**3. A concise summary of biological effects observed after ozone therapy**

### **3.1. Erythrocytes**

Although ozone is one of the most potent disinfectants, it cannot inactivate bacteria, viruses and fungi in vivo because, paradoxically, the pathogens are well protected, particularly inside the cells, by the powerful antioxidant system. Thus, as it was proposed a long time ago [22,23], ozone acts as a mild enhancer of the immune system by activating neutrophils and stimulating the synthesis of some cytokines (2,5–7). Once again, the crucial messenger is hydrogen peroxide, which after entering into the cytoplasm of blood mononuclear cells (BMC) by oxidizing selected cysteines, activates a tyrosine kinase, which then phosphorylates the transcription factor nuclear factor kB [24], allowing the release of a heterodimer (p50+p65).

164 Pharmacology and Nutritional Intervention in the Treatment of Disease

This complex moves on to the nucleus and switches on some hundred genes eventually responsible for causing the synthesis of several proteins, among which are the acute-phase reactants and numerous interleukins. In the past, it was measured the release of several cytokines from ozonated blood upon in vitro incubation (2–7). Once the ozonated leukocytes return to the circulation, they home in lymphoid microenvironments and successively release cytokines acting in a paracrine fashion on neighboring cells with a possible reactivation of a depressed immune system [25]. This process, described as the physiological cytokine response,

is part of the innate immune system and helps us to survive in a hostile environment.

During ozonation of blood, particularly if it is anticoagulated with heparin, we have noted an ozone dose-dependent increase of activation of platelets [8, 26] with a consequent release of typical growth factors, which will enhance the healing of chronic ulcers in ischemic patients. Whenever possible, the use of heparin as an anticoagulant is preferable to sodium citrate because, by not chelating plasmatic Ca++, it reinforces biochemical and electric events.

During reinfusion of the ozonated blood into the donor, the vast expanse of the endothelial cells will be activated by LOPs, resulting in an increased production of NO, plasma Snitrosothiols and S-nitrosohemoglobin [15, 27]. Whereas NO has a half-life of less than 1 sec, protein-bound-NO can exert vasodilation also at distant ischemic vascular sites with relevant

Moreover, on the basis of the phenomenon of ozone tolerance that says the exposure of an organism to a low level of an agent, harmful at high levels, induces an adaptive and beneficial response [28,29], it is postulated that LOPs, by acting as long-distance messengers, can transmit to all organs the information of an acute oxidative stres [10, 11]. The bone marrow is particu‐ larly relevant because it can upregulate antioxidant enzymes during erythrogenesis and allows the release of staminal cells for possibly regenerating infarcted organs. Moreover, the stimu‐ lation of the endocrine and central nervous systems may help to understand why most patients during prolonged ozone therapy report a feeling of euphoria and wellness, probably due to an improved metabolism as well as to an enhanced hormonal or neurotransmitter release.

The paradoxical concept that ozone eventually induces an antioxidant response capable of reversing a chronic oxidative stress is common in the animal and vegetal kingdom and there is good experimental evidence [30–34] that this phenomenon is present in the animal and vegetal kingdom. Moreover, it is already supported by findings of an increased level of antioxidant enzymes and HO-1 during ozone therapy [10,11]. It also suggests that a judicious use of ozone, in spite of acting as an oxidant, enhances the antioxidant capacity, which

therapeutic effect.

These cells respond with an activation of glycolysis due to activation of the pentose hosphate pathway. It is found that increased adenosine triphosphate levels (from 13899/260 to 19689/232 mM) in patients with age-related macular degeneration (ARMD) (atrophic form) after a therapeutic cycle (14 sessions) of O3-AHT.26 Moreover, Viebahn [27] reported the same effect in athletes and elderly patients after rectal insufflation of O2\_/O3. Ozonation implies a small but consistent oxidation of GSH to glutathione disulfide, and GSH reductase utilizes the reduced form of the coenzyme nicotinamide adenine dinucleotide phosphate supplied by G6PDH to reduce glutathione disulfide to GSH, which indeed returns rapidly to the original level. [28] The increase of 2,3-diphosphoglycerate varies depending on the basic level in ARMD patients and only those who had a low level showed a marked increase with therapy. Viebahn, [27] after a longer cycle of therapy in elderly people, observed a significant increase. An increase of 2,3-diphosphoglycerate level in oxyhemoglobin shifts to the right (p50 value increases); its dissociation curve implies an increased delivery of O2 into the hypoxic tissues. The life-span of ozonated 99Tc-labeled erythrocytes and their uptake by liver and spleen are comparable with oxygenated erythrocytes.(3)

