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08 Dec '17

The dual action of ozone on the skin

Posted by Marin Crangaci
  The aim of this brief review is to summarize the recent literature on the effect of ozone (O3) on cutaneous tissues. Recently it has been reported that a chronic contact with O3 can be deleterious for the skin. G. Valacchi, V. Fortino and V. Bocci group and others have shown a progressive depletion of antioxidant content in the stratum corneum and this can then lead to a cascade of effects resulting in an active cellular response in the deeper layers of the skin. Using an in vivo model we have shown an increase of proliferative, adaptive and proinflammatory cutaneous tissue responses. On the other hand the well known activity of O3 as a potent disinfectant and oxygen (O2) donor has been also studied for therapeutic use. Two approaches have been described. The first consists of a quasi-total body exposure in a thermostatically controlled cabin. This treatment has proved to be useful in patients with chronic limb ischaemia. The second approach is based on the topical application of ozonated olive oil in several kinds of skin infection (from soreness to diabetic ulcers, burns, traumatic and surgical wounds, abscesses and skin reactions after radiotherapy). G. Valacchi, V. Fortino and V. Bocci and other authors have observed a striking cleansing effect with improved oxygenation and enhanced healing of these conditions. It is now clear that, on the skin, O3, like other drugs, poisons and radiation, can display either a damaging effect from a long exposure or a beneficial effect after a brief exposure to O2 and O3 or to the application of ozonated oil to chronic wounds.

  Christian Friedrich Schonbein discovered ozone (O3) in 1839 and in 1853 he made the first measurement of O3 in the Austrian mountains. Today, we know that some gases such as O3, carbon monoxide, nitric oxide and carbon dioxide can have dual actions, behaving either as useful or as harmful agents(1). The O3 layer is located at an altitude of about 22 km. Approximately 90% of the O3 in the atmosphere resides in the stratosphere. The O3 concentration in this region is about 10 parts per million by volume. O3 absorbs the bulk of solar ultraviolet (UV) radiation in the wavelengths from 290 to 320 nm. These wavelengths are harmful to life because they can be absorbed by the nucleic acid in cells and damage it. Increased penetration of UV radiation to the planet’s surface would damage plant life and have harmful environmental consequences. Appreciably increased amounts of solar UV radiation at the Earth’s surface would result in a host of biological effects, such as a dramatic increase in cancer; it seems that a 10% drop in the level of the O3 layer may cause a 25% increase in skin carcinoma and melanoma(2). Moreover this risk has recently been enhanced by excessive pollution with O3 in the troposphere, particularly evident during summertime in large cities(3). Thus the strong oxidative power of O3 in
association with other contaminants, can be harmful for plants and animals. The human bronchopulmonary system and the skin are the most accessible targets; they are vulnerable owing to the paucity of local antioxidant defences. O3 toxicity for the pulmonary system has been extensively examined while attention to the skin problem is more recent but no less important. An interesting difference that we would like to point out here is that while the pulmonary system is absolutely intolerant to O3 and this gas should never be inhaled, the skin, for anatomical and biochemical reasons, is somewhat more resistant.
  Recent literature points out that although a long exposure is certainly deleterious, transitory exposure at low and precisely controlled O3 concentrations can have useful effects. The damage to the respiratory tract by oxidative environmental pollutants such as O3 and nitrogen oxides have already been reviewed(4) while recent literature has focused only on the damaging interaction between long exposures to O3 and cutaneous tissues(5–10). We believe that it is now also correct to discuss the unexpected therapeutic effect of a brief exposure of patients to O3 or the use of ozonated oil for cutaneous infections.
Skin responses to environmental stress
  The skin consists of two main layers, the epidermis and the dermis, of which the latter is superficial to the subcutaneous fat tissue. Dermal fibroblasts synthesize a complex extracellular matrix containing collagenous and elastic fibres. Blood capillaries reach the upper part of the dermis. The epidermis contains mostly keratinocytes that rise to the skin surface as they differentiate progressively to form the non-nucleated corneocytes that comprise the superficial part of the epidermis, the stratum corneum (SC). The skin, as an interface between the body and the environment, is chronically exposed to stress from both UV radiation and environmental oxidative pollutants such as diesel fuel exhaust, cigarette smoke, halogenated hydrocarbons, heavy metals and O3 (one of the most toxic of these compounds)(11) The skin is protected against oxidative stress by a variety of antioxidants; these include enzymatic antioxidants such as glutathione peroxidase, superoxide dismutase, catalases and nonenzymatic low-molecular weight antioxidants such as vitamin E isoforms, vitamin C, glutathione (GSH), uric acid and ubiquinol(7).
 Recently, the presence of a- and c-tocopherol, ascorbate, urate and GSH has been shown also in the SC(10). Interestingly, the distribution of antioxidants in the SC follows a gradient with higher concentrations in deeper layers(12) This may be explained by the fact that SC layers move up in time as a part of the physiological turnover of skin cells and are replaced by freshly differentiated keratinocytes. Therefore, the superficial layer is exposed to chronic oxidative stress for a longer time than the deep layer. Compared with the SC, the surface lipids contain high levels of a- and c-tocopherol because of the secretion of vitamin E by sebaceous glands(13) Eventually, the uppermost layer of the SC will desquamate and the remaining antioxidants and reacted products will be eliminated from the body. In general the outermost part of the skin, the epidermis, contains lower concentrations of antioxidants than the dermis. In the lipophilic phase, a-tocopherol is the most prominent antioxidant, while vitamin C and GSH are the most abundant in the aqueous phase.
 
  Skin responses to ozone
   It is generally understood that the toxic effects of O3 are mediated through free radical reactions, although O3 is not a radical species per se(14) They are achieved either directly by the oxidation of biomolecules to give classical radical species (hydroxyl radical) or by driving the radical-dependent production of cytotoxic, nonradical species (aldehydes)(15) Furthermore, the formation of the oxidation products characteristic of damage from free radicals has been shown to be prevented by the addition of the antioxidants vitamin E and C, though the mechanism is not fully understood. The target specificity of O3 towards specific compounds together with its physicochemical properties of fairly low aqueous solubility and diffusibility, must be taken into account when a target tissue (lung and skin) is exposed to O3(16). 
Polyunsaturated fatty acids
 
  Cell membranes and their lipids are relevant potential targets of environmental stressors such as UV and O3. Using a spin trapping technique, the formation of radicals in the SC upon exposure to O3 and⁄or UV was detected (L. Packer, unpublished observation). The spin adduct could arise from an alkoxyl radical formed during lipid peroxidation.           Furthermore, lipid radicals (LÆ) are generated in epidermal homogenates that have been exposed to environmental stressors. The organic free radical LÆ reacts with O2, forming peroxyl radical LOOÆ and hydrolipoperoxides (LOOH). Transition metals and in particular iron, play a key role in the reactions of LOOH and in the subsequent generation of alkoxyl radicals (ROÆ can amplify the lipid peroxidation process).
The stratum corneum as the first target of environmental stressors
  
