Ozone acting on human blood yields a hormetic dose-response relationship
© Bocci et al; licensee BioMed Central Ltd. 2011
Received: 29 November 2010
Accepted: 17 May 2011
Published: 17 May 2011
The aim of this paper is to analyze why ozone can be medically useful when it dissolves in blood or in other biological fluids. In reviewing a number of clinical studies performed in Peripheral Arterial Diseases (PAD) during the last decades, it has been possible to confirm the long-held view that the inverted U-shaped curve, typical of the hormesis concept, is suitable to represent the therapeutic activity exerted by the so-called ozonated autohemotherapy. The quantitative and qualitative aspects of human blood ozonation have been also critically reviewed in regard to the biological, therapeutic and safety of ozone. It is hoped that this gas, although toxic for the pulmonary system during prolonged inhalation, will be soon recognized as a useful agent in oxidative-stress related diseases, joining other medical gases recently thought to be of therapeutic importance. Finally, the elucidation of the mechanisms of action of ozone as well as the obtained results in PAD may encourage clinical scientists to evaluate ozone therapy in vascular diseases in comparison to the current therapies.
Ozone is a double-faceted gas. It has a crucial protective relevance in partially blocking mutagenic and carcinogenic UV radiations emitted by the sun (wavelengths of 100-280 nm) in the stratosphere , while its increasing concentration in the troposphere causes severe pulmonary damage and increased mortality [2, 3]. In spite of this drawback, there are growing experimental and clinical evidences about the medical use of ozone [4–11]. Since XVI Century, Paracelsus had ingeniously guessed that "all things are poison and nothing is without poison and only the right dose differentiates a poison from a remedy". In 2005, Timbrell reiterated the concept in his book: "The poison paradox; chemicals as friends and foes" . During the Earth evolution, harnessing oxygen by metazoans has allowed a fantastic biodiversity and growth but it has also created a slow acting "poison". It is reasonable to believe that the antioxidant system slowly evolved and specialized during the last two billion years for counteracting the daily formation (3-5 g in humans) of anion superoxide in the mitochondria and the release of H2O2 by ubiquitous NADPH oxidases. However, there is a general consensus that the physiological production of H2O2 is essential for life. Olivieri et al. and Wolff  were the first to describe the effect of either low concentrations of radioactive thymidine or of a very low dose of radiation inducing an adaptive response in human cells in comparison to a high dose. Goldman  introduced the term "hormesis" to mean "the beneficial effect of a low level exposure to an agent that is harmful at high levels". It goes to the merit of Calabrese [16–19] to have experimentally controlled this concept and to have presented a number of examples of stimulatory responses following stimuli below the toxicological threshold. Until 2002 ozone therapy was pharmacologically conceived as a therapy where low ozone doses were stimulatory, while high doses were inhibitory. This conception, reflecting the classical idea that a low antigen dose is stimulatory, where an antigen overdose is inhibitory, was vague and unsuitable because ozone acts in a complex way and a high dose can still be effective but accompanied by side-effects. Indeed, one of us in 2002 amply delineated the sequence of biochemical reactions elicited ex vivo after the addition of a certain volume of O2-O3 gas mixture to an equal volume of human blood . First of all, mixing blood with an oxidant implies a calculated and precise oxidative stress, i.e. a homeostatic change with production of highly reactive messengers. The oxidative stress, like many others, induces a biological response leading to an adaptive phenomenon. The teleological significance of this response is universal, from bacteria to plants and Mammals, and small repetitive stresses induce an extremely useful adaption response represented by the revival of critical defense mechanisms [20–22]. At the same time, Calabrese and Baldwin described the "overcompensation stimulation hormesis" (OCSH) as the result of a compensatory biological process following an initial disruption in homeostasis . After a reviewer's information also Re later on had expressed this possibility . Ozone presents some subtle differences that will be explained by clarifying the biochemical reactions occurring between the organic compounds of plasma and this gas.
Ozone is a Strong Oxidant Gas
Such an aspect has generated the idea that ozone will donate its energy to the organism by reacting with specific body compartments . However, after having ascertained the complexity of the mechanism of action, the conclusion is that ozone dissolved in the water of plasma acts as a pro-drug, generating chemical messengers which will accelerate transfer of electrons and the overall metabolism. It goes to the merit of Hans Wolff (1927-1980), a German physician, to have developed the O3-AHT by insufflating ex vivo a gas mixture composed of medical oxygen (95%) and ozone (5%) into the blood contained in a dispensable ozone-resistant and sterile glass bottle .
