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Ozone In Medicine
V. Bocci a; C. Aldinucci a; E. Borrelli b; F. Corradeschi a; A. Diadori c; G. Fanetti d; G. Valacchi a
a Institute of General Physiology, University of Siena, Siena b Institute ofThoracic and Cardiovascular Surgery,
c Department of Ophthalmology of the University of Siena, d Servizio Trasfusionale, Azienda Ospedaliera Senese, Siena, Italy
Online Publication Date: 01 January 2001
To cite this Article Bocci, V., Aldinucci, C., Borrelli, E., Corradeschi, F., Diadori, A., Fanetti, G. and Valacchi, G.(2001)'Ozone In Medicine',Ozone: Science & Engineering,23:3,207 — 217
To link to this Article: DOI: 10.1080/01919510108962004
URL: http://dx.doi.org/10.1080/01919510108962004

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OZONE SCIENCE & ENGINEERING 0191-9512101 53.00 + .OO
Vol. 23. pp. 207-217 lntcrnational Ozone Association
Rintcd in the U.S.A. Copright @ 2001
Ozone In Medicine
V.Bocci4, C.Aldinucci, E.Borrelli,' F.Corradeschi, ~ . ~ i a d o r iG' ,.F anetti3 and G.Valacchi
Institute of General Physiology, University of Siena, Via A. Moro, 53100 Siena, Tel: 0039 0577 234226;
Fax: 0039 0577 234219 ; email: Fisaen@unisi.it
'1 Institute of Thoracic and Cardiovascular Surgery
Department of Ophthalmology of the University of Siena
') Servizio Trasfusiodale, Azienda Ospedaliera Senese, Siena. Italy, Tel: 0039 577 585070, Fax: 0039 577 5861 67
') Corresponding author
Received for Review: 9 March 2000
Accepted for Publication: 6 December 2000
Abstract
Ozone therapy has been used as a complementary medical approach for half a century but it has
encountered skepticism by orthodox medicine because, particularly in the past, it has been used by
practitioners and others without a rational basis and appropriate controls. With the advent of modem
medical ozone generators incorporating a photometer, it has become possible to obtain precise ozone
concentrations and to evaluate some mechanisms of action and possible toxicity. In contrast with the
respiratory tract, human blood exposed to appropriate ozone concentrations is able to tame its strong
oxidant properties and neither acute, nor chronic side effects have ensued in millions of patients treated
with ozonated autohaemotherapy (0,-AHT). This review summarizes our studies aimed at clarifying
biological effects, defining any possible damage, the therapeutic window and suitable doses able to express
a therapeutic activity. A very interesting and promising aspect is the induction of the so-called heat stress
proteins (HSP) leading to adaptation to a chronic oxidative stress. The use of ozone in human therapy has
been reviewed but so far very few controlled clinical studies have been reported. Mostly on the basis of
anecdotal results, ozone therapy appears usehl in infectious diseases, immune depression, vascular
disorders, degenerative diseases and orthopedics.
Key Words
Ozone; Medical Applications; Reactive Oxygen Species; Antioxidants; Hemotherapy; Ozone Tolerance;
Introduction
Although ozone has been used as a potent
disinfectant since the first World War (I), its
validity in medicine still remains controversial, even
though the National Health Institutes of several
countries, namely Germany, Italy, Austria, Russia
and some of the United States now include ozone
therapy and bio-oxidative therapy among the
pharmacological approaches of complementary
medicine. In most of the United States, the problem
of ozone, as one of the worse pollutants in large
cities, has acquired such a preeminent consideration that
it practically denies its use in medicine. Studies in vitro
and in vivo (2-5), confirming its toxicity for the
respiratory tract have led to the co.nclusion that ozone is
"always" toxic for humans, animals and plants. The
authors believe that the generalization of this conclusion
is, at least in part, unjustified because ..we have
demonstrated that judicious use of ozone can be
therapeutically useful and atoxic (6-10). There is no
doubt that ozone is intrinsically toxic (1 I ) , but as any
other drug, when used properly, has a definite
therapeutic window. Moreover, every year millions of
207
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208 V. Bocci et al.
patients all over the world undergo some sort of
ozone therapy and minimal, if any, side effects have
been noted. Some charlatans, mostly without any
medical qualifications, have caused a few deaths
because they inject directly the gas intravenously, a
procedure prohibited since 1986 in Europe (7;8; 10).
It is unfortunate that even today a few physicians
and many naturopaths and others, owing to the fact
that they cannot practice the classical hemotherapy,
predicate that intravenous injection of oxygen-ozone
is "the only effective way". This crucial problem
will be discussed in order to clarify the danger and
its basic irrationality.
The purpose of this brief review is four fold: firstly,
to present data from our Laboratory that show how
ozone, coming in contact with biological fluids,
decomposes and generates reactive oxygen species
(ROS), secondly, to define how ozone's messengers
can activate biochemical and immunological
mechanisms leading to biological effects, thirdly, to
show that we are now able to determine a
therapeutic window or, in other words, a range of
biologically active concentrations below which
ozone is practically inactive and above which can be
toxic. Fourthly, we will attempt to analyze the
results regarding therapeutic efficacy in five main
areas: infectious diseases, immune depression,
vascular disorders, degenerative diseases and
orthopedics. The breadth of ozone therapy, rather
than arising the suspicion of a "panacea", ought to
be envisaged as due to the multiform action of
ozone on cells with different functions.
The knowledge recently acquired allows one today
to plan rational clinical applications in different
diseases and to evaluate the therapeutic activity and
side effects. Future breakthroughs can be achieved
only if we are able to grasp firstly, the biological
activity of lipid oxidation products (LOPS),
secondly, the practical implications of the ozone
tolerance by clarifying the role of heat-stress
proteins (HSP) and, thirdly, if we will be able to
cany out randomized, double blind clinical trials
possibly performed in several medical centers.
The present paper intends to give a general
overview of the results so far achieved and therefore
technical details can be found in previous papers
(I 2- 15).
Ozone Mechanism
Progress in this field is expected only if we are able to
clarify precisely the mechanisms of action because it
will allow defining the therapeutic dose and possible
toxicity. We will examine separately the possible
mediators broadly defined as reactive oxygen species
(ROS) and the cell targets that are ultimately responsible
for the therapeutic response.