  Within the skin, the SC has been identified as the main target of oxidative damage(17,18). As the outer skin barrier, the SC has important functions, limiting transepidermal water loss and posing a mechanical barrier to penetration by exogenous chemicals and pathogens. It comprises a unique two-compartment system of structural, non-nucleated cells (corneocytes) embedded in a lipid enriched intercellular matrix, forming stacks of bilayers that are rich in ceramides, cholesterol and free fatty acids.(19,20)
   The effects of O3 on cutaneous tissues have recently been evaluated using a murine model. While no effect of O3 on endogenous antioxidants was observed in full thickness skin (dermis, epidermis and SC), it could be demonstrated that a single high dose of O3  significantly depleted topically applied vitamin E.(21) When the skin was separated into upper epidermis, lower epidermis and papillary dermis, and dermis, O3 induced a significant depletion of tocopherols and ascorbate followed by an increase in the lipid peroxidation measured as malondialdehyde (MDA) content. O3 is known to react readily with biomolecules and does not penetrate through the cells; therefore, it was hypothesized that O3 mainly reacts within the SC.(17) This hypothesis was supported by further experiments, where hairless mice were exposed to varying levels of O3 for 2 h. Depletion of SC lipophilic (tocopherols) as well as hydrophilic (ascorbate, urate, GSH) antioxidants was detected upon O3 exposure and it was accompanied by a rise in lipid peroxidation as an indicator of increased oxidative stress.(22) Furthermore, a recent study has shown the increase of 4-hydroxylnonenal (4-HNE) content in murine SC using both Western blot and immunohistochemical analysis.(23)
 Skin cellular responses to ozone exposure
   As mentioned above, O3 exposure was shown to induce antioxidant depletion as well as lipid and protein oxidation in the SC. Recent studies have investigated the effects of O3 in the deeper functional layers of the skin.(23–25) 
   To evaluate the effect on cutaneous tissues of O3 exposure, hairless mice were exposed for 6 days to   )1 for 6 h day)1 and the homogenized whole skin was analysed. Under these experimental conditions an increase of proinflammatory marker cyclooxygenase-2 (COX-2) expression was detected confirming the role that O3 can play in skin inflammation. This induction was accompanied by an increase in the protein level of heat shock protein (HSP)(32), also known as haem oxygenase-1 (HO-1), confirming that HSPs are sensitive markers of O3-induced stress in cutaneous tissues.  Our group was the first to document the upregulation of HSPs 27, 32 and 70 in homogenized murine skin upon O3 exposure   HSP27 showed the earliest (2 h) and highest (20-fold) response to O3 compared with the delayed induction (12 h) of HSP70 and HO-1. Increased expression of HSP27 has been demonstrated following heating of both keratinocyte cell lines and organ-cultured human skin.(26,27) HSP27 is expressed predominantly in the suprabasal epidermis in human skin,(28) whereas HSP70 predominates in the dermis compared with the epidermis.
  These differences in location between HSP27 and HSP70 might explain the different time course of induction of these stress proteins upon O3 exposure. Interestingly, O3 induction of HO-1 showed a delayed time course compared with that for HSP27 and 70, in line with a previous study, which showed a peak of HO-1 induction at 18–24 h in rat lungs after O3 treatment.(29) It is therefore possible that bioactive compounds generated by products of O3 exposure may be responsible for the induction of HO-1 as was also shown after UV irradiation.(30,31) As HSPs are involved in cell proliferation, apoptosis and inflammatory response, O3-mediated HSPs induction can affect normal skin physiology. Thus, HSPs might provide an adaptive cellular response to O3; enhancing the expression of HSPs might turn out to be a new way to deal with the immediate and long-term consequences of O3 exposure. A prerequisite for the utilization of this concept is the development of nontoxic HSP inducers and their evaluation for clinical efficacy and safety. Furthermore, increased levels of metalloproteinase-9 (MMP-9; mRNA and activity) was observed after O3 exposure  ) MMPs have been associated with the degradation of the basal membrane and play important roles in wound healing and in tumour development. In addition, MMPs may contribute to the enhancement of skin ageing and formation of wrinkles.(32) O3 is also able to modulate proliferative responses in mouse skin.(23) Proliferating cellular nuclear antigen (PCNA) is a protein identified as the polymerase-associated protein synthesized in the early G1 and S phases of the cell cycle involved in DNA replication and repair. PCNA is induced by stress responses that cause DNA damage;(33) it has been reported that PCNA gene expression can be induced in the lungs by diesel exhaust particles, another form of oxidative lung damage,(34) suggesting that oxidation can affect proliferative behaviour in target tissues.
   O3 exposure can also affect cell differentiation. In skin tissue, we detected an increase of keratin 10 (K10) production after O3 treatment;23 K10 is a keratin produced in well differentiated, suprabasal keratinocytes; O3-induced changes in K10 suggest that O3, induces keratinocyte proliferation and differentiation.(35) It is not clear how O3 displays its effects, but recent studies have shown that it is able to induce the activation of the transcription factor, NF-KB, by phosphorylation of the kinase, IKBalfa-gama.(23) 
   Changes in the redox state have been shown to activate the NF-jB intracellular signalling pathway; this cascade includes several kinases and transcription factors. NF-jB-mediated signal transduction has been implicated in the regulation of viral replication, autoimmune diseases, tumorigenesis and apoptosis, and in the inflammatory response. In this regard, the activation of NF-KB is known to play a crucial role in COX-2 gene activation,(36) suggesting that O3 plays a role in the expression of numerous proinflammatory and adaptive inflammatory responses. It is not surprising that exposure of the skin to O3 can trigger several biochemical pathways leading to inflammation and affecting skin biology. On the other hand basic and clinical work developed during the last 15 years has shown that transient treatment and small O3 doses can reactivate useful body functions and might display therapeutic activity.(37)
  
Topical application
     Interestingly, in spite of its instability, the O3 molecule can be stabilized as an ozonide between the double bonds of a monounsaturated fatty acid such as oleic acid.37 As a consequence, ozonated olive oil remains stable for 2 years at 4 Celsius. This preparation is proving to be ideal for the topical use of O3 in the treatment of chronically infected cutaneous and mucosal areas of the body. O3 is widely recognized as one of the best bactericidal, antiviral and antifungal agents and therefore it is profitably and practically employed as ozonated olive oil with well defined peroxide contents. The ozonated oil is now used topically for the treatment of war wounds, anaerobic infections, herpetic infections (HHV I and II), trophic ulcers and burns, cellulitis, abscesses, anal fissures, decubitus ulcers (bed sores), fistulae, fungal diseases, furunculosis, gingivitis and vulvovaginitis.(41) Matsumoto et al. tested the efficacy of the ozonated oil in the treatment of fistulae and chronic surgical wounds and, in a series of (28) patients, the ozonated oil was fully effective in 27 cases without side-effects.(42) Even radiodermatitis lesions in patients with cancer have been found to be beneficially influenced by exposure to O3(43) but far better results could be achieved with the simple application of ozonated oil. 

