Ozone and nitrogen dioxide. A study on mechanisms of toxic action and cellular defense.

Ozone and nitrogen dioxide are major toxic components of photochemical smog. They arise from the combustion of fossil fuels (traffic, industrial processes) and from solar radiation-catalyzed reactions in polluted atmospheres.The morphological, physiological and biochemical effects of ozone and nitrogen dioxide on the respiratory system of man and experimental animals have been investigated over the last decades.More recently the development of <strong>i)</strong> isolation and cell culture procedures for different types of lung cells and <strong>ii)</strong> model systems for <u>in</u><u>vitro</u> exposure of cells to gaseous compounds, offers new possibilities to study the mechanism of toxic action of ozone and nitrogen dioxide. An advantage of the cell model is that changes in homogeneous cell populations can be studied, although it is recognized that effects found in isolated cell cultures always need <u>in</u><u>vivo</u> validation.The experiments described in this thesis were undertaken to further elucidate <strong>i)</strong> the mechanisms of action of ozone and nitrogen dioxide, as well as <strong>ii)</strong> the mechanisms of cellular protection against both gaseous compounds.<u>In</u><u>vitro</u> exposure of cells: was achieved by using a system in which cells are grown on a thin teflon membrane and exposed by means cif gas diffusion through this membrane.Experiments were carried out using either cells from the A549 cell line or primary cultures of alveolar macrophages or alveolar type II pneumocytes, isolated from the lungs of control and in some cases (chapter 6a + b) from ozone or nitrogen dioxide exposed rats.Part I of this thesis starts with a review of literature data on historical backgrounds, physical characteristics, concentrations encountered in the environment and toxic effects of ozone and nitrogen dioxide (chapter 1).This is followed by a review on the current theories with regard to the mode of toxic action of ozone and nitrogen dioxide as well as to the mechanisms of cellular protection against these compounds (chapter 2).Finally a review of <u>in</u><u>vitro</u> exposure models is presented, including a description of the gas diffusion mediated exposure model applied in this thesis (chapter 3).Part II of this thesis deals with the experiments carried out to obtain additional insight into <strong>i)</strong> the mode of toxic action of ozone and nitrogen dioxide and <strong>ii)</strong> the mechanisms providing protection against both gases in an intact cell system.First experiments are described in which the mechanisms of toxic action of ozone and nitrogen dioxide were compared (chapter 4). In the <u>in</u><u>vitro</u> exposure model applied ozone appeared to be 10 times more toxic than nitrogen dioxide. This difference is comparable with the difference in toxicity reported for <u>in</u><u>vivo</u> exposures.In addition it was demonstrated that the cellular antioxidant compounds, vitamin E ( α-tocopherol), vitamin C (ascorbic acid) and glutathione, are all involved in the protection of cells against ozone or nitrogen dioxide.The protection of α-tocopherol was shown to be dependent on its 6-hydroxyl group and not to be mediated by a structural stabilizing membrane effect of the compound arising from its strong physicochemical association with for example sensitive arachidonyl fatty acid residues. This could be concluded from the observations that <strong>i)</strong> α-tocopherol, at concentrations that provided optimal protection against the oxidative compounds, did not influence cell membrane fluidity and that <strong>ii)</strong> phytol and the methyl ether of α-tocopherol, two structural α-tocopherol analogues, could not provide protection against ozone or nitrogen dioxide comparable to the α-tocopherol protection.Furthermore chapter 4 presents <u>in</u><u>vitro</u> data which clearly demonstrate that the reaction pathways involved in ozone and nitrogen dioxide induced cell damage must be different. This conclusion is based on the observations that <strong>i)</strong> vitamin C enrichment of cells provided significantly better protection against nitrogen dioxide than against an equally toxic amount of ozone, that <strong>ii)</strong> glutathione depletion increased the cellular sensitivity towards ozone to a greater extent than the sensitivity towards nitrogen dioxide and that <strong>iii)</strong> α-tocopherol dependent protection of the cells was accompanied by a significantly greater reduction in cellular α-tocopherol upon nitrogen dioxide than upon ozone exposure.The observations referred to above are compatible with the hypothesis that ozone damage proceeds by the ionalr mechanism for ozonide formation, whereas nitrogen dioxide induced cell damage is induced by a radical mediated lipid peroxidative pathway.This hypothesis includes the involvement of oxidation of unsaturated membrane lipids in the mechanisms of ozone or nitrogen dioxide induced cell damage.Evidence for the involvement of lipid oxidation in the mechanism of ozone or nitrogen dioxide induced cell damage is presented in the next chapter, (chapter 5), in which experiments are described that investigated the influence of polyunsaturated fatty