Ozone's active messengers
Both oxygen (about 97%) and ozone (no more than 3%)
dissolve in biological fluids according to their solubility,
relative concentrations, partial pressure and temperature
(1). However, there is a critical 'difference between these
two gases because oxygen is fairly stable in solution
while ozone decomposes immediately by avidly reacting
with polyunsatured fatty acids (PUFA) (1 1). This
implies that ozone does not obey Henry's law and
therefore an extremely dynamic equilibrium arises
between the ozone in the gas phase and the ozone
reacting and disappearing in the aqueous solution. Thus,
we can envisage a continuous flow of ozone into the
solution from the gas phase until the latter is exhausted.
It is felt that this crucial instability has not been fully
appreciated by cell biologists, who examine ozone
toxicity in tissue cultures maintained in a gas phase
where concentration of ozone, although very low (0.2 -
1 pprn), remains stable for several hours or days of
incubation. The final results are misleading because it is
obvious that overall cell toxicity cannot be simply
attributed to the low ozone concentration, but to the
uncalculated total sum of ozone that during every
millisecond has passed into the solution. In other words,
a cell layer in culture exposed to an ozone concentration
as low as 0.1 ppm may not be damaged if the exposure
lasts only ten min whereas total cell death may ensue
after 60 min exposure because ozone will continue to
dissolve during the following 50 min reaching the lethal
amount.
It has been shown (1 1) that the reaction between a mole
of an unsaturated fatty acid containing a cis-double bond
and 0, in water generates two moles of aldehyde and
one mole of hydrogen peroxide (H20,).
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Ozone in Medicine 209
H20, is also generated when O, reacts with
physiological saline.
The reduction potential is smaller (0.682 volt) in the
semireaction towards H,02 than directly towards
H20 (1.229 volt). Indeed we have demonstrated (14)
that after ozonation of either saline or human
plasma, H,02 is formed in both liquids with the
important difference that H,O, in the plasma has a
very short half-life (about 2.5 min) due to the
presence of traces of enzymes such as glutathione
peroxidase (GSH-Px) and catalase, which are able to
degrade H202. Appropriate enzyme inhibitors are
able to prolong the lifetime of H20, while addition
of catalase, as it was expected, accelerates its decay
(14). The other important reaction is that 0,, by
reacting with PUFA, will generate a number of
LOPs such as hydroperoxides, isoprostanes,
platelet-activating factor (PAF) and terminal
products such as malondialdehyde and 4-
hydroxyponenal (HNE) (162 1 ) The latter
compound is becoming particularly interesting
because, depending upon its final concentration
(>lo ph4 or < I pM), may either be harmful or act
as a physiological messenger, respectively (22;23).
Owing to the wealth and heterogeneity of PUFA,
several types of LOPs may be generated and their
biological activities, including potential toxicity,
remain to be explored in vivo. Once again,.results
obtained in vitro by using apparently toxic LOPS
may not be applicable in vivo owing to their rapid
turnover and, as an example, enzymes such as
glutathione transferases and aldehyde
dehydrogenases are involved in the metabolism of
HNE (16). Phospholipases and sphyngomyelinase
are likely to be activated by LOPs and this may lead
to an amplification of some biological processes.
Furthermore LOPS have a short half-life but, upon
reinfusion of ozonated blood, may reach specific
sensors situated in critical organs such as bone
marrow, spleen, liver and other sectors of the
immune system. If this is true, .LOPS may be
responsible for transmitting the information of
peroxidative stress and possibly inducing the
upregulation of antioxidant enzymes, hence the
tolerance to 0,. This dubbed as
"oxidative stress adaptation" or oxidative
preconditioning (7;24-26) is extremely interesting
because it could allow a reversal of chronic
oxidative stress typical of degenerative diseases. We
have already demonstrated an increase of
antioxidant enzymes (7) and we are examining.
levels of heme oxygenase (HO) activity. The
isoform 1 of the latter enzyme (HO-I), also known as
heat shock protein 32 @sp 32), is inducible and is
responsible for the conversion of heme into biliverdin,
carbon monoxide (CO) and free iron (27-29). ,
Cytochrome P450 constitutes another source of heme
undergoing degradation via HO-1. We would like to
emphasize that the above products, until recently
regarded as toxic waste destined only for excretion, are
compounds with great physiological and a possible
therapeutical role: bilirubin (via biliverdin reductase) is
a crucial lipophylic antioxidant and CO may function as
a gaseous regulator of endothelial tone in synergy with
nitric oxide (NO). Indeed we have just demonstrated
that human endothelial cells exposed to ozonated
plasma increase the release of NO (30). Nitrosothiols
such as S-nitrosocysteine, S-nitrosoglutathione and Snitrosoalbumin,
formed in human plasma to buffer NO'S
concentration, have physiological significance because
function as a reservoir for NO (31). Another important
mechanism of activation that has been partly clarified
(13) is the opening of Ca2' channels somehow related to
ROS acting on the external part of the cell membrane
leading to a sudden increase of intracellular Cal'
concentration with consequent enzymic activation. So ,
far we have only indirect evidence of this phenomenon
by either chelating tjle extracellular Ca2+ with citrate
used as a blood anticoagulant or by adding from 5 up to
25 rnM Ca2+ in heparinized blood (13) but obviously it
will be important to measure the actual intracellular
increase of Ca2+
Effectors and the biochemical targets
It is now clear that ozone works indirectly in different
ways: owing to' the fact that H,O, is an unionized
molecule and its passage through the cell membrane is
free, its sudden increase in the extracellular water is
immediately transferred into the intracytoplasmic water,
but the intracellular environment counteracts this
potentially toxic increase by quenching it with reduced
glutathione (GSH) coupled to GSH peroxidase. This
causes an increase of oxidized glutathione (GSSG) and a
decrease of the GSWGSSG ratio, which is rapidly
reconstituted by the action of GSH reductase in turn
exploiting the NADPWNADP reservoir. Lowering the
NADPH level enhances the activity of glucose 6
phosphate dehydrogenase (G6PD) that, particularly in
the erythrocytes, leads to the activation of. the hexose
monophosphate shunt. When necessary an excessive
increase of intracytoplasmic Y,02 is also double
checked by catalase. There is a concomitant activation
of glycolysis with increased ATP and a still
controversial increase of 2-3 diphosphoglycerate (2-3
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210 V. Bocci et al.
DPG) production as key enzymes involved in this
process have not yet been shown to be activated
(32;33). Obviously the shift to the right of the HbO,
dissociation curve would favor an increased oxygen
delivery to hypoxic tissues (7;lO). It has also been
claimed (34) that the erythrocytic membrane
becomes more fluid and more negatively charged,
that blood viscosity decreases due to
hypofibrinogenemia and to a decreased level of low
density lipoproteins (LDL). However Morgan et al.