Conclusions
   Biological and clinical studies on the effects of O3 on the skin have shown that O3 can be either toxic, or safe at the point of use as a real drug, depending upon its dosage, length of exposure and the antioxidant capacity of the tissue exposed. The ambivalent character of O3 has been likened to the Latin god Janus;(44) indeed O3 is useful in the stratosphere but is toxic in the troposphere because of its chronic effects on the respiratory system, skin and mucosae.(45)
   On the other hand, it has recently been observed that olive oil, which during ozonation traps O3 in the form of a stable ozonide, when applied to all sorts of acute and chronic cutaneous infections, slowly release O3 which, in comparison with conventional creams, displays effective disinfectant and stimulatory activities that lead to rapid healing. The dual behaviour of O3 fits well the concept of ‘hormesis’ that says the exposure of a living organism to a very low level of an agent harmful at high or chronic levels induces an adaptive and beneficial response.(46,47)
2005 British Association of Dermatologists • British Journal of Dermatology 2005
01 Dec '17

Therapeutic Effects of Topical Application of Ozonated Olive Oil on Acute Cutaneous Wound Healing

Posted by Marin Crangaci

   In 2009 a group of scientists (Hee Su Kim, Sun Up Noh, Ye Won Han, Kyoung Moon Kim, Hoon Kang, Hyung Ok Kim and Young Min Park) from Department of Dermatology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea, undertook a study in which they researched  the therapeutic effects of topical ozonated olive oil on acute cutaneous wound healing in a guinea pig model and also to elucidate its therapeutic mechanism. After creating full-thickness skin wounds on the backs of guinea pigs by using a 6 mm punch biopsy, they examined the wound healing effect of topically applied ozonated olive oil (ozone group), as compared to the pure olive oil (oil group) and non-treatment (control group). The ozone group of guinea pig had a significantly smaller wound size and a residual wound area than the oil group, on days 5 (P<0.05) and 7 (P<0.01 and P<0.05) after wound surgery, respectively. Both hematoxylin-eosin staining and Masson-trichrome staining revealed an increased intensity of collagen fibers and a greater number of fibroblasts in the ozone group than that in the oil group on day 7. Immunohistochemical staining demonstrated upregulation of platelet derived growth factor (PDGF), transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) expressions, but not fibroblast growth factor expression in the ozone group on day 7, as compared with the oil group. In conclusion, these results demonstrate that topical application of ozonated olive oil can accelerate acute cutaneous wound repair in a guinea pig in association with the increased expression of PDGF, TGF-β, and VEGF.

  

INTRODUCTION

   The closure of cutaneous wounds involves complex tissue movements such as hemorrhage, inflammation, re-epithelization, granulation tissue formation, and the late remodeling phase of repair . These events involve coordination of dozens of types of cells and matrix proteins, which are all important to control stages of the repair process. Previous studies have demonstrated that endogenous growth factors, such as fibroblast growth factors (FGF), platelet derived growth factors (PDGF), transforming growth factor-β (TGF-β) and vascular endothelial growth factors (VEGF) are the important regulatory polypeptides for coordinating the healing process. They are released from macrophages, fibroblasts, and keratinocytes at the site of injury and they participate in the regulation of re-epithelization, granulation tissue formation, collagen synthesis and neovascularization.

   Ozonated oil has been widely recognized as one of the best bactericidal, antiviral and antifungal agents and it has been used empirically as a clinical therapeutic agent for chronic wounds, such as trophic ulcers, ischemic ulcers and diabetic wounds. The beneficial effects of ozonated oil on wound healing might be assumed to be due to decreased bacterial infection, ameliorated impaired dermal wound healing or increased oxygen tension by ozonated oil exposure in the wound area.

   It was reported that O3 exposure is associated with activation of transcription factor NF-κB; this is important to regulate inflammatory responses and eventually the entire process of wound healing. It was shown that huge amounts of PDGF and TGF-β1 were released from platelets in the heparinized plasma of a limb ischemia patient after ozonation. It has been revealed that there were substantial increases of steady-state mRNA levels of TGF-β1 in the fibroblasts that were co-cultured with bronchoepithelial cells after O3 exposure. A recent study has shown that hydrogen peroxide (H2O2) potently induced the VEGF expression in human keratinocytes which can stimulate wound healing. From these previous studies of O3, we hypothesized that O3 might enhance acute cutaneous wound healing, and this could be associated with growth factors such as FGF, PDGF, TGF-β and VEGF.

    Nowadays, O3 is profitably and practically employed as ozonated olive oil; this contains the O3 molecule stabilized as an ozonide between the double bonds of a monounsaturated fatty acid such as oleic acid, which is ideal for the topical use of O3 to treat chronically infected cutaneous and mucosal areas of the body. Ozonated materials referred to as ozonides are formed by the reaction of olefins with ozone. Any olefin can be treated with gaseous ozone to form an ozonide. The ozonide compositions have the capacity to deliver nascent oxygen deep within the lesion without causing primary skin irritation.

     Ozonated oil has been used topically for the treatment of chronic wounds, but there have been few studies concerning with the therapeutic effects of ozonated olive oil on acute cutaneous wound healing in animal models. The present study was designed to evaluate the therapeutic effect of topical ozonated olive oil on acute cutaneous wound healing in a guinea pig model, and to elucidate its therapeutic mechanisms that are associated with such growth factors as FGF, PDGF, TGF-β, and VEGF.

    For the experiment, the researchers team used sixteen female guinea pigs (400-450 g), aged 8-9 weeks, were placed in a room that was under a 12-hr light and 12-hr dark cycle. The animals were placed individually in separate cages with ad libitum access to food and water. The animal care, handling, and experimental procedures were carried out in accordance with a protocol approved by the Animal Care and Use Committee of the Catholic University of Korea.

   After 7 days of acclimation, the guinea pigs were anesthesized with ketamine and their backs were shaved and then sterilized with normal saline. The skin was then pinched and folded, and a sterile biopsy punch (6 mm in diameter, Stiefel Co., Offenbach, Germany) was used to make a full-thickness hole in the skin. Two wounds were created on both sides of the back for a total 4 circular wounds per animal.

Application of topical ozonated olive oil

   Two drops (about 0.1 mL) of ozonated olive oil were applied everyday to two sites of the four wounds (Ozone group). Olive oil as a pure base was applied to a third wound (Oil group). As a control group, nothing was applied on the fourth wound. The wounds were then dressed with Opsite® to cover them without dryness. An elastic bandage was wrapped around the area to prevent further injury.  

   On days 3 (n=4), 7 (n=4), and 11 (n=8) after wounding, guinea pigs were euthanized using ketamine and the four wounded tissues from each guinea pig were excised. Then the tissues were cut into halves; one half was placed in formalin (10% formaldehyde in phosphate-buffered saline) for hematoxylin-eosin and Masson-trichrome staining, and the second half was quickly kept as a frozen state (-80℃) for immunohistochemistry.