A problem still under study regards the generation of biochemically improved erythrocytes during prolonged ozone therapy. While ROS have an extremely short life, LOPs, during the reinfusion of ozonated blood, return into the donor's circulation. While they are fairly stable in vitro, they rapidly disappear from blood in vivo owing to considerable dilution into body fluids, degradation by aldehyde dehydrogenases, excretion into bile and urine, and uptake in various organs including bone marrow cells. This process is crucial for explaining the mech‐ anism of ozone tolerance: during erythrogenesis, submicromolar LOP concentrations can upregulate the synthesis of antioxidant enzymes and indeed, after appropriate density gradient separation, it is found that young (lighter) erythrocytes contain more G6PDH than older (heavier) cells generated before the therapy. [25] This result suggests that ozone therapy enhances the generation of erythrocytes with improved metabolic characteristics, a sort of 'supergifted erythrocytes' able to correct hypoxia in vascular diseases.

### **3.2. Leukocytes**

These were the cells that were examined first as it is hypothesized that ozone could act as an IFN-g inducer.[29] Since then it is shown [30,31] that ozone behaves as a weak (compared with mitogens) cytokine (such as tumor necrosis factor-a, interleukin-2, interleukin-6, interleukin-8, transforming growth factor-b [TGF-b]) inducer. Several studies [32 - 35] have confirmed that ozone can stimulate bronchoalveolar cells to release proinflammatory cytokines and eicosa‐ noids. Thanks to parallel progress in understanding the role of antioxidants and redox regulation of gene transcription, it has been clarified that, among several signals, H2O2 is one of the most significant cytokine inducers.[36] As already mentioned, after ozonation H2O2 freely diffuses into the leukocyte cytoplasm and activates specific protein kinases that, by phosphorylating IkB bound to the nuclear factor-kB allows the migration of the transcription heterodimer p50\_/p65 into the nucleus where it activates gene expression.[37] Obviously H2O2 must reach a concentration able to activate the kinase without being instantaneously reduced by intracellular antioxidants. [38] Therefore the relevance of the response depends on the levels of H2O2, which can act as either 'life or death' signals. The fact that ozone can either be a toxic or a useful signal depends on the minimal antioxidant capacity of the respiratory tract lining fluid, whereas blood has a very potent capacity. The data in fact indicate that too little ozone (hence H2O2) is ineffective and too much (or too little antioxidants) can be toxic. During recent years it is addressed the following questions: first, as ozone acts as a mild cytokine inducer, does reinfusion of ozonated blood modify the plasma cytokine level in vivo? Second, does the induction of oxidative stress proteins, particularly of heme-oxygenase I (HO-I), and of adaptation to the therapeutic oxidative stress have an immunomodulatory effect? And third, can we devise an optimal schedule for improving the immune reactivity in immunodepressed patients?

trations (20, 40 and 80 mg/ml). Because the plasmatic Ca2\_ level potentiates the ozone effect, we were not surprised to observe a rapid platelet aggregation in heparinized plasma particu‐ larly at the highest concentration. [47] Consequently the release of several growth factors like platelet-derived growth factor AB, TGF-b1, interleukin-8 and thromboxane\_/2 were signifi‐ cantly higher from heparinized platelets than Ca2\_-free platelets. [48] These results taught us that it is better to chelate Ca2\_ for performing a safe autohemotherapy. Nonetheless the release of growth factors from Ca2\_-free platelets is still important because the reinfusion of ozonated blood implies an elevation of plasma levels of TGF-b that may explain why the healing of necrotic ulcers in hind limb ischemia due to atherosclerosis and diabetes markedly quickens during treatment with both parenteral (O3-AHT) and topical treatments with ozonated water

General Aspects of Ozone Therapy http://dx.doi.org/10.5772/57470 167

During the reinfusion of ozonated blood, the endothelium comes in contact with traces of LOPs that soon disappear in vivo. It is [51] investigated the effect of addition of ozonated (40 and 80 mg/ml) human plasma to human endothelial cells in culture and it is measured a significant