(35) found that erythrocytes from ozone-exposed
mice exhibited decreased deformability and
therefore all of these claims must be controlled
because we must be sure if indeed ozone can
improve, blood rheology in ischemic diseases.
As far as the activation of cytokine synthesis in
leukocytes is concerned, it is now well accepted that
the sudden surge of intracytoplasmic H,O, is finally
responsible for the activation of the nuclear
transcription factor (NF-kl3). Briefly, H20,, by
activating specific protein kinases, would
phosphorylate the I-KB subunits that detach from
the NF-k8 complex. The free heterodimer (p50-p6S
proteins) can then move into the nucleus where,
after binding to DNA control elements, activates
gene expression and the successive synthesis of
interferons and interleukins as shown by us (12-
15;36) and others (37;38).
The transient rise of intracytoplasmic H,O, prompts
a few considerations: the first one is that the 0,
concentration must be adequate to allow a sufficient
H,O, generation for the activation of transducer
molecules and to counteract, the simultaneous
degradation, and the second is that H,02
concentration must reach a critical threshold. If it is
below the liminal value, activation will not occur
but if it is excessive, damage may result implying
the relevance of having identified the therapeutic
window between about 20 and 80 pglml of gas per
ml of blood. If the 0, concentration is below 20
pglml, 'most of the oxidant power of 0, will be
quenched by the natural antioxidants (between 1.28
and 1.83 mM plasma) (39) and therefore the
necessity of measuring precisely the 0,
concentration to avoid either a placebo or a toxic
effect is of crucial importance. On the experimental
basis of progressively increased hemolysis, ozone
concentrations higher than 80 pglml are more likely
detrimental than beneficial.
Little is known about the biological activity of LOP
such as hydroperoxides, isoprostanes, malondialdehyde
and 4-hydroxyalkenals produced during blood
ozonation. Aggregation of platelets, as we have
observed in platelet rich plasma anticoagulated with
heparin (40), is at least in part attributable to released
PAF (21). While some of these can act as physiological
messengers (18;20-23) they appear to be, particularly in
vitro, very toxic (17;22;23). Their production and
consequent plasma levels are somewhat related to the
ozone dose and it is conceivable that in vivo a low
ozone dose may express a more favorable
activityltoxicity ratio than a higher ozone dose. Thus,
once again, we should aim to define in different
pathologies the optimal dose that may be either in the
low (20-40 pg/ml per ml of blood), or in the mediumhigh
range (30-80 pglml per ml of blood).
Moreover LOPS may exert the overlooked and yet
crucial function for transmitting the information of on
ongoing peroxidative stress to distant organs with the
purpose of inducing the "oxidative stress adaptation" or
ozone tolerance (24-26;41-48). This can be achieved
only by slowly activating gene expression towards the
synthesis of heat-shock proteins, antioxidants enzymes
(GSH-Px, catalase, superoxide dismutases etc), DNA
repair enzymes and, most important, heme-oxygenase
(27-29). This may lead to increased bilirubin levels (49)
and local release of CO that, associated to increased
endothelial production of NO (30) may well explain the
vasodilation and consequent clinical improvement
observed in limb ischemia treated with 0, AHT. It is
almost needless to say that upregulating the production
of antioxidant enzymes in patients with degenerative
diseases (favored or caused by a life-long oxidative
stress) is the simplest way to readjust the redox balance,
possibly leading to a stabilization of the disease.
Administration of antioxidant compounds may be
helpful (50;Sl) but, most likely, not so effective for
neutralizing ROS as the intracellular increase of
antioxidant enzymes.
Applications of Ozone Therapy in Medicine
Today, a better understanding of the basic reactions of
ozone able to activate different biological functions
allows the dispelling of skepticism surrounding ozone
therapy. Although its application is extremely versatile
there are two important limitations: firstly, ozone should
never be inalated as the fluid film lining the tracheobronchial
mucosa is too thin to protect it from the .
oxidative insult (11) and secondly, the gas mixture of
OJO, should never be injected intravenously (IV) either
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Ozone in Medicine 211
because it can cause oxygen embolism and because
no meaningful blood /ozone ratio can be ever
calculated. We will never get tired of repeating that
human organism, although composed of almost 66%
water should not be compared and treated as a water
sterilization plant. Were we to allow the IV gas
administration, it would cause severe side effects
and many deaths each year.
On the other hand the approach consisting in the
exposure of a precisely measurable volume of the
patient's blood (200-250 ml) to an equal volume of
gas (1 to l), of which the ozone concentration can
be accurately measured in real time by photomeby,
is by far the most scientific, simple, inexpensive and
side-effects free procedure. Most of the merit goes
to Wolff (52) who applied the ozonated
autohemoterapy (0,-AHT) in the late 70s. The
optimized procedure that must be can-ied out in
neutral glass and ozone-resistant tubing where the
inlet is separated from the outlet equipped with a
standard blood, filter has been recently described in
detail (53). Standard autotransfusion bags made of
polyvinyl chloride (PVC) additioned with about
40% additives have been banned by the Italian
Ministry of Health after our demonstration (53) that
ozone causes the release of significant amounts of
plastic microparticles and phthalates into the blood.
Other routes of administration of ozone can be
allowed for selected applications: the subcutaneous
(SC) route for treating lipodistrophy; the
intramuscular (LM) route into the paravertebral
muscles aAer locating the point(s) triggering low
back pain; the intradiscal- intraforaminal andfor the
epidural route for treating a herniated disc; the
intraarticular or periarticular route for treating acute
and chronic arthrosis. Knoch et al. (54), Carpendale
et al. (55) and ourselves (56) have evaluated pros
and cons of the rectal insufflation of 0,-0, as a
possible option when 0,-AHT cannot be used for
difficult venous access. This route has been used in
human. immunodeficiency virus (HIV) infection
( 5 9 , chronic hepatitis, ulcerative colitis and
Crohn's disease with apparently satisfactory results
(54) using up to 800 ml of 0,-0, at a maximal 0,
concentration of 40 pg/rnL administered within 5
minutes. In the case of chronic bacterial and
parasitic infections becoming resistant to antibiotics,
low 0, concentration (3-5 pg/mL) have been also
insufflated into the oral, nasal, tuba1 (during 30 sec
apnea), vaginal, urethral, vesical, pleural and
peritoneal cavities. Obviously the technique of gas
insufflation is a very empirical and approximate one but
it can be useful, as ozone does not allow bacterial
resistance.
