Ozonides: intermediates in ozone-induced toxicity : a study on their mechanism of toxic action and detoxification by antioxidants

Ozone is a major constituent of photochemical smog. The toxicity of ozone is well documented and has been related to its strong oxidative potential. The principal target organ for ozone toxicity is the respiratory system. Unsaturated fatty acids, which are present in both the lipids of the lung lining fluid and the cell membranes of the cells that line the airways, are thought to be primary target molecules for ozone. Ozone reacts with unsaturated fatty acids via the so-called Criegee mechanism. Along with aldehydes and hydroxyhydroperoxides, Criegee ozonides are the main products of the reaction of ozone with unsaturated fatty acids. It is generally assumed that these lipid ozonation products act as secondary toxins in ozone-induced toxicity. However, very little is known about the reactivity and fate of ozonides and the role they play in ozone toxicity. Therefore, the aim of the present study was to gain insight into the mechanism of the toxic action of ozonides in relation to ozone-induced lung toxicity.For the investigations the ozonide from the polyunsaturated fatty acid methyl linoleate was used. On ozonation of methyl linoleate the major product formed appeared to be the trans -9,10-methyl linoleate ozonide (MLO).It has been suggested that ozonides are a kind of peroxide, and it is, therefore, believed that ozonides are a source of free radicals. This implies that ozonides might be capable of initiating the chain autoxidation of other non-ozonated polyunsaturated fatty acids in the membrane bilayer, and consequently of producing a cascade of damage. This hypothesis was investigated by comparing the in vitro cellular toxic action of MLO with a model peroxidative agent, i.e., cumene hydroperoxide. The ozonide was shown to be three times more toxic towards alveolar macrophages than the peroxide. On the basis of the cellular protection of antioxidants it was clearly shown that the ozonide and the hydroperoxide exert their toxic effects using different mechanisms. Whereas the results with the model hydroperoxide, cumene hydroperoxide, confirmed the mechanism by which peroxides exert their toxic effects, namely by lipid peroxidation and/or depletion of intracellular glutathione (GSH) levels, the investigations regarding the potency of MLO to induce lipid peroxidation revealed that the main toxic mechanism of MLO did not proceed via a radical-mediated mechanism. Nevertheless, suppletion of cells with the lipid-soluble radical scavengerα-tocopherol resulted in a significant protection towards ozonide exposure. In addition, preincubation of MLO withα-tocopherol resulted in a detoxification of the ozonide. This suggests thatα-tocopherol is able to interact directly with the ozonides themselves, thus scavenging these reactive intermediates.Investigations regarding the chemical characteristics of the detoxification of MLO byα-tocopherol revealed that the main products formed were the aldehyde nonanoic 9-oxo methyl ester and the acid nonanedioic acid monomethyl ester. This finding is in agreement with the general opinion that the decomposition of ozonides results in the formation of aldehydes and acids. In general, it is believed that thermal decomposition of ozonides proceeds via homolytic cleavage of the peroxide bond to yield the oxy bi-radical followed by a rearrangement. A modification on this mechanism includes concerted homolysis and intramolecular hydrogen atom abstraction. In contrast to the results obtained at elevated temperatures (≥50 °C), in previous studies ozonides have been shown to be stable compounds at 37 °C. No radical formation could be detected when MLO was incubated for 30 min at 37 °C using spin traps and electron spin resonance (ESR). Taking into account the fact that peroxide bond homolysis is an essential part of ozonide decomposition, one might postulate thatα-tocopherol, being an efficient hydrogen atom donor, facilitates the process of O-O bond homolysis, thereby inducing aldehyde and acid formation already at relatively low (i.e.≤37 °C) temperatures.An additional interesting finding was that the degradation products of MLO, i.e., nonanoic 9-oxo methyl ester and nonanedioic acid monomethyl ester (azelaic acid monomethyl ester), were not toxic towards alveolar macrophages at concentrations where MLO showed complete loss of cell viability. This observation is especially of importance because it is often suggested that Criegee ozonides will be formed in relatively small amounts (ca. 10%) when ozone reacts with unsaturated fatty acids in the lung lining fluids and may, therefore, play a minor role in ozone-induced toxicity. However, the results of the present study clearly indicate that ozonides are far more toxic than their aldehyde and acid type degradation products, which are generally observed as major products resulting from the reaction of ozone with fatty acids under physiological conditions.In addition, the mechanism underlying the reaction of another cellular antioxidant, namely glutathione, with polyunsaturated fatty acid ozonides was investigated. In previous studies it has been shown that preincubation of MLO with glutathione caused a significant detoxification of the ozonide. Furthermore, the detoxification reaction was shown to be catalysed by glutathione S -transferases leading to the formation of oxidised glutathione and aldehydes. The reaction of ozonides and peroxides with glutathione was investigated using molecular orbital calculations and the frontier orbital theory. In addition to the results obtained in the comparative study on the toxicity of ozonides and peroxides, the reaction of ozonides with the nucleophilic agent glutathione appeared also to be different when compared with the reaction with peroxides. On the basis of semi-empirical molecular computer calculations the nucleophilic attack by glutathione on the ozonide is expected to occur at one of the carbon atoms of the ozonide ring instead of at one of the peroxidic oxygen atoms as in the case of peroxides. A mechanism for the glutathione S -transferase-mediated detoxification of ozonides, different from that of the reaction with hydroperoxides has been proposed.Furthermore, the in vivo toxicity of ozonides was investigated. Methyl linoleate ozonide (MLO) (0.07 mmol/100 g body wt) was administered to female Wistar rats either intravenously or intraperitoneally. After 24 h the rats were killed and the effects were examined. MLO was found to be toxic only after intravenous administration. The major effects were observed in the lungs. The lungs became enlarged from edema and showed severe haemorrhages. Furthermore, the total thiol level was depleted in serum and lung tissue, accompanied by a decrease in the activity of thiol-dependent enzymes. The vitamin E levels in serum and lung tissue were also reduced. The malondialdehyde (MDA) concentrations in serum and lung tissue were elevated suggesting that in vivo oxidation had occurred. On intraperitoneal administration of MLO, no effects on enzyme activities, thiol and vitamin E content in lung tissue were observed. In serum, however, as on intravenous administration, an increase in the MDA levels and decreases in total thiol and vitamin E levels were found. In view of the route of administration it is to be expected that the ozonide is partially cleared by the liver, and the ozonide and its potentially toxic products are further detoxicated by vitamin E and thiols in the serum before they reach the lung. The above data show that the main target organ for ozonides is the lung, and that the effects caused by MLO in vivo are in many respects similar to the effects found after acute ozone exposure.In short, the most important conclusions of the present studies are:Ozonides exert their toxic effects by a mechanism different from that from the structurally related peroxides.The protective action ofα-tocopherol against ozonides proceeds via a direct interaction by whichα-tocopherol facilitates ozonide degradation through peroxide bond homolysis already at 37 °C.The involvement of ozonides in ozone-induced toxicity may be more important than currently suggested, since they are far more toxic than their aldehyde-type degradation products, which are generally observed as major products resulting from the reaction of ozone with unsaturated fatty acids under physiological conditions.Overall, the results presented in the thesis provide new insights into the toxic mechanism of ozonides and their implications for ozone-induced lung toxicity.