The induction of nitric oxide synthase and the release of NO+was reinforced in the presence of 20 mm of arginine and was abolished by the addition of 20 mM of L-N-omega-nitro-Larginine methyl ester. In physiological conditions the endothelium regulates the vascular tone [52] by producing some 1\_/10 mM of NO+and 1 nM of anion superoxide (one of the contracting factors). The intravascular half-life of NO+is about 2 msec with a strictly localized consumption so that the likelihood of improving vasodilation in remote ischemic areas (the macula or the limbs) seems negligible. However, NO+readily reacts with GSH, cysteine, albumin and hemoglobin (cysteine residue b 93) and the formed S-nitrosothiols and S-nitrosohemoglobin have half-lives of 5\_/50 min, allowing a pharmacological effect at distant sites. [53 – 54] It remains to be ascertained whether ozonated blood enhances the release of prostacyclin (PGI2)

increase of the critical relaxing factor NO+that was ozone dose dependent.

and angiopoietins, both important factors for improving ischemic vasculopathies.

**1.** Neuro-endocrine responses explaining the reported feeling of wellness, [55]

Upon reinfusion of ozonated blood, LOPs can reach other organs such as the hypothalamus, endocrine glands, liver, kidneys and bone marrow. The phenomenon of adaptation to the repeated and acute oxidative stress imposed by O3-AHT is most interesting and able to elicit crucial therapeutic responses. During prolonged treatment, cells throughout the body receive

**2.** The upregulation of antioxidant enzymes in several cell types that is an excellent way to

**3.** Inducing a number of stress or heat shock proteins (HSPs) such as HSP27, HO-1 (HSP 32),

and oil. [49 – 50]

**3.4. Endothelial cells and the vascular system**

**3.5. Parenchymal cells in other organs**

HSP72 and HSP [90 – 56 – 59]

small and gradual pulses of LOPs that are responsible for:

re-equilibrate the oxidant\_/antioxidant unbalance

The classical O3-AHT, usually consisting of 225 ml of blood (plus 25 ml of 3.8% sodium citrate solution) treated with 225 ml of gas (O2\_/O3) with ozone concentrations ranging from an initial 20 mg/ml slowly scaled up to 40\_/50 mg/ml per ml of blood, continued for several months, twice weekly, is ideal for this purpose. A probable explanation is that, after each blood reinfusion, a small percentage of immune cells are activated and home in several organs: these cells release into the microenvironment cytokines that, in turn, prime or activate neighboring cells thus slowly reinforcing immune responses. Modifications of cytokine plasma levels are hardly detectable so that side effects like the flu-like syndrome, typically observed after administration of immunoadjuvants, are absent [39] and actually most of patients report a sense of well-being during the therapy. There is a wealth of experimental data [40 - 44] showing that both animals and plants can develop ozone tolerance by upregulating the expression of antioxidants, which can correct a chronic imbalance between excessive endogenous oxidation due to viral infections, cancer, chronic inflammations and depressed antioxidants. Both chronic hepatitis C virus and cancer patients have shown a marked improvement of their clinical conditions after several months of O3-AHT treatments, suggesting that this 'calculated and brief oxidative stress' truly merits the term 'therapeutic shock'.

#### **3.3. Platelets**

It is known that ROS can induce platelet activation and it was obvious to assume that blood ozonation, by generating H2O2, could cause it. [45 – 46] Moreover H2O2 or other ROS can activate phospholipase C, phospholipase A2, cyclo-oxygenases and lipo-oxygenases and thromboxane synthetase, allowing a step increase of intracellular Ca2\_, release of prostaglan‐ din E2, prostaglandin F2a and thromboxane A2 with irreversible platelet aggregation. For these reasons it is studied the behavior of either human platelet rich-plasma anticoagulated with heparin or citrate, either untreated or simply oxygenated, or ozonated at three concen‐ trations (20, 40 and 80 mg/ml). Because the plasmatic Ca2\_ level potentiates the ozone effect, we were not surprised to observe a rapid platelet aggregation in heparinized plasma particu‐ larly at the highest concentration. [47] Consequently the release of several growth factors like platelet-derived growth factor AB, TGF-b1, interleukin-8 and thromboxane\_/2 were signifi‐ cantly higher from heparinized platelets than Ca2\_-free platelets. [48] These results taught us that it is better to chelate Ca2\_ for performing a safe autohemotherapy. Nonetheless the release of growth factors from Ca2\_-free platelets is still important because the reinfusion of ozonated blood implies an elevation of plasma levels of TGF-b that may explain why the healing of necrotic ulcers in hind limb ischemia due to atherosclerosis and diabetes markedly quickens during treatment with both parenteral (O3-AHT) and topical treatments with ozonated water and oil. [49 – 50]