Which are the diseases likely to benefit from the
application of ozone therapy? It appears reasonable and
ethical to use ozone especially when conventional
therapies are ineffective or not available as too often
occurs in poor countries. Obviously, by considering the
potent disinfectant action of 03, top priority goes to all
sorts of bacterial, viral and fungal infections. Either gas,
or ozonated water, or ozonated oil display a cleansing
and disinfectant effect (1 ;57-63).
Moreover 03-mT, combined with topic therapy, can be
helpful because, as previously ,discussed, it activates cell
metabolism and the immune system. Indeed various
immunodeficiencies associated with chronic viral
diseases and metastatic cancer, particularly after highintensity
chemotherapy, may benefit from a long cycle
(about 50 treatments, twice weekly for six months) of
0,-AHT that, in comparison to interferon, highly active
antiretroviral therapy (HAART) and cytostatics does not
procure acute or chronic side effect (64;65). Actually
the majority of patients reports an unusual feeling of
well-being that should not be neglected.
Unfortunately, for the time being, we have to rely on
anecdotal reports (65). One clinical study in HIV
infection (66) yielded doubtful results because blood
was badly mistreated by heat, W irradiation and 0, in
unknown concentration.
In western countries several circulatory disturbances
,bind-limb ischemia, heart-brain-retinal ischemia) due
to atherosclerosis, diabetes, smoking, aging and a too
intense lifetime oxidative damage represent a
formidable medical problem that cannot. be entirely
coped by orthodox medicine.
0,-AHT has shown therapeutic effects particularly in
patients refractory to conventional treatments because,
as it has been mentioned, expresses multiple actions
such as vasodilation, increased delivery of oxygen in
hypoxic tissues and release of wound healing factors
(67). Clinical results in acute cerebro-vascular disorders,
chronic ischemic cardiopathy and even in the 111--1V
stages of hind-limb ischemia have been remarkable,
particularly, when a systemic treatment was combined
with a topical one on torpid ulcers and incipient necrosis
(33; 68-70). Two randomized, placebo controlled (0,-
AHT) cross-over studies have been performed to
evaluate the efficacy of 0,-AHT in patients with age-
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212 V. Bocci et al.
related macular degeneration (ARMD) (33) and
with mild hypertension (71). Significant clinical
improvement was achieved in both trials although it
faded 2-4 months after the end of the treatment.
However, as it happens with other medications, this
is to be expected and can be minimized by
continuing the treatment at a slow pace.
As far as degenerative diseases are concerned,
preliminary studies by using 0,-AHT and 0, rectal
insufflation cairied out in patients with cardiac
infarction (72), neurodegenerative disease (73) and
ARMD (33) have shown clinical improvement and
interestingly a progressive increase in GSH Px,
glucose-6-phosphate dehydrogenase and superoxide
dismutase in erythrocytes. However there is an
urgent need for programming controlled studies in
order to show that ozone therapy can induce a state
of oxidative stress adaptation, possibly capable of
stabilizing the disease.
Finally injections of small volumes of 0,-0, at a 0,
concentration below 30 'pg/ml ire being used in
orthopedic pathology, via peri, or intrarticular, or
intradiscal injection (74; 75). It appears that the
treatment that is occasionally painful for a few
minutes has no side effects and in about 70 % of
patients allows pain relief, decongestion,
reabsorption of edema and improved mobility (74;
75). How ozone works remains hypothetical: after
intradiscal injection, ozone generates hydrbxyl
radicals (OH') measured by electron spin resonance
(Bocci et al, manuscript in preparation) that can
degrade proteoglycans in the degenerate nucleus
pulposus leading to its reabsorption with consequent
reduction of herniated material responsible for
radicular pain. In the synovial membrane ozone
therapy may either induce the release of
immunosuppressive cytokines andlor
proinflammatory cytokine antagonists as well as the
over-expression of antioxidant enzymes able to
block excessive ROS formation. In regard to the
injection of 5-10 mL 0, -0, (15-20 pg/ml) into the
trigger points of paravertebral muscles
correspondent to the metamers of the hernial disc,
we have proposed (76) that the "chemical
acupuncture" due to the needle and ozone inhibits
amyelinic nociceptors fibers and activate the
antinociceptive system. This explanation appears
plausible because the successive analgesia permits
muscle relaxation and vasodilation with consequent
improvement of local muscular physiology and
disappearance of pain. Brayda-Bruno and Cinnella
(77) have reported that about 70 % of patients improve
after a few session of this easy, risk free procedure. It is
worth noting that lower back pain syndrome is very
common and it is advantageous to try this minimally
invasive treatment. However, as it was proposed in 1998
(76), it is impellent to compare this procedure against a
wait-list control, two placebo controls (one with 0,
alone and another without any gas) and a standardtreatment
control.
Conclusions and Perspective
On the basis of experimental results obtained in the last
decade (6;7;91;2 - 15;24-26;36-38;40), we have selected
a range from 20 up to 80 pg/ml of ozone per ml of blood
to be used for different pathologies, within which, no
damage to blood components has been noticed. An
orientative scheme of dosages has been previously
reported for different diseases (10) depending upon
whether the therapeutic activity is mainly exerted by
either erythrocytes or leukocytes (7; 8;10). In order to
avoid toxicity and allow oxidative stress adaptation, we
are applying the "start low, go slow" principle: that is
0,-AHT is performed starting with very low ozone
concentrations (20-25pg/mL per ml of blood) to be
increased in single steps of 5 pg/mL to the highest level
between 40 and 80 pglmL depending upon the disease
and the state of the patient (10).
Although we do not yet have unequivocal clinical data
based upon controlled double-blind studies, we have
encouraging evidence suggesting that ozone therapy can
be useful in vascular, infectious and degenerative
diseases (1;7;10;34;55;57-63;68-73). Whether ozone,
therapy can be useful in metastatic cancer (65) and
surprisingly in orthopedics (74-77), respiratory and
immune diseases remains to be seen and it should be
ascertained starting with cautious and controlled
experimentation.
Even if, theoretically, ozone therapy implies always an
oxidative insult, this must be carefully calculated on the
basis of a precise ozone dose and brief time of exposure.