Histology

   The specimens for histological examination were collected from each group by the full-thickness excision. Intensity of the collagen fibers and the fibroblast proliferation were examined under microscope in hematoxylin-eosin and Masson-trichrome staining. The staining intensity of the collagen fibers was graded under ×200 magnification as follows: -, completely negative staining intensity; ±, lower staining intensity; +, moderate staining intensity; ++, slightly higher staining intensity; +++, considerably higher staining intensity. The number of fibroblasts was counted in the 5 randomized fields per specimen under ×400 magnification. Three dermatologists, who were "blinded" to which groups the specimens were in, independently analyzed all the specimens and then the mean numbers and standard deviations of the fibroblasts in all groups of wound were calculated, respectively.

Result

The ozonated ozonated oil significantly enhances the acute cutaneous wound healing

As shown in Fig. 1, there was an enhanced wound closure in the ozone group of wounds, the left two of the four wounds, as compared to the oil group as well as the control group. On day 11, all of the wounds completely re-epithelized irrespective of treatments. The ozone group showed a significantly smaller wound size than the oil group on days 5 (P<0.05) and 7 (P<0.01). The ozone group showed 58%, 46.3%, and 16.4% in residual wound area on days 3, 5 and 7, respectively. On the other hand, the oil group revealed 61.8%, 53.7% and 25.6% in residual wound area, respectively (Table 1). Thus, there was a significant difference in the residual wound area between the ozone group and the oil group on days 5 (P<0.05) and 7 (P<0.01). On repeated-measures of ANOVA, the ozone group showed a significantly decreased residual wound area as compared to the oil group, as well as the control group (P<0.05).

fig.1 The effects of ozonated olive oil on clinical wound closure. The photomicrographs demonstrate the enhanced wound closure, in the ozone group, on the left two wounds (a and b) on the back of the guinea pig, as compared to the oil group (c) as well as the control group (d). (Bar=5 mm).

Tabel 1 Comparison of the average wound size and residual wound area on post-operation days 0, 3, 5, 7, and 11

The ozonated ozonated oil promotes collagen synthesis and fibroblast proliferation

  On the hematoxylin-eosin staining, epithelization, infiltration of inflammatory cells and vascular proliferations were commonly seen on all of the wounds; but there were proliferation of fibroblasts and collagen fibers noted in the ozone group as well (data not shown). We performed the Massontrichrome staining in order to determine whether ozonated OLO's ability to accelerate wound closure was associated with collagen synthesis and fibroblast proliferation at the wound bed and at the edge of the injury site. The staining intensity of collagen fibers and the number of fibroblasts were evaluated on days 3 and 7. On day 3, the ozone group did not show a significant difference in the staining intensity of collagen fibers and the number of fibroblasts as compared to the oil group. In contrast, on day 7, the ozone group revealed an increased staining intensity of collagen fibers at the wound bed and at the edge of the entire dermis in comparison to the oil and control groups (Fig.2). On day 7, the numbers of fibroblasts in the ozone group were 62.3 and 35.6 at the wound bed and edge, respectively; but, the numbers of fibroblasts in the oil group were 48.5 and 22.7, respectively (Table 2). Thus, there was a significant difference in the staining intensity of collagen fibers and the number of fibroblasts between the ozone group and the oil group on day 7 (P<0.05), but not on day 3.

Fig. 2.

Masson-trichrome staining of the wound bed and the edge of the injury site on days 3 and 7. The ozone group revealed the increased staining intensity of collagen fibers and the number of fibroblasts at the wound bed and edge, in comparison to the oil group and the control group on day 7, but not on day 3 (original magnification ×400, Bar=50 µm).
Table 2.
Comparison of staining intensity of collagen fibers and the number of fibroblasts by the Masson-trichrome staining on days 3 and 7

 

The ozone group revealed increased expressions of PDGF, TGF-β, and VEGF, but not FGF

  In order to determine which growth factors play an important role for the accelerating wound closure associated with the proliferation of fibroblasts and collagen fibers by the ozonated OLO, we evaluated the immunohistochemical staining intensity of FGF, PDGF, TGF-β, and VEGF on day 7 (Fig.3). FGF expressions were identified in dermal fibroblasts and collagen fibers, but this was barely detected in the epidermis of the control group. There were little differences in the FGF expression between the ozone group and the oil group (Table 3). PDGF was expressed in dermal inflammatory cells, fibroblasts, epidermal cells and keratinocytes of hair follicles of the control group. The ozone group revealed a relatively higher PDGF expression, as compared to the oil group. There was a relatively distinct difference in the dermis between the former and the latter. TGF-β expressions were detected in the dermal fibroblasts, epidermal cells and keratinocytes of the hair follicles of the control group. Likewise, the ozone group showed a relatively increased expression of TGF-β, as compared to the oil group. VEGF expression was identified in dermal fibroblasts, endothelial cells and collagen fibers, but it was barely detected in the epidermis of the control group. The ozone group revealed a relatively increased VEGF expression in both the dermis and the epidermis, as compared to the oil group. These findings demonstrated that the ozone group revealed relatively higher expressions of PDGF, TGF-β, and VEGF, but not FGF than the oil group on day 7.

Fig. 3 Immunohistochemical staining for FGF, PDGF, TGF-β, and VEGF on day 7. The ozone group revealed the relatively increased expressions of PDGF (BFJ), TGF-β (CGK), and VEGF (DHL), but not FGF (AEI) as compared to the oil group on day 7 (×100, original magnification, inlet; ×400, Bar=500 µm).

Table 3.

Comparison of the expression of growth factors on day 7

Discussion

   Based on the results from the wound closure between the ozone group and the oil group on days 0, 3, 5, 7, and 11, the ozone group showed a significantly smaller wound size and residual wound area than the oil group in the guinea pig model on days 5 and 7. Thus, these results demonstrated that O3 could enhance acute cutaneous wound healing. Especially, on day 7, both the wound size and residual wound area of the ozone group were much more significantly decreased. This implies that topical exposure of O3 may affect granulation tissue formation of the wound healing process rather than affecting immediate formation of blood clot and recruitment of inflammatory cells during the inflammation phase.

  On Masson-trichrome staining on day 3, there was not much difference in the staining intensity of collagen fibers and fibroblast proliferation between the ozone group and the oil group. However, on day 7, the ozone group revealed about one and half times increased staining intensity of collagen fibers and significantly increased fibroblasts at the wound edge as well as at the wound bed, compared to the oil group. These findings indicated that O3 may act on acute wound healing directly or indirectly via collagen synthesis and fibroblast proliferation during granulation tissue formation and the early tissue remodeling phase of wound healing.