#### **3.4. Endothelial cells and the vascular system**

regulation of gene transcription, it has been clarified that, among several signals, H2O2 is one of the most significant cytokine inducers.[36] As already mentioned, after ozonation H2O2 freely diffuses into the leukocyte cytoplasm and activates specific protein kinases that, by phosphorylating IkB bound to the nuclear factor-kB allows the migration of the transcription heterodimer p50\_/p65 into the nucleus where it activates gene expression.[37] Obviously H2O2 must reach a concentration able to activate the kinase without being instantaneously reduced by intracellular antioxidants. [38] Therefore the relevance of the response depends on the levels of H2O2, which can act as either 'life or death' signals. The fact that ozone can either be a toxic or a useful signal depends on the minimal antioxidant capacity of the respiratory tract lining fluid, whereas blood has a very potent capacity. The data in fact indicate that too little ozone (hence H2O2) is ineffective and too much (or too little antioxidants) can be toxic. During recent years it is addressed the following questions: first, as ozone acts as a mild cytokine inducer, does reinfusion of ozonated blood modify the plasma cytokine level in vivo? Second, does the induction of oxidative stress proteins, particularly of heme-oxygenase I (HO-I), and of adaptation to the therapeutic oxidative stress have an immunomodulatory effect? And third, can we devise an optimal schedule for improving the immune reactivity in immunodepressed

166 Pharmacology and Nutritional Intervention in the Treatment of Disease

The classical O3-AHT, usually consisting of 225 ml of blood (plus 25 ml of 3.8% sodium citrate solution) treated with 225 ml of gas (O2\_/O3) with ozone concentrations ranging from an initial 20 mg/ml slowly scaled up to 40\_/50 mg/ml per ml of blood, continued for several months, twice weekly, is ideal for this purpose. A probable explanation is that, after each blood reinfusion, a small percentage of immune cells are activated and home in several organs: these cells release into the microenvironment cytokines that, in turn, prime or activate neighboring cells thus slowly reinforcing immune responses. Modifications of cytokine plasma levels are hardly detectable so that side effects like the flu-like syndrome, typically observed after administration of immunoadjuvants, are absent [39] and actually most of patients report a sense of well-being during the therapy. There is a wealth of experimental data [40 - 44] showing that both animals and plants can develop ozone tolerance by upregulating the expression of antioxidants, which can correct a chronic imbalance between excessive endogenous oxidation due to viral infections, cancer, chronic inflammations and depressed antioxidants. Both chronic hepatitis C virus and cancer patients have shown a marked improvement of their clinical conditions after several months of O3-AHT treatments, suggesting that this 'calculated and

It is known that ROS can induce platelet activation and it was obvious to assume that blood ozonation, by generating H2O2, could cause it. [45 – 46] Moreover H2O2 or other ROS can activate phospholipase C, phospholipase A2, cyclo-oxygenases and lipo-oxygenases and thromboxane synthetase, allowing a step increase of intracellular Ca2\_, release of prostaglan‐ din E2, prostaglandin F2a and thromboxane A2 with irreversible platelet aggregation. For these reasons it is studied the behavior of either human platelet rich-plasma anticoagulated with heparin or citrate, either untreated or simply oxygenated, or ozonated at three concen‐

brief oxidative stress' truly merits the term 'therapeutic shock'.

patients?

**3.3. Platelets**

During the reinfusion of ozonated blood, the endothelium comes in contact with traces of LOPs that soon disappear in vivo. It is [51] investigated the effect of addition of ozonated (40 and 80 mg/ml) human plasma to human endothelial cells in culture and it is measured a significant increase of the critical relaxing factor NO+that was ozone dose dependent.

The induction of nitric oxide synthase and the release of NO+was reinforced in the presence of 20 mm of arginine and was abolished by the addition of 20 mM of L-N-omega-nitro-Larginine methyl ester. In physiological conditions the endothelium regulates the vascular tone [52] by producing some 1\_/10 mM of NO+and 1 nM of anion superoxide (one of the contracting factors). The intravascular half-life of NO+is about 2 msec with a strictly localized consumption so that the likelihood of improving vasodilation in remote ischemic areas (the macula or the limbs) seems negligible. However, NO+readily reacts with GSH, cysteine, albumin and hemoglobin (cysteine residue b 93) and the formed S-nitrosothiols and S-nitrosohemoglobin have half-lives of 5\_/50 min, allowing a pharmacological effect at distant sites. [53 – 54] It remains to be ascertained whether ozonated blood enhances the release of prostacyclin (PGI2) and angiopoietins, both important factors for improving ischemic vasculopathies.