Luckily this is possible owing to the large antioxidant
potential of blood (39;40;50;5 1). that is practically
impossible to overwhelm with the indicated ozone
concentrations. Moreover, during the course of therapy
the total antioxidant status must be sustained. by daily
administration of antioxidant vitamins (0.5 g of vit C, 10
mg vit E, Se, etc and at least 0.6 g of N-acetylcysteine as
a precursor of GSH) accompanied by a diet rich in fresh
vegetables and fruits.
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Ozone in Medicine 213
The concept of "oxidative stress adaptation" must
be thoroughly evaluated because it is expected to
lead to great improvements. If this idea will prove to
be correct against all the most pessimistic views of
ozone as a therapeutic agent (ll), we will have
demonstrated that ozone is indeed a paradoxical
molecule and that prejudices are the worst foes of
biology and medicine. In order to achieve a suitable
and smooth adaptation, the best strategy seems to
start with low and slowly increasing ozone dosages.
Two to three weeks may be necessary before
measuring a substantial increase of antioxidant
enzymes in erythrocytes. One must also take into
account that erythrocytes have a fairly slow turnover
(78) and therefore it takes a few weeks before the
newly "super-gifted" erythrocytes, released from
the bone marrow, can progressively substitute the
old ones. Thus the application of the "start low, go
slow" principle (10) appears reasonable for
demonstrating the validity of the concept.
In conclusion, in spite of our efforts during the last
decade to give a solid scientific basis to ozone
therapy, much work remains to be done. Ozone
therapy is in the middle of a schizophrenic situation:
on one hand, if one reads the weekly reports in the
oxgists, one remains appalled by wonderful
therapeutic achievements obtained in most cases by
charlatans without any medical qualification. This is
very detrimental for the real progress of ozone
therapy as desperate patients searching a hopeful
treatment are not in the position to distinguish
between the truth and the fake. On the other hand, in
the age of molecular medicine and gene therapy,
ozone therapy appears at best as an obsolete,
empirical and still doubtful approach. It reminds the
well-hown Indian story about the blind men and
the elephant. We touch it, we smell it but we still do
not see it. However, as-it happens in Science, even
gene therapy that seemed so promising present great
problem (79). Against all the odds, I firmly believe
that if we can continue with an appropriate
biological and clinical experimentation, ozone can
become an important therapeutic agent because it
can reactivate a variety of biological hctions
crucial for regaining health and is very cheap, easy
to use, versatile and atoxic, if used properly.
Acknowledgements
This work was partly supported by MURST grants
(ex40%). The editorial assistance of Mrs. Helen Carter
and Patrizia Marrocchesi is gratefully acknowledged.
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Ozone in Medicine

Overview and Future Directionsby Gérard V. Sunnen, M.D.

Abstract:
Ozone, an allotropic form of oxygen possesses unique properties which are being defined and applied to biological systems as well as to clinical practice. As a molecule containing a large excess of energy, ozone, through incompletely understood mechanisms, manifests bactericidal, virucidal and fungicidal actions which may make it a treatment of choice in certain conditions and an adjunctive treatment in others.
Introduction
Ozone, best known for its protective role in the earth's ecological harmony, and for its interaction at ground level with industrial pollutants, has unique biological properties which are being investigated for applications in various medical fields.
As early as the First World War, ozone's bactericidal properties were used to treat infected wounds, mustard gas burns and fistulas. These first treatment attempts, however, were hampered by technological difficulties. Medical ozone generators have since been developed and refined. They differ from industrial generators in their capacity to deliver the purest ozone-oxygen mixtures in precise dosages. A critical advance in medical ozone technology was the development, in the early 60's, of plastics which can adequately conduit this mixture and permit proper interfacing with patients. In the last few years ozone treatment has seen growing interest from diverse medical disciplines, and research is in progress to delineate its effects on biological systems and to define its clinical applications.
Historical Perspectives
The history of ozone's discovery is intrinsically entwined in the evolution of the earliest concepts in chemistry. Priestly and Cavendish noted that electrical sparks fired in a closed volume of air resulted in volume compression.[1,2] In 1785, Martinus Van Marum, subjecting oxygen to electrical discharges, noted "the odor of electrical matter" and the accelerated oxidation of mercury. In 1840, Schonbein repeated these experiments, concluded that this odor was due to a gas which he named ozone, from the Greek ozein (odorant), and described several of its properties.[3] Numerous researchers since that time have worked to elucidate the nature and actions of ozone. Still today, theoretical issues remain regarding its electron structure, the varieties of its molecular configurations and its kinetics. Mariniak and Delarive showed that it is an allotropic form of oxygen, and Mulliken and Dewar clarified its molecular architecture.[4]
In the latter part of the 19th century, ozone was found to oxidize a spectrum of organic compounds and to interact with double bonds. Chemists made use of these properties to study complex molecules by cleaving them into smaller fragments. Harries, by such methods, discovered the structure of natural rubber.[4]
The ability of ozone to destroy toxic or noxious industrial impurities (phenols, cyanides, tetraethyl lead among others) and to inactivate bacterial contaminants in sewage has made it an attractive alternative to chlorination. Wiesbaden, Germany became the first city to use ozonation for purification of its drinking water (1901), followed by Zurich, Florence, Brussels, Marseille, Singapore and Moscow (the largest installation in the world), among others. The history of ozone's medical applications has nebulous and anecdotal beginnings. Kleinmann is said to have carried out the first bacteriological studies on pathogenic organisms using the Siemens tube, shortly after its invention.[5] Payr,[6] and Fisch and Wolff[7] were clinician pioneers, and J. Hansler developed one of the first reliable models of medical ozone generators.[5,8]
Physico-Chemical and Biochemical Properties
The oxygen atom exists in nature in several forms: (1) as a free atomic particle (O), it is highly reactive and unstable; (2) oxygen (O2) its most common and stable form, is colorless as a gas and pale blue as a liquid; (3) ozone (O3), has a molecular weight of 48, a density one and a half times that of oxygen and contains a large excess of energy in its molecule (P3--) 3/2 O2 + 143 KJ/mole. It has a bond angle of 127 [3], which resonates among several forms, is distinctly blue as a gas and dark blue as a solid; (4) O4 is a very unstable, rare, nonmagnetic pale blue gas which readily breaks down into two molecules of oxygen.