  Fibroblasts have been known to play important roles in reepithelization, collagen fiber synthesis, extracellular matrix regeneration, remodeling of wounds, and for the release of such endogenous growth factors as FGF, PDGF, TGF-β, and VEGF. In this study, increased expressions of PDGF and TGF-β were seen in the ozone group on day 7, which is correlated with increased staining intensity of collagen fibers and fibroblast proliferation on the same day. There were also increased expressions of PDGF and TGF-β in the epidermal keratinocytes and the hair follicular cells adjacent to the injury. These findings suggest that O3 might induce expressions of PDGF and TGF-β from epidermal keratinocyte as well as from the dermal fibroblast at the injury site. In the present study, there was a relatively increased expression of VEGF in the epidermal keratinocytes of the ozone group on day 7; this was consistent with the previous study showing that VEGF was gradually increased from the 1st day to the 7th day of normal wound healing process. On hematoxylin-eosin staining, the relatively increased vascularity in the ozone group might be due to the increased expression of VEGF by ozonation. These findings may be associated with the generation of H2O2 through ozonation, which can directly induce a VEGF expression and/or indirectly induce it by the induction of heme oxygenase-1. Because VEGF is main cytokine of vascularization in the late phase of wound healing, further study will be needed to clarify the effect of O3 on the neovascularization of cutaneous wound healing. In contrast to the increased expressions of PDGF, TGF-β, and VEGF in the ozone group, there was little difference in the FGF expressions between the ozone group and the oil group on day 7. One of the possible explanations for this result is that FGF had already been up-regulated within 24 hr after wounding.

In conclusion, these results demonstrate that application of ozonated olive oil , the topical form of O3, can accelerate acute cutaneous wound repair in the guinea pig model by promoting collagen synthesis and fibroblast proliferation at the injury site and by increasing the expression of growth factors such as PDGF, TGF-β, and VEGF. Taken together, we can infer that topical O3 may be regarded as an alternative therapeutic modality to enhance cutaneous wound healing.

Sources:

Therapeutic Effects of Topical Application of Ozone on Acute Cutaneous Wound Healing

Hee Su Kim, Sun Up Noh, Ye Won Han, Kyoung Moon Kim, Hoon Kang, Hyung Ok Kim and Young Min Park

Department of Dermatology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

 

 

24 Nov '17

Germicidal Properties of Ozonated Oil

Posted by Marin Crangaci

Germicidal Properties of Ozonated Oil  

Ozone is a well-known allotropic form of oxygen and a very strong oxidative agent. Due to this property it is one of the strongest disinfectants. Its biocidal activity was discovered by Fox in 1873. Ozone was first used in medicine in 1894 by Labbe and Oudin for tuberculosis treatment. The 20th century brought many new applications for ozone germicidal properties. A gaseous mixture of ozone and air was used in 1915 for wound disinfection by Wolff. In 1935 it was used in dental medicine by Fisch and in surgery by Payr (Viebahn-Hansler, 2002).

  Ozone germicidal action was widely proved on a broad group of microorganisms including gram-positive and gram-negative bacteria as well as spores and vegetative cells (Guzel-Seydim et al., 2004). Many studies comparing ozone with popular disinfecting agents have been carried out (Khadre and Yousef, 2001; Telles Silveira et al., 2005; Filippi, 2005; Orta de Velasquez et al., 2005; John et al., 2005). The results of the studies suggest that ozone is the best disinfectant. Special attention is deserved by studies by Telles Silveira et al., who confronted germicidal action of ozone and sodium hypochlorite on Enterococcus sp. (gram-positive bacteria) which are often found in hospital sewage. Ozone turned out to be effective against these microorganisms, even the vancomycin-resistant Enterococcus (VRE), whereas sodium hypochlorite proved to be less effective (Telles Silveira et al., 2005).

  Another significant study was conducted by Filippi, who investigated the survivability of the pathogens in dentistry equipment disinfected with ozone and hydrogen peroxide. Samples from devices disinfected with hydrogen peroxide showed the presence of Pseudomonas aeruginosa, whereas in samples from devices disinfected with ozone the occurrence of P. aeruginosa was not observed (Filippi, 2005). Another study comparing the biocidal action of ozone and hydrogen peroxide against foodborne Bacillus spp. spores was conducted by Khadre and Yousef. They proved that hydrogen peroxide is less effective than ozone even when it was used in concentrations 10,000-fold higher than those applied for ozone (Khadre and Yousef, 2001).

   Ozone kills microbes by acting on cell walls and semipermeable membranes. This leads to changes in the chemical structure of the cell and interferes with cell metabolism products exchanging with the environment (Bin´, 2002; Shiota et al., 2005). Furthermore it causes cell envelope disintegration leading to leakage of the cell contents (Guzel-Seydim et al., 2004; Pascual et al., 2007). Bactericidal, fungicidal and virucidal properties of ozone are attributed to its ability to destroy many of the enzymatic structures. Naturally each microorganism has specific sensitivity to ozone. Bacteria are more sensitive than yeast and fungi. As a result of differences in structure of the cell walls, gram-positive bacteria are more sensitive to ozone than gram-negative organisms (Guzel-Seydim et al., 2004; Pascual et al., 2007).

  Nowadays methods exploiting medical applications of ozone are called ozone therapy, which may be employed in different fields of medicine, e.g., general medicine, dermatology, surgery, dental medicine, orthopedics, immunology (Viebahn-Ha¨nsler, 2002). Since ozone is a gas and there are not many medicines available in gaseous phase, special techniques had to be developed for the safe use of ozone (Viebahn-Ha¨nsler, 2002). It can be administered in the form of ozone-oxygen mixtures, ozonized water or ozonized vegetable oils. Although gaseous ozone is used in medicine for treating patients, that does not mean that ozone is harmless. When breathing ozone in at high concentrations it can be a very harmful substance, especially for the pulmonary tract. Hence it is extremely important to use ozone in small and safe doses just above the threshold level (Bocci, 2006).

 Vegetable oils have found application in wound disinfection and improve the process of healing. They are interesting because they can be used outside a surgery. Their disinfecting effect is much slower than in the case of ozone-oxygen mixtures or even ozonated water. Ozonated oils need hours rather than seconds to achieve a satisfactory reduction in the amount of microorganisms (Viebahn-Ha¨nsler, 2002). Ozonated oils are quite popular in many countries especially in Cuba where OLEOZONE is produced according to the process developed at the Ozone Research Center. It is a registered drug in Cuba and is obtained through the ozonation of sunflower oil. 

  New methods of pathogenic microorganism elimination have aroused much interest recently; that is why ozone and its application as a disinfection agent has become a popular object of scientific research. The aim of this study (Kinga Skalska , Stanisław Ledakowicz , Jan Perkowski & Barbara Sencio (2009) was to determine minimum ozone doses in ozonated oil for killing various microorganisms like Bacillus subtilis, Escherichia coli, Candida albicans. First, five different vegetable oils were ozonated and their properties, such as iodine number, peroxide value, acidity value and viscosity were compared. Afterwards one chosen vegetable oil was subjected to further microbiological studies.

RESULTS AND DISCUSSION

Physical and Chemical Properties

  The first stage of this research pursued a goal of assessing the changes in chemical properties of ozonated vegetable oils. Five vegetable oils with different compositions of fatty acids were tested: olive oil, sunflower oil, rapeseed oil, corn oil and grape seed oil. All five oils were ozonated until reaching the absorbed ozone dose around 10 mg O3/g oil. 

