the culture medium and cells to the gas stream for 10 min, fMLP, a chemotactic factor, was added and the mobility of the macrophages measured by superimposing sequential analogue images captured by a CCD camera that were digitised and analysed using a software developed for this purpose. For example, the effect of ozone on macrophage-like cell (THP-1) was investigated. After exposure to 0.1 and 0.5 ppm, cells lost, respectively 79% and 90% of their mobility, compared to the control sample.