Ozone is a powerful oxidant, surpassed in this regard only by fluorine. Shonbein,3 in 1855, discovered that it reacts with ethelene. Exposing ozone to organic molecules containing double or triple bonds yields many complex and as yet incompletely configurated ephemeral transitional compounds (zwitterions, molozonides, cyclic ozonides), which may be hydrolyzed, oxidized, reduced or thermally decomposed to a variety of substances, chiefly aldehydes, ketones, acids or alcohols. Ozone reacts with saturated hydrocarbons, amines, sulfhydryl groups and aromatic compounds.
Of importance to biological systems is ozone's interaction with tissue (especially blood) constituents. The most studied is lipid peroxidation although interactions have yet to be more fully investigated with complex carbohydrates, protein, glycoproteins and sphingolipids. These dynamics are especially relevant for medical applications because some of the most practiced methods in ozone therapy involve the mixing of a small volume of whole blood with a pure oxygen ozone mixture and subsequently returning it to the patient. In this manner, it is calculated that the dose of ozone administered will perform its therapeutic functions without disrupting blood constituents.
Since there are a variety of lipid components in whole blood, it is of more than theoretical interest to determine the end products of ozone per oxidation and their effects, not only on physiological systems but on the integrity of ambient patholgenic organisms, since one of the mechanisms of viral inactivation is thought to be through this modality. Cholesterol accounts for 120 to 220 mg/100 ml, of which 60% to 75% are cholesterol esters; phospholipids 9 to 16 mg/100 ml; triglycerides 40 to 150 mg/100 ml, and free fatty acids 6 to 16 mg/100 ml. Given a total lipid concentration of 450 to 1000 mg/100 ml and the large variety of lipid constituents, the possible end products of ozonation are bountiful.[9,10]
This question is further complicated by the presence of systems to buffer lipid peroxidation, including vitamin E, uric acid,[11] and enzymes such as superoxide dismutase, catalase, and the glutathione peroxidase system which has gathered the most experimental attention.[12]
Several agents derived from lipid peroxidation include free radical, singlet oxygen, hydrogen peroxide, hydroperoxide, ozonides, carbonyls, alkanes and alkenes. Of these, lipid hydroperoxides, the most extensively studied, are known in sufficient concentrations to manifest their toxicity by altering cell membranes. Acted upon by glutathione peroxidase, they are reduced to their corresponding alcohols.
Method of Manufacture and Precautions
The production of ozone-oxygen mixtures for human and veterinary applications is subject to important technical consideration and standards. Clinical ozone generators which regulate the flow of medical grade oxygen through high voltage tubes with outputs ranging from 4000 V to 14000 V are capable of producing precise ozone-oxygen mixtures within concentration ranges extending to 5%, predicated on three variables: (1) the voltage applied; (2) the oxygen flow rate; and (3) the electrode separation distance. The purity of the oxygen source is especially emphasized since nitrogen, in the presence of high energy fields, forms toxic nitric oxides.
Since the half life of ozone is 45 minutes at 20C (68F), losing its concentration to 16% of its initial value in two hours, it must be freshly generated for immediate use at the treatment site. The maximum dose generated, 5% ozone to 95% oxygen, is well below the explosive limit (15 to 20%). Caution is needed not to appose ether and an ozone, an especially reactive mixture.
Listed contraindications to ozone treatment[5] include acute alcohol intoxication, recent myocardial infarction, hemorrhage from any organ, pregnancy, hyperthyroidism, thrombocytopenia and ozone allergy.
Methods of Administration, Dosage, and Clinical Applications External Ozone Gas Application
Historically, ozone was first administered by application to external body surfaces to determine its effects on a variety of lesions, A. Wolff,[13] in 1915, is credited for using local ozone treatments for wounds, fistulas, decubitus ulcers and osteomyelitis. Like natural rubber which cracks and fritters when exposed to oxygen-ozone mixtures, early materials caused ozone to "bag" around skin surfaces and met with early oxidation disuse. Today, specially designed plastics (Teflon) enable extremities or portions of the head or torso to be comfortably encased in a space where a determined dosage ratio of oxygen to ozone is administered at a chosen flow rate. In this way, the walls of the transparent bags do not touch the patient, an important consideration in burn treatment.
Indication for external ozone application include poorly healing wounds, burns,[14] staphylococcal infections, fungal and radiation lesions, herpes simplex and zoster, and gangrene (diabetic or Clostridium). Dosage is adjusted to the condition treated. Gas perfusions may last from 3 to 20 minutes, ozone concentrations varying from 10 to 80 ug/ml (maximum five parts of ozone to 95 parts of oxygen). High ozone concentrations are used for disinfection and cleaning (or debridement), while low concentrations promote epithelialization and healing.[6,15]
Ozone Insufflation
Payr in 1935[6] and Aubourg in 1936[16] first used ozone-oxygen mixtures in rectal insufflation to treat ulcerative colitis and fistulae. The list of indications has expanded to include proctitis and hemorrhoids. It is reported that in inflammatory diseases of the bowel, ozone promotes healing and restores the flora balance disturbed by pathogenic organisms. In a typical treatment for ulcerative colitis, daily insufflations are applied starting with 50 ml in severe cases, increasing as tolerated in increments (till 500 ml), high concentrations administered initially (75 ug/ml) to achieve hemostasis, followed by low concentrations to promote resolution.[5] This technique may have some promise in the treatment of bowel infections associated with AIDS.
Microsporidia, a tiny, rarely detected parasite may be responsible for many cases of AIDS wasting illness,[17] and studies await determination of its susceptibility to ozone treatment.
Major Autohemotherapy (AHT)
Whereas it can be readily understood that external ozone applications produce local effects such as disinfection, wound healing or local circulatory enhancement, the technique of introducing ozone into the circulation poses more complex theoretical issues. In the technique of major autohemotherapy, 50 to 100 ml of blood is drawn from the patient, mixed with a dose of ozone-oxygen of a predetermined concentration, then returned via the same intravenous catheter (butterfly). Returned to the patient, the ozonated blood is rapidly distributed to all tissues.
In the treatment aliquot of blood, it is gauged that the dose of ozone given not only will exert therapeutic actions locally (virucidal activity, oxygenation, increased red cell fluidity), but will determine beneficial systemic actions.[18]
The duration of time that ozone remains in solution and its effects on endocrine, neurological, and immunological systems are not known. Clinically, some patients, upon receiving their own ozonated blood, report a faint background taste of ozone, which may be an indication of its survivability in solution for at least a few seconds.