  The highest initial iodine number was observed for sunflower oil, the lowest value was found in olive oil. This is connected with the high amount of unsaturated, especially polyunsaturated, fatty acid in sunflower oil and their low amount in olive oil. For all vegetable oils examined, a decrease in iodine number after ozonation was observed, obviously as a result of reaction between ozone and unsaturated fatty acids.

  It varied depending on the tested oil, which again is related to the composition of oils. Another analyzed parameter was peroxide index which increased during the ozonation process. The highest values were obtained for ozonated olive oil and ozonated sunflower oil. High peroxide index is connected with the reaction of ozone with carbon-carbon double bonds in unsaturated fatty acids. It produces ozonides, hydroperoxides, aldehydes, peroxides, diperoxides and polyperoxides according to well-known Criege´e mechanism (Diaz et al., 2005; Jardines et al., 2003). Furthermore according to Tamoto et al. the major component of ozonated olive oil is triozonide of triolein. 

The increase of peroxide index for sunflower oil was higher than for olive oil due to lower amounts of double bonds in olive oil. Iodine number analysis for ozonized sunflower oil showed a decrease of iodine number with an increasing ozone dose. This shows that almost all double bonds present in fatty acids were saturated in the reaction with such a strong oxidative agent as ozone.

 From the germicidal point of view, the most important is peroxide number since it indicates the amount of peroxide compounds formed. As Diaz et al. suggested, these are responsible for the biocidal activity of ozonized vegetable oils (Diaz et al., 2005a, 2005b, 2006). Peroxide number increases almost linearly with the increase of ozone dosage as shown in figure. Peroxide number for the ozone dosage 40 mgO3/g oil rose forty times compared to pure sunflower oil, whereas for 300 mgO3/g oil it rose more than 100 times.  

 Chemical analyses of ozonized sunflower oil proved that acidity number increases with increase of ozone dosage. Even for short ozonation times, the acidity number increased more than 30 times. This relation is presented in figure. Conventionally, acidity number is used to assess if the studied oil is fresh or rancid, and if it was well stored. The growth in the value of the acidity number indicates the correctness of the ozonation process. Fats show neutral reaction, however partial hydrolysis takes place during the ozonation process and unbound fatty acids give fats an acidic reaction. Apart from chemical and biological properties, viscosity also was analyzed. Results showed increases in viscosity of ozonated sunflower oil with increasing ozone dose. This phenomenon could be used for process monitoring in order to estimate ozonation reaction progression. The two ozonated sunflower oils with the highest ozone doses had the consistency of gel (viscosity achieved app. 0.28 Pas). 

Microbiological Studies

  

  Microbiological studies for ozonated sunflower oil were carried out with the following ozone doses: 20, 40, 70, 100, 200, 250 and 300 mg O3/g oil. Bactericidal and fungicidal effectiveness of ozonized sunflower oil was investigated. The examination of the results obtained allowed determination of the ozone dose in preparation that causes 100% growth inhibition of the examined microorganisms. Results were gathered in the table. 

  The examination of the results obtained allowed determination of the ozone dose in preparation that causes 100% growth inhibition of the examined microorganisms. Results were gathered in Table 4. The findings suggest that microbes have diversified sensitivity to ozonated sunflower oil. Gram-positive bacteria B. subtilis turned out to be the least resistant, because no growth on the plates for preparation with the ozone dose of 200 mg O3/g oil and above was observed. Both gram-negative bacteria E. coli and yeast C. albicans were more resistant to ozonated sunflower oil than B. subtilis, since these germs needed preparation with the ozone dose higher by 50 mg O3/g oil to be totally destroyed. This could suggest that ozonated sunflower oil, similarly to gaseous ozone, acts stronger on grampositive bacteria than it does on gram-negative bacteria and fungi. As explained in the introduction, this is connected with the cell wall structure. However, after conducting a further literature survey, it is obvious that such an approach would be too simplified. Since the Diaz et al. studies on antimicrobial action of ozonated vegetable oils, not all gram-positive bacteria are more sensitive to ozonated oils than gram-negative ones. This suggests that the mechanism of biocidal action of ozonated oils may be more complex.

  Exemplary fungicidal effects of ozonated sunflower oil is shown in figure. Ozonized sunflower oil with the ozone dose below 200 mg O3/g oil shows no germicidal properties. Then, 100% growth inhibition of the studied microorganisms was obtained for ozonated sunflower oils with high values of peroxide number (more than 630 milliequivalent/kg). For ozone doses lower than 200 mg O3/g oil and the peroxide number lower than 630 milliequivalent/kg, no disinfecting effect was gained. Our studies confirmed the results of the earlier research by Diaz et al. which indicated that only ozonized sunflower oils with sufficiently high concentrations of peroxide compounds show toxic influences on microorganism growths (Diaz et al., 2005a, 2005b, 2006). Results presented in this paper suggest that ozonated sunflower oils inhibited expansion and growth of microbes.

Therapeutic Effects

  Additionally, therapeutic effects of ozonized sunflower oil with ozone doses around 300 mg O3/g oil were tested with the cooperation of the Dermatology Clinic of the Medical University of Ło´dz´. The preparation was used for one month for topical treatment of two patients, one with a wound ulcer and the other with nail psoriasis. The wound ulcer before treatment can be observed, with a high amount of fibrous tissue and only few areas with granular tissue, which is the sign of healing. After a month of therapy  a significant increase in amount of granulation is observed. In the case of nail psoriasis, presented, the improvement of patient condition is easily noticeable.

 

CONCLUSIONS

  Among the studied microorganisms, the most resistant against ozonated oil appeared to be gram-negative bacteria E. coli and yeast C. albicans, which required around 250 mg O3/g oil to become inhibited. Whereas gram-positive bacteria B. subtilis was shown to be the least resistant because no growth on the plates for preparation with the ozone dose 200 mg O3/g oil was observed. Diaz suggested that there exists a connection between the value of the peroxide number and the disinfecting properties of ozonized sunflower oils. The results indicated that ozonated sunflower oils can inhibit the development and growth of the microorganisms tested. Ozonated sunflower oils gain recognition since they have one significant advantage over gasous ozone and ozonized water. It can be used outside a surgery by the patient himself. It is also easy to manufacture and to be preserved. Furthermore, its gelled consistency makes it easy to apply locally. In order to provide the required effectiveness of topical therapy for scarcely healing wounds, it is important to apply ozonated oil with the ozone dose exceeding the minimal dose for the specific pathogen.

 

Sources:

Germicidal Properties of Ozonated Sunflower Oil

Kinga Skalska a , Stanisław Ledakowicz a , Jan Perkowski b & Barbara Sencio a a Department of Bioprocess Engineering , Technical University of Łódź , Łódź, Poland b Institute of Radiation Chemistry, Technical University of Łódź , Łódź, Poland Published online: 27 May 2009.

17 Nov '17

How Does Ozone Act? How and Why Can We Avoid Ozone Toxicity? 2

Posted by Marin Crangaci

 In previous article we started to speak about ozone mechanism of action and how we can avoid ozone toxicity. We spoke about “ozone messengers” and their pharmacodynamics.   Today we will continue with  biological effects elicited during exposure of human blood to ozone. 