acid (PUFA) supplementation on the sensitivity of cells towards ozone and nitrogen dioxide.The results showed that cells enriched in their PUFA content demonstrate an increased sensitivity towards both ozone and nitrogen dioxide. It was also shown that this increased sensitivity was not caused by an increased membrane fluidity but really by the increased number of unsaturated fatty acids. Therefore these results clearly point to the involvement of lipid oxidation in the mechanism of action of both ozone and nitrogen dioxide.In addition to the evidence for a difference in the mechanisms of toxic action of ozone and nitrogen dioxide observed in the <u>in</u><u>vitro</u> experiments, evidence suggesting a difference in their toxicity was also obtained in <u>in</u><u>vivo</u> experiments (chapter 6a + b). Exposure of rats to doses of both gaseous compounds that equally induced glucose-6-phosphate dehydrogenase activity in whole lung homogenates and in isolated alveolar macrophages and type II pneumocytes, resulted in a greater increase in the activity of glutathione peroxidase in cell material derived from ozone than from nitrogen dioxide exposed rats.In addition it was shown that the increased activities of the enzymes of the glutathione peroxidase system, observed in lung homogenates of ozone or nitrogen dioxide exposed rats were caused by cell proliferation as well as by the increase of enzyme activities within individual cells.In the literature these inductions have often been coupled to a glutathione dependent mechanism of cellular defense. To test this hypothesis alveolar macrophages and type II pneumocytes isolated from exposed rats were exposed to ozone or nitrogen dioxide <u>in</u><u>vitro</u> . From these studies it appeared that cells derived from exposed animals revealed no increased resistance to the oxidative compounds as compared to the cells isolated from non-exposed animals. This in spite of a significantly increased glutathione peroxidase activity in the cells derived from exposed animals. Hence it can be concluded that an increased cellular glutathione peroxidase activity is not related to an increased cellular resistance towards ozone or nitrogen dioxide.Data presented in the next chapter (chapter 7) demonstrated even more clearly that the glutathione dependent protection of cells against ozone is not mediated by the glutathione peroxidase catalyzed detoxification of fatty acid hydroperoxides. A549 cells showed a significantly increased sensitivity towards ozone upon depletion of their cellular glutathione content, which clearly points to a glutathione dependent mechanism of cellular protection, although these cells do not contain a detectable glutathione peroxidase activity. This observation obviously excludes the glutathione peroxidase catalyzed detoxification of lipid hydroperoxides as a main mechanism for glutathione dependent cellular protection against ozone.Additional results demonstrated the loss of glutathione from the cytoplasm of ozone exposed cells. This loss of glutathione from the cytoplasm of exposed cells was caused by leakage and/or active transport of glutathione out of exposed cells to the surrounding medium. The loss could not be ascribed to incorporation of a substantial amount of glutathione into mixed disulfides. This observation excludes a second hypothesis for the glutathione dependent protection of cells against ozone, viz. its incorporation into mixed disulfides, thus protecting cellular thiol groups from irreversible oxidation by ozone or its reactive intermediates.The results presented in the next chapter (chapter 8), indicate that the increased ozone sensitivity of glutathione depleted cells is not caused by an impaired regeneration of α-tocopherol in these cells. This follows from the observations that <strong>i)</strong> glutathione depleted, ozone exposed cells did not contain decreased levels of α-tocopherol and that <strong>ii)</strong> vitamin E supplementation could not diminish the increased ozone sensitivity of the glutathione depleted cells.So far all three hypotheses mentioned in the literature for a glutathione dependent cellular protection against ozone, had been excluded. From this, and from the observation that vitamin C supplementation abolished part of the increased ozone sensitivity of glutathione depleted cells, it is concluded that the most likely mechanism for a glutathione dependent protection of cells against ozone is provided by its action as a direct scavenger of reactive initial and/or intermediate species.Experiments described in the final chapter of part II (chapter 9), actually demonstrate the ability of glutathione to detoxify a possible ozone intermediate; the ozonide derived from methyl linoleate. Methyl linoleate ozonide appeared to be toxic towards alveolar macrophages at concentrations between 10 to 100 μM, and showed characteristics with regard to cellular antioxidant protection that were similar to those for ozone itself.In addition the detoxification of methyl linoleate ozonide by glutathione appeared to be even more pronounced when c atalyzed by glutathione S-transferase.Hence the glutathione S-transferase catalyzed detoxification of fatty acid ozonides provides a new point of view on the protective role of glutathione in ozone exposed cells