Which are the Blood Components Reacting with Ozone?
The Biochemical Reactions of Ozone with Blood
As all of these reactions happen in a few seconds, ozone, until present in the gas phase, continues to dissolve in the plasmatic water and instantly reacts. Within the canonical 5 min, ozone is fully extinct with both a rather small depletion of hydrosoluble antioxidants and the simultaneous plasmatic increase of ROS and LOP. The ozonated blood is then infused into the donor patient.
What is the Significance and Fate of These Ozone Messengers?
First of all the brief life-span of H2O2 will be discussed. During the 5 min of mixing blood with the gas ex vivo, H2O2 will dynamically increase its concentration: rapid at first and progressively slowing down as ozone is being depleted. With the therapeutically high ozone concentration of 80 μg/ml per ml blood, the H2O2 concentration measured in plasma after 2.5 min is at most 40 μM because the rate of synthesis is equilibrated by multiple degradation routes. Some H2O2 is reduced by free soluble antioxidants including traces of catalase and GSH-Px. As the hemolysis is negligible (<0.5%), free Fe2+ or Cu+ are not present and it is unlikely that hydroxyl ions are ever formed by either the Fenton-Jackson or the Haber-Weiss reactions. As H2O2 is unionized, it freely diffuse into all blood cells although the bulk is mopped up by erythrocytes. The establishment of a dynamic, yet transitory, H2O2 gradient between the plasma and the cytoplasmatic water of blood cells makes this oxidant a very early effector. Its final intracellular concentration may be not higher than 10%, hence 3-4 μmoles, as it has been demonstrated in other studies [34–39]. The smartness of this system is that the H2O2 concentration, though small, is enough to trigger several crucial biochemical reactions without toxicity because the internal cell environment contains a wealth of GSH, thioredoxin, catalase and GSH-Px, which do not allow a dangerous increase. In spite of a threshold of only a few micromoles, it has a critical relevance and means that an ozone amount below 0.42 μmol for each ml volume of the gas mixture (medical grade O2 ≥95% and O3 ≤5%) reacting in a 1:1 ratio with autologous blood may be ineffective, resulting in a therapeutic failure of O3-AHT. It is also necessary to remind that the ozonation process greatly differs whether it occurs either in plasma or in blood. In plasma, TAS levels was, as expected, ozone-dose dependent and decreased between 46 and 63% in relation to ozone concentrations of either 0.84 μmol/ml or 1.68 μmol/ml per ml of plasma, respectively. On the other hand, in blood taken from the same donors, after being treated with the same ozone concentrations, TAS only decreased from 11 to 33% in the first minute after ozonation, respectively. Moreover, it was surprising to determine that they both recovered and returned to the original value within 20 min, indicating the capacity of blood cells to quickly regenerate dehydroascorbate and GSH disulfide . It has been also brilliantly demonstrated that, thanks to erythrocytes, dehydroascorbate was recycled back to Aa within 3 min . On the same way, only about 20% of the intraerythrocytic GSH had been oxidized to GSSG within one min after ozonation and promptly reduced to normal after 20 min . Aa, alpha-tocopherol, GSH and lipoic acid undergo an orderly reduction by a cooperative sequence of electron donation continuously supplied by NADPH-reducing equivalents to GSH-Rd and thioredoxin reductase  (Figure 1). These data, by showing that the therapeutic ozonation only temporarily and reversibly modifies the cellular redox homeostasis were reassuring regarding the safety of ozone as a medical drug. In summary, the initial disruption of homeostasis due to ozone oxidation is followed by the rapid reestablishment of homeostasis with two main advantages: the first being the value of triggering several biochemical reactions in blood cells and the second mediated by LOP compounds, the induction of an adaptive process due to the up-regulation of the antioxidant enzymes. This is in line with the temporal sequence of the OCSH dose-response relationship.
What is the Action of Ozone in the Blood Cells?