  There is an ample literature regarding the cytotoxicity of LOPs . These compounds, when tested either in tissue culture, or examined in the context of the delicate respiratory system, are toxic even at a concentration of 1 μM. Surprisingly, submicromolar concentrations (0.01–0.5 μM) tested in several cell types can stimulate proliferation and useful biochemical activities. These findings lead to believe that toxicity of ozonated lipid products depends upon their final concentrations and tissue-localization, so that they can act either as injurious or useful signal.  Blood, in comparison to the lungs, is a much more ozone-resistant “tissue” and has never been observed any damage. 

However, when we reinfuse ozonated blood, what is the fate of LOPs?

It was often measured the pharmacokinetics* of their disappearance from blood and their half-life in six patients with age-related macular degeneration (ARMD) was equivalent to 4.2 ± 1.7 min. On the other hand, if the same ozonated blood samples were incubated in vitro, levels of LOPs hardly declined during the next 2 h, a result clarifying their toxicity in static cell cultures. As far as cholesteryl ester hydroperoxide is concerned, Yamamoto (2000)
has emphasized the role of the enzymatic degradation and hepatic uptake. Thus
LOPs toxicity in vivo is irrelevant for the following processes:

  1. FORMATION OF ALBUMIN-4-HNE ADDUCTS. Assuming to ozonate
    200 ml of blood with an ozone dose of 8 mg, the presence of about 5 g of
    albumin (Cys 34) is sufficient to form adducts with 4-HNE. Moreover in a total
    body pool of about 320 g of albumin, the ozonated aliquot is less than 1%
    (Aldini et al., 2006).
  2. DILUTION (up to 150–200 folds) of these compounds in blood and body fluids rapidly lowers their initial concentration to pharmacological, but not toxic
    levels. Obviously the ozone dose must be within the therapeutic range.
  3. NEUTRALISATION of LOPs due to the antioxidant capacity in body fluids
    and cells.
  4. DETOXIFICATION of LOPs due to the interaction with billions of cells endowed with detoxifying enzymes such as aldehyde- and alcoholdehydrogenases, aldose reductase and GSH-transferases (GSH-T) (Siems and
    Grune, 2003; Awasthi et al., 2005)
  5. EXCRETION of LOPs into the urine and bile after hepatic detoxification and
    renal excretion (Alary et al., 2003).
  6. BIOACTIVITY without toxicity. As already mentioned, submicromolar concentrations of LOPs can act as physiological messengers able to reactivate
    a biological system gone awry. 

*Pharmacokinetics( the dynamics of drug absorption, distribution, metabolism, and elimination)-in order to understand and control the therapeutic action of drugs in the human body, one must know how much drug will reach the site(s) of drug action and when this will occur. The absorption, distribution, metabolism (biotransformation), and elimination of drugs are the processes of pharmacokinetics. Understanding and employ ing pharmacokinetic principles can increase the probability of therapeutic success and reduce the occurrence of adverse drug effects in the body.

 

The interrelationship of the absorption, distribution, binding, metabolism, and excretion of a drug and its concentration at its sites of action. Possible distribution and binding of metabolites in relation to their potential actions at receptors are not depicted.

 From a pharmacokinetic point of view, trace amounts of LOPs, can reach all organs and particularly the bone marrow and the Central Nervous System. LOPs are extremely important in informing cells of a minimal and calculated oxidative stress eliciting the adaptive response. In regard to erythrocytes, LOPs can influence the erythroblastic lineage, allowing the generation of cells with improved biochemical characteristics. These “supergifted erythrocytes”, due to an induction of glucose-6-phosphate dehydrogenase, a higher content of 2,3-DPG and antioxidant enzymes, during the following weeks, are able to deliver more oxygen into ischemic tissues.

The consequence of repeated treatments, obviously depending upon the volume of ozonated blood, the ozone concentration and the schedule is that, after a few initial treatments, a cohort (about 0.8% of the pool) of “supergifted erythrocytes” will enter daily into the circulation and, relentlessly, will substitute old erythrocytes generated before the therapy. This means that, during prolonged ozonetherapy, the erythrocyte population will include not only cells with different ages but, most importantly, erythrocytes with different biochemical and functional capabilities.

In the course of ozone therapy, we have already measured a marked increase of G-6PD and other antioxidant enzymes in young erythrocytes (Bocci, 2004). The process of cell activation is very dynamic and don’t last for ever because blood cells have a definite life-time and a limited biochemical memory; therefore, the therapeutic advantage MUST BE MAINTAINED WITH LESS FREQUENT TREATMENTS.

The antioxidants system

 

 The interaction among antioxidants, enzymes and the metabolic system is very important as it allows their rapid regeneration and the maintenance of a normal antioxidant status.
 

The following scheme beautifully illustrates the cooperation among various antioxidant system in order to neutralize a lipoperoxide radical ROO• (shown on the left hand side) to a less reactive hydroperoxide, ROOH. The reducing activity is continuously generated by cellular metabolism via the continuous reduction of NAD(P)+ to NAD(P)H.

 It suffices here to say that, during the transient exposure of blood to appropriate concentrations of ozone, the antioxidant reservoir decreases no more than 35% in relation to ozone doses between 10 and 80 mcg/ml of gas per ml of blood.

 It is important to add that this partial depletion is corrected in less than 20 min thanks to the recycling of dehydroascorbic acid, GSSG, alpha-tocopheryl
radical to the reduced compounds. 

 

Conclusions

 The concept that ozonetherapy is endowed with an acute oxidative stress bothers the opponents of this approach because they consider it as a damage inflicted to the patients, possibly already under a chronic oxidative stress. THEY DO NOT BELIEVE THAT OZONETHERAPY INDUCES A MULTIVARIED THERAPEUTIC RESPONSE ALREADY WELL DOCUMENTED IN SOME DISEASES. Moreover THEY DO NOT DISTINGUISH THE CHRONIC OXIDATIVE STRESS (COS) DUE TO AN ENDOGENOUS AND UNCONTROLLED HYPEROXIDATION LINKED TO SEVERAL PATHOLOGIES WITH THE SMALL AND TRANSIENT OXIDATIVE STRESS that we can precisely perform EX VIVO with the ozone dose.

The THERAPEUTIC RESPONSE achieved after these repeated oxidative
stresses can be envisaged as a PRECONDITIONING EFFECT eventually able to
reequilibrate the redox system altered by pathogenetic stimuli.

 

Sources:
Velio Bocci
Ozone a new medical drug.

 

 

 

10 Nov '17

How Does Ozone Act? How and Why Can We Avoid Ozone Toxicity?

Posted by Marin Crangaci
How Does Ozone Act? How and Why Can We
Avoid Ozone Toxicity?
Therefore it should be clear that some of ozone dose is neutralized by the antioxidants present in plasma and only the reaction with PUFA is responsible for the biological and therapeutic effects. This should clarify why a very low ozone dose can be ineffective or equivalent to a placebo. Moreover, after ozonation of human blood, the antioxidant capacity measured in plasma decreases no more than 30% after about 5 min but returns to the normal value during t he following 15 min, thanks to the rapid r eduction of t he oxidized antioxidants operated by erythrocyte. This result emphasizes that even the higher ozone dose (80 mcg/ml gas per ml of blood) never overwhelms the antioxidant capacity of plasma and insures against any damage to blood cells.

