At the airspace level, the alveolar cells are constantly overlaid by a film composed of water, salts and a myriad of biomolecules such as a profusion of surfactant phospholipids and a very small amounts of proteins, lipophilic and hydrophilic antioxidants. Any inspired gas, depending upon its relative concentration and pressure, must first dissolve into the aqueous layer before reaching the alveolar microcirculation and the erythrocytes. This process implies a physical transport regulated by a pressure gradient and a diffusion process. On the other hand, it is known that ozone, in contact with biological water, does not follow Henry's law and, although it is ten fold more soluble than oxygen, it is not transferred into the alveolar capillaries because it reacts immediately with the biomolecules present in the Alveolar Lining Layer (ALL).
It must emphasized that the average thickness of ALL is only 0.2 micron [
26]. As it was hypothesized [
11], ozone does not penetrate the cells but oxidizes available antioxidants and reacts instantaneously with surfactant's polyunsaturated fatty acids (PUFA) present at the interface to form Reactive Oxygen Species (ROS), such as hydrogen peroxide and a mixture of heterogenous LOPs including lipoperoxyl radicals, hydroperoxides, malonyldialdeyde, isoprostanes, the ozonide and alkenals, particularly 4-HNE [
27‐
29]. As cholesterol is a component of the epitherial lining fluids (ELF) and because its double bond is readily attacked by ozone, it can give rise to biologically active oxysterols [
30,
31] of which 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (CSeco) has been implicated in pulmonary toxicity, Alzheimer's disease and atherosclerosis.
The antioxidant capacity present in the human ALL is extremely limited and, although different portions of the respiratory tract may have different antioxidant levels, these are always irrelevant in comparison to the amount of antioxidants that, in blood, easily tame the ozone reactivity. First of all, by considering the expanse of the alveolar surface (1 m
2/kg body weight) in a 70 kg human, it can be calculated that the normal volume of ALL ranges only between 17 and 25 ml, whereas 5 L of blood include about 2.7 L of plasma. Moreover, the erythrocyte mass, amounting to about 2.3 kg, has an enormous antioxidant capacity due to hydro-lipophilic antioxidants and enzymes able to reduce any antioxidant in a few minutes [
32]. Erythrocytes, via glucose-6-phosphate dehydrogenase activity in the pentose cycle, can continuously supply NADPH-reducing equivalents. The amount of plasma albumin acting as a "sacrificial compound" against oxidants is impressive (99.9% higher than in ALL). Moreover erythrocytes have a GSH content of about 2.2 mM (almost 800-fold higher than plasma) and therefore they contain a huge reserve. In the course of evolution, aerobic organisms have developed a sophisticated antioxidant system against oxygen and, although about 2% of the inhaled oxygen generates superoxide anion, this is normally neutralized at an alveolar pO
2 pressure of 100 mm Hg. It is useful, however, to bear in mind that rats inhaling pure oxygen (alveolar pressure at about 700 mmHg) die within 60-66 h [
33], Ozone is far more reactive than oxygen, and breathing air containing 10.0 ppm ozone causes death within 4 h in rats. In order to understand the effects of a daily 8-hour ozone exposure (April-October), we need to know the average environmental ozone levels that vary considerably for many reasons. The US Clean Air Act has set an ozone level of 0.06 ppm as an 8-h mean concentration to protect the health of workers (U.S. Environmental Protection Agency, 2005). Evaluation of recent studies [
34,
35] allows establishing an average environmental ozone concentration of 0.09 ± 0.01 ppm. However, ozone concentration in urban air can exceed 0.8 ppm in high pollution conditions [
27]. For 8 h at rest (a tidal volume of about 10 l/min and a retention of inspired ozone of no less than 80%), the ozone dose amounts to 0.70-0.77 mg daily or 21.0-23.1 mg monthly. This is likely the minimal ozone intake because physical activity increases the volume of inhaled air and, at peak time, the ozone levels can easily augment to 200-300 ppb, reducing pulmonary functions and enhancing the risk of cardiovascular death [
