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

Abstract

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.

Introduction

Ozone (O₃) is a major component of urban air pollution. Indeed, tropospheric ozone is a secondary pollutant mainly formed by photochemical reactions of nitrogen oxides with volatile organic compounds and/or carbon monoxide (Mustafa, 1990). Several studies have shown a significant negative association between ozone exposure and pulmonary function including the reduction of lung volume parameters (Blomberg et al., 1999), inflammatory response (Scannell et al., 1996), disruption of the epithelial barrier associated with altered vascular permeability (Bhalla, 1999, Bhalla and Gupta, 2000) and the release of toxic and chemotactic mediators (Chang et al., 1998, Klestadt et al., 2002). This pollutant is a powerful oxidant exerting its biological action either by direct reaction with target molecules or via free radicals formed in the process of peroxidation of polyunsaturated fatty acids (PUFA) and oxidation of proteins, amines and thiols (Pryor et al., 1982, Pryor, 1994). Therefore, a consequence of ozone exposure is the induction of oxidative stress which is responsible for the toxicity of this pollutant. The susceptibility to ozone can be estimated by evaluating the antioxidant/stress related response, such as an increase of 8-isoprostane concentration (a lipid peroxidation product of arachidonic acid) in exhaled breath condensate (Corradi et al., 2002, Montuschi et al., 2002). An induction of heat shock/stress protein (HSP) expression has also been observed in lavaged lung cells in guinea pigs (Su and Gordon, 1997), rat and mouse lungs (Wong et al., 1996, Valacchi et al., 2004), and in human alveolar macrophages (Hamilton et al., 1996, Hamilton et al., 1998). Concerning the antioxidant response to ozone, depletion of vitamin E has been reported in mouse lung tissue (Valacchi et al., 2004) and in rat lung, activities of antioxidant enzymes activities (superoxide dismutase, catalase and glutathione peroxidase) are increased after long term ozone exposure (0.7 ppm O₃ for five days) (Rahman et al., 1991). To determine the cell specific response to ozone, an in vitro approach using primary culture or established cell lines has proven to be useful, allowing the evaluation of the peroxidative damage induced by ozone on rat alveolar macrophages (Banks et al., 1990) or even the modification of glutathione contents (Rietjens et al., 1985a, Rietjens et al., 1985b).

In order to test the susceptibility of THP-1 cells, a human macrophage-like cell line, to oxidative stress induced by ozone, we submitted these cells to short time exposures (30 min at 0.5 ppm) followed by different incubation times ranging from 4 to 24 h. The system for short time in vitro exposures to low ozone concentrations has been developed previously, allowing the measurement of mobility decrease after intoxication (Laval-Gilly et al., 2000) in order to develop a biosensor to evaluate gases pollutant toxicity. The aim of this study was to determine ozone effects on several typical oxidative markers after short exposure time in our cell model using our in vitro system. The evaluation of the primary response consisted of the determination of lipid peroxidation whereas the delayed response was investigated by the measurement of HO-1 expression, glutathione and linked enzyme activities after exposure. Our results show that ozone exposure provokes an increase of lipid peroxidation and heme oxygenase-1 (HO-1) expression, as well as modifications of the redox status and of antioxidant enzyme activities in THP-1 cells using our in vitro model.