V. Bocci, A. Larini and L. Bianchi
Department of Physiology University of Siena, 53100 Italy
This was the very question that, by sheer coincidence, posed myself in 1998: after a few years, we discovered that indeed careful ozonation of blood may have immuno modulatory activities due to release of cytokines. Ozone is only the reaction trigger and, among several molecules included in the family of reactive oxygen species, ROS (HOCl, NO•, OH•, 1O2, ROO•, O2−•, O=NOO−) H2O2 is one of the most important. Ozone, by reacting with double bonds present in PUFA, generates H2O2 and 2 moles of R-CHO. The sudden increase of H2O2 concentration in the plasma during lipid peroxidation causes the rapid diffusion of H2O2 into the cellular cytoplasm. Here, its increase is checked by antioxidants (GSH, AH− and enzymes such as GSH-Px and catalase) so that its concentration is 20-40% less than in plasma. Nonetheless the sudden increase of H2O2 acts a crucial signal for specific kinases (IKK-1/2) that, by phosphorylating the protein IKBα, causes its detachment from the inactive transcription factor called NF-KB and allows the rapid migration of the heterodimer (p50-p65) into the nucleus where it binds to specific sequences in the promoter regions of several genes, including those inherent to cytokines, haematopoietic growth factors, adhesion molecules and acute phase proteins. This implies that some leukocytes, once activated, can synthetize a variety of proteins including IFNs, ILs, TNFα, chemokines, orosomucoid, C-reactive protein growth factor, MHC proteins etc.
Although this is a significant mechanism due to an acute and transitory oxidative stress, in leukocytes is not necessarily the only one because there are other transcription factors that can be either activated via diacylglycerol (DAG activates protein kinase C), inositol 1,4,5-trisphosphate (IP3, opens Ca 2+ channel) or adenylate cyclase or inhibited by ceramide via activation of sphingomyelinase. However they still need to be investigated.
Another uncertain issue is the role played by other ROS and particularly compounds such as OCl−, OH•, and O=NOO− that can be responsible of cytotoxic effects when ozone concentrations overwhelm the antioxidant capacity. Similarly, we have just started to investigate the relevance of one typical aldehyde such as 4-hydroxy-2,3-trans-nonenal (4-HNE), while the activity of the heterogeneous group of other lipid oxydation products (LOP) remains unknown.
The effects of these compounds is likely quite different if the problem is examined in the whole blood, or in isolated leukocytes resuspended either in their own plasma, or in protein-free tissue culture media and, most critically, if the problem is examined in vitro or in vivo. Owing to the dogma that “ozone is always toxic”, orthodox medicine is very much against ozonetherapy and therefore the problem of cytotoxicity must be seriously addressed. We have taken the view that, although ozone is potentially toxic, low concentrations can be tamed by the physiological antioxidant capacity and can have beneficial effects. LOP have a relatively long half-life and in vivo the re-infusion of the blandly ozonated blood implies a considerable dilution in body fluids, an extensive breakdown by aldehyde-, alcohol-dehydrogenases and GSH S-transferase and a marked excretion via urine and bile. In this way LOP’s uptake by cells is minimized (probably at submicromolar levels) and while atoxic, it can serve as the crucial signal for inducing oxidative stress proteins (OSP), hence the adaptation to chronic oxidative stress and possibly activation of staminal cells. In recent years, even the most implacable supporters of free radical toxicity, have to admit that very low concentrations of ROS and LOP behave as physiological messengers and can be beneficial, while higher concentrations can obviously provoke detrimental consequences culminating in inflammation and cell degeneration. Our recent studies with Jurkat cells, maintained in media with variable antioxidant capacities, when tested against a very wide range of ozone and 4-HNE concentrations have beautifully demonstrated the validity of this thesis. Nonetheless there are still many outstanding scientists, who maintain that ozonetherapy is “a barbaric, unsafe procedure” and could be substituted by the injection of “a little lipid peroxide or LPS”. Obviously the problem ofcontraposition between molecular and “natural” medicine is a fascinating issue but it cannot be discussed here. In order to finally understand whether the exposure of whole blood to oxygen-ozone can lead to immune modulation and therapeutic effects, we have addressed the following problems:
1. How important is the maintenance of the plasmatic Ca2+ level during blood ozonation?
2. Is there any advantage in ozonizing a large number of isolated leukocytes rather than whole blood?
3. Within the “therapeutic window” is there a range of either immunostimulating or immunosuppressive O3 concentrations?
4. On the experimental basis that ozone can act as a modest cytokine inducer, does reinfusion of ozonized blood modify the plasma cytokine in vivo?
5. Is O3-AHT effective and is there an optimal schedule?
6. Does the induction of OSP and of adaptation to chronic oxidative stress have an immunomodulatory effect?
7. Which is the immunomodulatory role and relevance of granulocytes?
8. Can we select tests suitable for evaluating modifications of the immune status during ozonetherapy?
Unfortunately, owing to several reasons (lack of cooperation of oncologist and clinicians, lack of funds, scepticism towards ozonetherapy, etc.), so far we have only a few hints that repeated administration of ozonated blood can be therapeutically useful but, as now we have been able to open an ozonetherapy center at our University polyclinic, there is a hope to answer this question in the next couple of years.
Bocci V. (2002) Oxygen-ozone therapy. A critical Evaluation, 1-440, Kluwer
Academic Publishers, Dordrecht, The Netherlands.