Major autohemotherapy has been applied to the treatment of several conditions, including acute and chronic viral infections (hepatitis), some carcinomas, circulatory disturbances (diabetes, arteriosclerosis), and hyperlipidemia.[8,19-21] Added to a standard pharmacotherapeutic regimen for postmenopausal osteoporosis, this technique enhanced remineralization of bone.[22] Clinical reports however, need to be substantiated by properly designed studies. Of interest are the reports of some patients, who after receivng this treatment experience feelings of well-being lasting for a few minutes to several hours. Whether this represents a placebo effect, a metabolic alteration or possibly a neuro-psychiatric mechanism remains to be determined.
Miscellaneous Applications
Although the above techniques of ozone administration represent the majority of hospital or office-based procedures, others deserve mention.
Minor Autohemotherapy
In this technique, 10 ml of venous blood is drawn from the patient, mixed with ozone-oxygen, then injected intramuscularly. Listed indications include asthma, acne, some allergic conditions and some carcinomas.[18,23,24]
Direct Intra-arterial or Intravenous Administration
Mostly of historical interest, this method was first used by Iacoste in 1951[25] for circulatory compromise and its possible sequelae (gangrene). Up to 10 ml of pure ozone-oxygen may be slowly injected directly into the artery (usually femoral), or into a vein, without incurring embolization since both gases are readily soluble in blood.[20] Indications include intermittent claudication, leg ulcers and cerebral vascular insufficiency. Due to accidents produced by too rapid introduction of the gas mixture into the circulation, this technique is now rarely used.
Intramuscular Injection
Up to 10 ml of pure ozone-oxygen mixture is injected into the gluteus maximus muscle or the deltoid. This treatment along with major autohemotherapy is invoked as an adjunct to cancer therapy.[15,18,26,27]
Ozonated Water
Ozone is approximately 10 times more soluble in water than oxygen. Mixed into aqua bidestillata (pyrogen free) water, the half life of ozone is nine to ten hours (at pH 7 and 20C); and at 0C, it is doubled. Ozonated water finds applications in dental surgery where it is reported to promote hemostasis, enhance local oxygen supply and inhibit bacterial proliferation. Applied following tooth extraction or during dental surgery,[28] it may also be rinsed in conditions such as thrush and periodontal disease, swallowed in cases of gastritis or gastric carcinoma, or irrigated in chronic intestinal or bladder inflammation.
Ozone Ointments
Ozonated olive oil provides long term, low dose exposure of ozone and lipid peroxides to tissues. Decubitus ulcers and mycoses are indications for its use.[29,30]
Balneotherapy
Ozonated water bubbled in warm baths, provides stimulation of local circulation and disinfectant action to varicosities, peripheral circulatory disorders and dermatological conditions (eczema, ulcers).[5]
Blood Purification
The possibility of using ozone to sterilize blood supplies has been investigated by several authors.[7,31] The treatment of 500 ml of whole blood with 100ml of O3/O2 mixture (40 to 50 ug/ml) is reported to render it virus-free without injuring any cellular elements. One study [31] examined 10,000 samples and found no cases of hepatitis transmission. This technique may extend its efficacy to the HIV virus as one preliminary unpublished study indicates although once ensconced in the genetic cellular material, it is unclear how any agent could inactivate it without compromising cellular integrity.
Metabolic and Physiological Effects of Ozone
Most research on ozone's biological effects have concentrated on pulmonary responses with emphasis on its toxicity. Interest has been keen on ozone's role in ground level atmospheric pollution. Produced as a result of interactions between industrial gases, oxygen and ultraviolet rays, there is evidence of synergistic action on pulmonary compromise. The effects of pure ozone, however, need to be differentiated from those of smog.
The majority of studies have been performed on animals who show great interspecies variability in their response to inhaled ozone. Extrapolation to humans is difficult due to differences in pulmonary anatomy and physiology. Mice[32] seem to be the most sensitive (LD50, 22 ppm for 3 hrs) and birds[33] the least (turkeys survived 417 ppm ozone for 3 hrs). While overdose is marked by pulmonary edema and hemorrhage, long term, low level exposure produces poorly understood, sometimes contradictory findings.
Reported effects[34] include enhanced enzyme activity, as evidenced by increase in glucose utilization, lactate and CO2 formation and elevated glucose-6-phosphate dehydrogenase; an increase in the NADPH-cytochrome P-450 content in rat lung pointing to enhancement of metabolizing enzymes; increased lung fibroblast glucose uptake, and production of lactate and pyruvate.
Humans exposed to ambient ozone (0.24 ppm in room air for two hours) typically develop mild accelerated breathing in the context of symptoms such as tracheal or laryngeal irritation and chest tightness on inspiration. Large intersubject response differences are notable.[35] Athletes[36] performing moderate intermittent exercise show a 7% drop in Forced Vital Capacity (FVC) and a 15% reduction in Forced Expiratory Volume (FEV). The threshold for significant changes in respiratory compromise ranges from 0.15 ppm[37] to 0.25 ppm,[38] increasing ozone concentrations yield corresponding airway hyper-responsiveness through bronchoconstriction. Histological findings extrapolated from primate research points to ciliated cell inhibition and type 2 cell proliferation, increased membrane permeability and variable inflammatory response.[12] Reported biochemical alterations[39] include increased oxygen consumption and glucose utilization; activation of NADPH, superoxide dismutase, GSH peroxidase, GSH reductase and glutathione peroxidase. Pulmonary effects from ozone in low doses appear to include metabolic activation of lung cells while higher doses produce evidence of cellular metabolic compromise.
In the methodology of ozone treatment, care is given to avoid the escape of ozone into the treatment area and modern machines are equipped to catalytically convert excess ozone to oxygen during administration. Interestingly some studies point to possible beneficial effects of low dose ambient ozone.[40,41] The phenomenon of ozone tolerance or adaptation the response to ozone exposure decreasing with time and finally evolving to a plateau occurs in both humans and animals.[38] Its significance remains obscure.
For the reason that below 0.30 ppm the probability of ozone traversing the respiratory epithelium and entering the systemic circulation is so low, very few studies have attempted to measure these effects.[39] In the technique of major autohemotherapy and others that involve the direct introduction of ozone into the circulation, however, this question is of special relevance. Studies of human blood in young adult males exposed to 0.50 ppm ozone for 2-3/4 hours[42] show significant changes in erythrocytes (RBC) as well as in the serum. RBC membrane fragility, glucose-6-phosphate dehydrogenase and lactate dehydrogenase enzyme activities were increased, while RBC acetyl cholinesterase and reduced glutathione reductase were not significantly changed. Serum vitamin E and lipid peroxidation levels were significantly increased. These findings indicate that ozone exposure increases metabolic activation parameters in red blood cells.