21,
34‐
36]). Moreover, the toxicity is certainly augmented by the presence of NO
2, CO, SO
2 and particulate matters (PM10). On this basis, it appears clear how the ozone generated ROS and LOPs at the ALL level, after being minimally quenched by the insufficient, antioxidants will act as cell signals able to activate nuclear factor-kappa B (NF-κB), NO-synthase, some protein kinases, thus enhancing the synthesis and release of TNFα, IL-1, IL-8, IFNγ and TGFβ1 and the possible formation of nitrating species. With an increasing inflow into the alveolar space of neutrophils and activated macrophages, a vicious circle will start, perpetuating the production of an excess of ROS including also hypoclorous acid [
37,
38]. Moreover, during Summer, there is a continuous flow of ozone entering the respiratory space and also the very fact that ozone dissolves in the ALL and reacts immediately; thus, every second, more ozone reacts so that in a 6-month period the cumulative dose (likely up to 150-300 mg ozone) becomes really deleterious. Similarly several months exposure to ozone or to a prolonged oxidative stress due to a chronic inflammatory disease (atherosclerosis, diabetes, cancer) can possibly raise 4-HNE plasma levels up to 5-20 μM and, in spite of continuous detoxification, they can exert pathological effects as those observed in vitro studies performed with leukemic cells [
39], lens epithelial cells [
40], Jurkat T cells [
41] and when testing cholesterol secoaldehyde (CSeco) in cardiomyoblasts [
31]. Interestingly, tolerance to ozone or 4-HNE is far more easily achieved by small and repeated oxidative stresses than after a continuous and heavy oxidation [
42,
43]. On the other hand, a normal endogenous 4-HNE level (0.1- 0.7 μM) appears to act as a defensive agent against itself and other toxic compounds [
13,
44]. Thus, the biological behavior of HNE is an enlightening example of how the physiological serum level of a potentially toxic aldehyde produced by the normal peroxidation is proficiently used for maintaining homeostasis.
Finally, it is worthwhile to mention that the vast skin surface, possibly exposed for hours to ozone and UV radiation, can contribute to the overall toxicity: several studies performed by exposing hairless mice to ozone have shown not only depletion of the skin antioxidants but the induction of a remarkable oxidative stress [
22,
23]. As a consequence, humans, living in hot countries and during summer, become particularly susceptible to ozone and UV irradiation. On the contrary, a quasi-total (excluding the neck and the head) exposure of human volunteers to a very low ozone concentration in a sauna cabin for 20 min results in a very transient increase of LOPs in the peripheral circulation that exerts therapeutic effects in chronic limb ischemia's patients [
45,
46] interpreted as due to an induction of antioxidant enzymes and HO-1. In conclusion, although ozone is not the only culprit for adverse health effects, it significantly contributes to exacerbate respiratory illnesses and enhances mortality in about 40% of the total US population [
21]. The problem is linked to the abnormal ozone concentration of troposheric ozone and to the continuously increased production of noxious compounds due to the excess burning of coal, oil and virgin forests. During this century, if human activities continue to release in the atmosphere the actual amount of global emissions, the Earth will experience a new Paleocene-Eocene Thermal Maximum or
"a fever period" with dramatic consequences. The overall toxicity, due to the constant aggressiveness of ozone on lungs and partly on the exposed skin, associated with the relative efficiency of the detoxifying system, progressively overwhelmed by the perennial stress, favours pathological effects such as inflammation and cell degeneration particularly on lungs, liver (fibrosis), heart, kidneys and brain [
47,
48]. Consequently vital organs can be envisaged to be subjected to a kind of a "toxic rain" produced in the pulmonary system. Obviously, this knowledge has popularized the idea of ozone toxicity but, in the next section, it will be clarified that the generalization of this concept is incorrect.