ROS include several radicals as anion superoxide (O•−2 ), nitrogen monoxide (NO•), peroxynitrite (O=NOO–), the already mentioned hydroxyl radical and other oxidant compounds such as hydrogen peroxide and hypoclorous acid (HClO). All of these compounds are potentially cytotoxic luckily have a very short half-life (normally a fraction of a second) and both the plasma and cells have antioxidants able to neutralize them, if their concentrations do not overwhelm the antioxidant capacity.  This concept emphasizes why the ozone dose must be precise and well calibrated against the antioxidant capacity of blood thus capable of triggering useful reactions without procuring any damage.
The scheme intends to show that ozone dissolved in the plasmatic water reacts immediately with a number of biomolecules and disappears. The compounds generated during the reactions (ROS and LOPs) represent the “ozone messengers” and are responsible for the biological and therapeutic effects.
The multivaried biological response of the organism to ozonized blood can be envisaged
by considering that ozonized blood cells and the generated LOPs interact with a number of organs. Some of these represent real targets (liver in chronic hepatitis, vascular system for vasculopathies), while other organs are probably involved in restoring normal homeostasis. CNS: central nervous system, GIT: gastrointestinal tract, MALT: mucosal associated lymphoid tissue
LOPs generated after peroxidation of a great variety of PUFAs are heterogenous and briefly are represented by peroxyl radicals (ROO•), a variety of hydroperoxides (R–OOH) and a complex mixture of low molecular weight aldehydic end products, namely malonyldialdeyde (MDA), and alkenals, among which 4-hydroxy-2,3 transnonenal (4-HNE), which is potentially cytotoxic. The chemistry and biochemistry of these compounds has been masterfully described by Esterbauer’s group (1991). If one thinks about the wealth and chemical heterogeneity of lipids, glycolipids and phospholipids present in plasma, it becomes difficult to imagine how many potent, potentially noxious, compounds can be generated by the lipids reacting with ozone. During one of my several disputes with American referees, a distinguished scientist wrote: “It is grotesque to think that any Western World Drug Regulating Agency would condone infusing the hodgepodge of ozonized products to treat diseases, although it is probable that the products would initiate and/or modulate a wide spectrum of inflammatory-immune processes to varying degrees”.
In opinion of Italian Professor Velio Bocci: " This referee missed what I believe is the formidable strength of ozonetherapy: provided that we can control (by using precise ozone concentrations exactly related to the blood volume and antioxidant capacity) the amount of LOPs, we can achieve a multitude of biological effects unthinkable with a single drug. Indeed a great expert in antioxidants, Prof Lester E. Packer, University of California at Berkeley wrote me that the hypothesis that a small dose of ozone can elicit a number of antioxidant responses useful to the organism is quite reasonable and in line with current thinking.
The next simple scheme ought to fix in the reader’s mind this crucial point and the sequence of events eventually leading to the therapeutic results: ROS are produced only during the short time that ozone is present in the glass bottle, ex vivo, and they yield EARLY biological effects on blood, whereas LOPs, which are simultaneously produced, have a far longer half-life and, during the reinfusion of ozonated blood in the donor, they reach the vascular system and practically all the organs where they trigger LATE effects."
We have come to a critical point: how can we reconcile the production of toxic compounds with the idea that these compounds exert important biological and therapeutic effects?
Let us first examine the behaviour and pharmacodynamic of hydrogen peroxide, which in practical terms is the most important ROS. As soon as ozone dissolves in the plasmatic water and reacts with PUFAs, the concentration of hydrogen peroxide starts to increase but, just as rapidly, decreases because this unionized molecule diffuses quickly into erythrocytes, leukocytes and platelets, where it triggers several biochemical pathways.
Does the increased intracellular concentration of hydrogen peroxide become
toxic for the cell?
Absolutely no! Because, at the same time, it undergoes reduction to water in both plasma and intracellular water, thanks to the presence of powerful antioxidant enzymes such as catalase, glutathione-peroxidase (GSH-Px) and free reduced glutathione (GSH). Perhaps for a few seconds, the chemical gradient between plasma and the intracellular concentration of hydrogen peroxide has been estimated to range from 1 to 4–5 μM equivalent to about 10% of its plasma concentration, which avoids any toxicity.
The transitory presence of hydrogen peroxide in the cytoplasm means that it acts as one of the ozone chemical messengers and that its level is critical: it must be above a certain threshold to be effective but not too high to become noxious. In studies conducted by Velio Bocci and co, performed with human blood exposed to ozone concentrations ranging from 20 to 80 mcg/ml per ml of blood, the process of hydrogen peroxide generation, diffusion and reduction was found always extremely transitory (Bocci et al., 1993a, b, 1998a, b) even though Halliwell et al. (2000a, b) consider this molecule physiologically ubiquitos
in the body.
 
Moreover hydrogen peroxide is recognised as an intracellular signaling molecule able to activate a tyrosine kinase, which phosphorylates a transcription factor (Nuclear Factor KB, NFKB), which allows the synthesis of a number of different proteins . Basically hydrogen peroxide functions by oxidizing cysteines, and Velio Bocci and Others have found that it acts on blood mononuclear cells (Bocci and Paulesu, 1990; Bocci et al., 1993b, 1998a; Reth, 2002), on platelets (Bocci et al., 1999a), on endothelial cells (Valacchi and Bocci, 2000) and on erythrocytes (Bocci, 2002). ROS entering into the erythrocytes are almost immediately reduced (hydrogen peroxide to water and lipoperoxides to hydroperoxides) at the expense of GSH. The enormous mass of erythrocytes can easily mop up hydrogen peroxide and, within 10–15 min, marvellously recycle back oxidized antioxidants in reduced form. While glutathione reductase (GSH-Rd) utilises the reduced nicotinamide adenine dinucleotide phosphate (NADPH, this coenzyme serves as an electron donor for various biochemical reactions) to recycle oxidized glutathione (GSSG) to the original level of GSH, the oxidized NADP is reduced after the activation of the pentose phosphate pathway, of which glucose-6-phosphate dehydrogenase (G-6PD) is the key enzyme. Thus, glycolysis is accelerated with a consequent increase of ATP levels. Moreover the reinfused erythrocytes, for a brief period, enhance the delivery of oxygen into ischemic tissues because of a shift to the right of the oxygen-haemoglobin dissociation curve due either to a slight decrease of intracellular pH (Bohr effect) or/and an increase of 2,3 diphosphoglycerate (2,3-DPG) levels.
A summary of the biological effects elicited during exposure of human blood to oxygenozone, ex vivo and during its reinfusion in the donor.
To be continued...
Sources:
Velio Bocci
Ozone a new medical drug.


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