Probably the activation of phosphofructokinase accelerates glycolysis with a demonstrated increase of ATP and 2,3-DPG [4, 20]. Functionally, the oxyhemoglobin sigmoid curve shifts to the right owing to the Bohr effect, i.e. a small pH reduction (about 7.25) and a slight increase of 2,3-DPG. This metabolite increases only in patients who have a very low level but it remains to be clarified how the phosphoglyceromutase is activated. The shift to the right is advantageous for improving tissue oxygenation as the chemical bonding of oxygen to hemoglobin is attenuated, facilitating oxygen extraction from ischemic tissues. Rokitansky et al., had previously shown that the pO2 was lowered to 20-25 mm Hg in the femoral vein of PAD's patient throughout O3-AHT sessions . It seems obvious that erythrocytes ozonated ex vivo may be modified only for a brief period. Only repeated therapeutic sessions may allow to LOP compounds to reach the bone-marrow and activate a subtle development at the erythropoietic level, favouring the formation of new erythrocytes with improved biochemical characteristics, which provisionally were named "supergifted erythrocytes" . If this hypothesis is correct, every day, during prolonged ozonetherapy, the bone marrow may release a cohort (about 0.9% of the pool) of new erythrocytes with improved biochemical characteristics. In fact, the therapeutic advantage does not abruptly stop with the cessation of the therapy but rather persists for 2-3 months, probably in relation to the life-span of the circulating supergifted erythrocytes . It is interesting that during prolonged ozonetherapy, by isolating through a sedimentation gradient the small portion of very young erythrocytes, it has been demonstrated that they have a significant higher content of G6PD . Such a result strengthens the postulation that only a cycle of more than 15 treatments (not less than 3 liters of ozonated blood) could improve an ischemic pathology.
Human neutrophils are able to generate an ozone-like molecule  and volatile compounds  as a part of their phagocyte activity. Neutrophil phagocytic activity has been found enhanced during ozonetherapy . Moreover, H2O2 activates a tyrosin-kinase with subsequent phosphorylation of IkB, one of the trimeric components at rest of the ubiquitous transcription factor denominated NF-kB [48, 49]. The phosphorylated IkB detaches from the trimer and it is broken down in the proteasome. The remaining eterodimer p50-p65 is transferred into the nucleus, where it can activate about 100 genes up-regulating the synthesis of acute-phase proteins, several proinflammatory cytokines (IFN-γ, TNF-α, IL-8) and even HIV proteins . There is no doubt that H2O2 is the trigger as the activation is related to a cysteine oxidation that can be prevented by an excess of thiol. Although ozone is a very modest inducer of some cytokines , the consequent immunomodulatory effect may be useful in immune-depressed patients after chemotherapy, or in chronic infectious diseases. It must be clear that ozone in itself cannot exist in the circulation and moreover, due to the potent antioxidant capacity of plasma, it is unable to kill any pathogens in vivo whereas an activated immune system may be helpful .
During O3-AHT, the detection of PDGF-B, TGF-β1, IL-8 and EGF released in heparinized plasma in ozone- dose dependent quantities was not surprising because platelets are exquisitely sensitive to a progressive acute oxidative stress [20, 52]. The increased level of these growth factors in the circulation may have the beneficial effect of enhancing the healing of foot-related problems from diabetes or PAD.
The pleiotropic LOP activities
Another interesting aspect observed in about 2/3 of patients is a sense of wellness and physical energy throughout the ozonetherapy . It is not yet known whether these feelings are due to the power of the generated ozone messengers which can modify or improve the hormonal secretion. On the other hand, the feeling of euphoria may be due to improved oxygenation or/and enhanced secretion of growth hormone, ACTH-cortisol and dehydroepiandrosterone [26, 71]. Furthermore, when LOP reach the hypothalamic area they may improve the release of serotonin and endorphins, as it was observed after intense dynamic exercise . Experience acquired after thousands O3-AHT has clarified that there is neither objective nor subjective toxicity, or to use Calabrese's acronyms, there is no observable adverse effects (NOAEL). Moreover, neither structural nor enzymatic damages have been observed in blood components after ozonation of blood within the therapeutic window [73, 74]. On the other hand, patients with more advanced disease during the initial session especially if performed with a high ozone dosage, frequently report to feel very tired and sleepy. This is the lowest observed adverse effect level (LOAEL) that has been observed in about 10% of PAD's patients with stage III and IV of the Leriche-Fontaine's classification. Such a knowledge compels to begin always with low ozone dosage and carefully observe the patient's response.