#### **3.5. Parenchymal cells in other organs**

Upon reinfusion of ozonated blood, LOPs can reach other organs such as the hypothalamus, endocrine glands, liver, kidneys and bone marrow. The phenomenon of adaptation to the repeated and acute oxidative stress imposed by O3-AHT is most interesting and able to elicit crucial therapeutic responses. During prolonged treatment, cells throughout the body receive small and gradual pulses of LOPs that are responsible for:


It is observed that HO-1 is a protective enzyme allowing the formation of Fe2\_, bilirubin (an antioxidant) and carbon monoxide (CO), a vasodilator that, like NO+, increases the level of cyclic guanosine monophosphate, the reaction catalyzed by guanylate cyclase. Besides gases produced by the gut flora, it is truly remarkable that cells can release other gaseous molecules (NO+, CO and CO2), and it is even more surprising that even ozone can be produced by activated antibody-coated neutrophils. [60] These gases can now be considered as molecules able to deliver crucial physiological and pharmacological effects.

**4.1. Acute and chronic bacterial, viral and fungine infections**

**4.2. Ischemic diseases**

**4.3. Age-related macular degeneration**

**4.4. Orthopedic diseases**

Intuitively, ozone therapy is very useful in both acute and chronic bacterial, viral and fungine infections because the generated ROS are the natural and most effective agents to which even antibiotic resistant pathogens do not resist. [3-61] Moreover, improvement of metabolism and immunological functions contribute to a favorable outcome. Abscesses, anal fissures, fistulae, bed sores, furunculosis, inveterate osteomyelitis, vulvovaginitis, necrotizing fasciitis and torpid ulcers of various origin have been shown to improve rapidly, particularly using the combination of O3-AHT with topical treatment using either direct O2\_/O3 exposure or the cleansing and stimulating effect of ozonated water and oil. The activity of ozonated solutions in eliminating the infectivity and enhancing healing is almost unbelievable. However, in Western countries accustomed to the use of antibiotic creams (often with corticosteroids) there is no mental attitude to profitably use the inexpensive and most active ozonated oil. [62]

General Aspects of Ozone Therapy http://dx.doi.org/10.5772/57470 169

Chronic limb ischemia (atherosclerosis, diabetes, Burger's disease) is most effectively treated at stage II-b with complete disappearance of pain and claudication. Moreover, since 1981, Rokitansky et al.[49] demonstrated that a cycle of O3-AHT (usually 14 treatments) led to a very good improvement in 70.6% and 53.8% of either stage III or stage IV (Fontaine) patients,

These results have been amply confirmed by Giunta et al.,[63] Mattassi et al. [64] and Tylicki et al.[65] Preterminal patients with chronic heart ischemia and no further susceptibility to conventional treatments have shown marked improvement after a cycle of 14 treatments of extracorporeal circulation of blood against O2\_/O3.[66] A randomized controlled study is in progress for establishing the validity of this more invasive method than classical O3-AHT.

A 6-year study in 90 patients with the 'dry' form of ARMD has been carried out performing a cycle of 13\_/14 O3-AHT treatments. Mean distance best-corrected visual acuity was signifi‐ cantly improved in the treatment group of patients while in the control group, first treated with oxygenated autohaemotherapy, only a modest and not significant improvement in mean distance visual acuity was observed. No adverse effects have been noted and the patient's compliance has been excellent. [26] Owing to the constant increase of ARMD patients and the

Until recently it was unthinkable that a mixture of O2\_/O3 could be useful in orthopedics. Indeed lumbar disk herniation and osteoarthritis, although having different etiologies, have a common inflammatory background expressed by a localized chronic oxidative stress due to excessive production of ROS, release of proinflammatory cytokines and activation of cyclo‐ xygenases. Common sense would proscribe the use of ozone, a master generator of free radicals and, as it is well shown, [34 – 67] after pulmonary exposure, a superb inflammatory agent.

lack of an effective conventional treatment, this approach appears mandatory.

respectively. Amputation of toes and limbs could be avoided in pre-terminal phases.

Excessive amounts of these molecules are toxic, causing serious pathological events and possibly death. Nature teaches us that these gases, depending on their concentrations, can be either friends or foes and similarly ozone therapy can be either useful or toxic. If this reasoning is correct, ozone therapy, when judiciously performed, is a simple, inexpensive and atoxic approach with the advantage of activating several biomechanisms in different cells unusually leading to an integrated and often incredible response.