Which is the Most Suitable Term for Describing the Dose-Response Relationship Between Ozone and Blood?
Ozone Therapy in Oxidative-Stress Related Diseases
The metabolic syndrome is recognized as one of the most serious disease in Western countries caused by a number of metabolic alterations such as type-2 diabetes, hypercholesterolaemia, atherosclerosis and renal dysfunction with the common denominator represented by a chronic oxidative stress. Diabetic patients, particularly those with foot ulcers, are critical and today they still have a gloomy prognosis. This is because they need a multiform therapy aiming to eliminate the peripheral ischemia, the neuropathy and the infected skin lesions. The range of ozone concentrations between 15 and 35-50 µ;g/ml is safe also in individuals with a low TAS level and it appears to be particularly effective in PAD [43, 80–85]. Several clinical studies performed in different hospitals seem to establish the validity of the inverted U-shaped curve in this frequent pathology (Figure 3, panel B). In line with "the concept of a beneficial effect within the context of a dose-response study is difficult to determine due to considerable biological complexity and the fact that beneficial effects are often seen with reference to a specific and relative setting" , a word of caution is necessary. This is especially true when ozone therapy is performed in different patients within the variety of three PAD's II, III and IV stages, according to the Leriche-Fontaine classification . First of all it is necessary to trust the precision of ozone's dosages used by different clinicians and secondly, ozone activity cannot be compared with that expressed by a single compound (see, eg, Arsenic , and homocysteine ) in cultured cells. As it has been clarified, the real ozone messengers are H2O2 as a ROS and a variety of alkenals as LOP. These messengers act on different cells, have a quite different lifetime and alkenals are intrinsically toxic. Furthermore, each patient has his own medical history and his own psycho-physical reactivity. Consequently, ozone dosages between 0.42-0.84 µ;mol/ml generate less alkenals than dosages in the range 0.84-1.68 µ;mol/ml, and therefore patients with a low antioxidant capacity become more susceptible to a side effect like deep fatigue after the therapy session. Attention must be also paid to the type of pharmacological response achieved in different pathologies as either muscular-orthopedic or autoimmune diseases. So far, in the latter it remains unknown the ozone dosage, if any, able to increase the T-cell regulatory levels and activity. Consequently, at this stage the U-shaped curve remains meaningful only for PAD and only future trials will be able to define the ozone behavior in either stroke or chronic heart disease. Martinez-Sanchez et al. have also reported that the theoretical U-shaped curve fits the ozone therapy results . Blood ozonation, even if performed within the therapeutic range and for a few minutes, represents always a calibrated acute oxidative stress. In order to never harm the patient, the strategy: "start low-go slow" is a golden rule to induce a valid adaptation to the far more dangerous chronic oxidative stress, typical of inflammatory and degenerative diseases . Such an aspect implies that the final therapeutic effect is due to an average of progressively increasing ozone dosages.
The gas mixture medical grade oxygen-ozone can be proficiently used for the ozonation of blood because this incomparable liquid tissue contains an imposing array of antioxidants, which are able to tame not only its oxidant power but also its messengers (ROS and LOP) generated by the reactions with blood components. Therefore, if ozone is judiciously used within the established therapeutic window (0.42-1.68 μmol/ml per ml of autologous blood) in PAD, it can exert better therapeutic effects than the current therapy by prostacyclin analogue. Moreover, regarding the accompanying foot-related problems, both some ozone derivatives like ozonated water and different gradation of standardized ozonated vegetable oils will be used until complete healing [89, 90]. As stroke, heart infarction and PAD are cumulatively the first cause of death and disability, if it will become possible to use ozone therapy in the public hospitals of the developed Countries, it may be possible to enter a phase where ozone will become an extensive remedy. Moreover, there is no doubt that either infective or autoimmune glomerulo-nephritis as well as end stages of renal failure associated with hemodialysis are characterized, to a different extent, by an imbalance between pro- and antioxidative mechanisms . Moreover the kidney does not have the regenerative ability of liver and this is one of the reasons for explaining why too often "nephropaties lack a specific treatment and progress relentlessly to end-stage renal disease" . Another important reason is that till today a valid strategy to reduce oxidative stress in renal diseases is not available. Ozone therapy, not only may correct a chronic oxidative stress, but it may also stimulate untapped resources able to afford some improvement [9, 93]. It appears therefore reasonable to suggest the combination of conventional treatments with mild O3-AHT in any initial nephropathy for preventing the risk of progression towards a chronic disease.
In several Countries, among others Cuba, Russia, and Ukraine, treatments by ozone are already a reality, although different administration modalities, such as the infusion of ozonated saline and of the rectal insufflations of ozone, are in current use because inexpensive and applicable to thousands of patients every day . Nevertheless, it is hoped that adequate ozone-based therapeutic treatments for patients affected by oxidative-stress related diseases could be implemented in every public hospital.
During the last two decades the paradoxical behaviour of ozone has been clarified: when it is chronically inhaled, it is highly toxic for the pulmonary system because the enormous alveolar surface, unprotected by sufficient antioxidants, is exposed to the cumulative ozone dose, which causes a chronic inflammation. This is not surprising because even for oxygen , as well as for glucose and uric acid levels a modification of the physiological concentrations is deleterious.
it improves blood circulation and oxygen delivery to ischemic tissue owing to the concerted effect of NO and CO and an increase of intraerythrocytic 2,3-DPG level;
by improving oxygen delivery, it enhances the general metabolism;
it upregulates the cellular antioxidant enzymes and induces HO-1 and HSP-70;
it induces a mild activation of the immune system and enhances the release of growth factors from platelets;
it procures a surprising wellness in most of the patients, probably by stimulating the neuro-endocrine system. However, ozone dosages must be calibrated against the antioxidant capacity of the patient's plasma, or otherwise the "start low-go slow" strategy must be used evaluating the subjective feeling of the patient after each session.
It remains to be clarified whether some messengers present in the ozonated blood are able to stimulate the release of staminal cells in the patient's bone marrow.
The evaluation of results obtained in several clinical trials performed in PAD has allowed to establish that the dose-response relationship in PAD can be depicted as an inverted U-shaped hormetic model with a brief, initial lack of effect due to the potency of blood antioxidants. A mild acute oxidative stress induced by ozone in blood ex vivo, as several other mild stresses due to either heat or cold exposure, a transient ischemia, other chemicals and physical exercise are able to induce a sort of "preconditioning response" often leading to both a repair and an increased defense capacity well within the "overcompensation stimulation hormesis". This new achievement, added to an increasing wide consensus in carefully using gases as NO, CO, H2S, N2O and H2 as real medical drugs , suggests that also ozone may be soon included into this category. One of the basic functions of ozone, after dissolving in the water of plasma is to accelerate the exchange of protons and electrons or, in simple words, to reactivate the metabolism all over the body. In this way, crucial biological functions gone astray can recover indicating that ozone operated as both a biological response modifier and an antioxidant inducer.
It is hoped that this paper will elicit the interest of clinical scientists in evaluating ozone therapy in vascular, renal and diabetic diseases, thus translating the laboratory results to the patient's bed.
VAB, M.D., Emeritus professor of Physiology, Department of Physiology, University of Siena, Viale Aldo Moro, 2, 53100, Siena, Italy
IZ, in charge as post-doc position at the Department of Pharmaceutical Chemistry and Technology, Viale Aldo Moro, 2, 53100, Siena, Italy
VT, Associate professor in Pharmaceutical Technology and Chief of the Post-Graduate School of Hospital Pharmacy, University of Siena, Viale Aldo Moro, 2, 53100, Siena, Italy
central nervous system
epidermal growth factor
human immunodeficiency virus
heat shock proteins (70 kDa)
inhibitor of NF-kB
lowest observed adverse effect level
lipid oxidation products
nicotinamide adenine dinucleotide phosphate
nuclear factor kappa-light-chain-enhancer of activated B cells
no observable adverse effect level
overcompensation stimulation hormesis
partial pressure of arterial oxygen
partial pressure of oxygen
peripheral arterial diseases
platelet-derived growth factor, subunit B
polyunsaturated fatty acids
reactive oxygen species
total antioxidant status
transforming growth factor β1
tumor necrosis factor.
This paper is dedicated to Mrs Helen Carter Bocci who for decades has generously linguistically corrected our papers.
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