According to other researchers,[20,24,43] the direct intravascular injection of pure oxygen-ozone mixtures results in the following responses: (1) an activation of enzymes involved in peroxide or erythrocytes, an outgrowth of which is (2) stimulation of the [2,3] Bisphosphoglycerate cycle, shifting the oxyhemoglobin dissociation curve to the right thus releasing oxygen to the tissues. Further physiological effects include (3) an enhanced oxidative decarboxylation of pyruvate with the formation of Acetyl-CoA, and consequent citric acid cycle activation, (4) a direct influence on the mitochondrial transport system with reduction of NADH and oxidation of cytochromes, and (5) an increase in RBC pliability, blood fluidity, and arterial PO2.
Mechanisms of Bactericidal, Virucidal and Fungicidal Action
Although the inhibitory and lethal effects of ozone on pathogenic organisms have been observed since the latter part of the 19th century, the mechanisms for these actions have not yet been satisfactorily elucidated. Ozone is a strong germicide needing only a few micrograms per liter for measurable action. At a concentration of 1 g/m3 H2O at 1C, ozone rapidly inactivates coliform bacteria, staphylococcus aureus and Aeromonas hydrophilia.[44]
The inactivation rate of enteroviruses[45] is more rapid than for E. coli, takes place in relatively small concentrations of ozone, and is influenced by pH, temperature, and the presence of ambient organic compounds.
Viruses differ in their susceptibility to destruction by ozone. The resistance of polio virus type 2 was 40 times that of coxsackie AS,[46,47] and in an experiment using a continuous flow mixed reactor under controlled laboratory conditions, relative resistance in descending order was found to be: polio virus type 2, echovirus type 1, polio virus type 1, coxsackie virus type B5, echovirus type 5, coxsackie virus type A9. In pure water, at maximal solubility of ozone and room temperature, Echovirus type 29 is inactivated in one minute, polio virus type 1 in two, type 3 in three and type 2 in seven minutes.
The cell envelope of Gram negative microorganisms such as E. coli is a complex multilayer system composed of an inner cytoplasmic membrane made of phospholipids and proteins invaginating into the cytoplasm, a peptidoglycan layer, and an outer membrane of poly polymers such as polysaccharides. Gram positive cells have a less complex, three layer envelope with a thick peptidoglycan middle layer.
The most cited explanation for ozone's bactericidal effects centers on disruption of envelope integrity through peroxidation of phospholipids and lipoproteins. There is evidence for interaction with proteins as well.[48] In one study[49] exploring the effect of ozone on E. coli, evidence was found for ozone's penetration of the cell membrane, reacting with cytoplasmic substances and converting the closed circular plasmid DNA to open circular DNA, which would presumably lessen the efficiency of bacterial proliferation. It is notable that higher organisms have enzymatic mechanisms to restabilize disrupted DNA and RNA, which could provide a partial explanation for why, in clinical treatment with ozone at doses prescribed, ozone appears to be toxic to infecting organisms and not to the patient.[50]
Ozone possesses fungicidal effects, through poorly understood mechanisms. In one study, Candida utilis cell growth inhibition with ozone was greatly dependent on phases of their growth, budding cells exhibiting the most sensitivity to its presence.[51] Interestingly, in another study,[52] low doses of ozone stimulated the growth and development of Monilia fructagen and Phytophtora infestans, while higher doses were inhibitory.
Viruses are parasites at the genetic level, separated into families based on their structure, type of nucleic genome and mode of replication. Many virions contain a phospholipid envelope with glycoprotein spikes, encasing the nucleocapsid which contains nucleic acids (DNA or RNA), and structural proteins (including enzymes).
Lipid-containing viruses are sensitive to treatment with ether, assorted organic solvents, and ozone, indicating that disruption or loss of lipids results in impaired or destroyed infectivity. Viruses containing lipid envelopes include the Herpes viridae a large family grouping the Simplex, Varicella-Zoster, Cytomegalovirus and Epstein-Barr viruses; the Paramyxoviridae (mumps, measles); the Orthonyxoviridae (influenza); the Rhabdoviridae (rabies); and the Retroviridae (HIV). The HIV virus has an outer envelope made of a double layer of lipids penetrated by proteins of several types encasing two molecules of RNA.[53]
Many of the above viruses have complex, sometimes baffling life cycles and replicative strategies with progressions from host cell attachment of the virus particle, to penetration, uncoating of the viral envelope, synthesis of molecular components, and release of new generations of virions to the surrounding medium, most often through cell lysis. Many chronic viruses have eclipse phases alternating with phases of viremia, when waves of viral particles flood the bloodstream.
In view of the above considerations, what part can ozone play as an antiviral agent? In one study,[46] polio virus 1 was exposed to 0.21 mg/liter of ozone at pH 7.2. After 30 seconds 99% of the viruses were inactivated (lost their ability to replicate within host cells), but appeared to maintain their structural integrity. Analysis of viral components showed damage to polypeptide chains and envelope proteins, which could result in attachment capability compromise, and breakage of the single-stranded RNA into two parts, producing replicating dysfunction at its root level. Other researchers[54] in similar experiments concluded that in ozonation, it is the viral capsid which sustains damage. It is to be noted however, that the polioviridae (Picornavirus family) contain four structural proteins encapsulating a single RNA strand and are devoid of lipids.
In those clinical applications which make use of external (or body cavity) application of ozone, it can be appreciated that in view of the fact that a direct ozone-organism contact exists, inactivation of micro-organisms, bacteria, viruses or fungi, proceeds by any one of a variety of different mechanisms. The treatment of burns, superficial mycotic infection, decubitus ulcers and abscesses is applied by this method. Theoretical issues present themselves, however, when examining treatment strategies aimed at systemic infections, notably viral afflictions which make use of introducing ozone-oxygen mixtures into the bloodstream (usually major AHT). The ozone-treated aliquot of blood which is reported to be rendered viral-free through direct contact with ozone and ozone peroxides,[5] is reintroduced into the circulation. Since very little free ozone remains in solution due to its high reactivity, it is its products mainly lipid compounds, possibly others which are thought to interact with circulating as well as tissue-bound virions, thus inactivating them.
Within the dose ranges prescribed (up to 10 mg (O3/100 ml of blood), we may be curious to measure this overflow antiviral capacity. Although unproven to be outright curative for any viral illness, ozone blood treatment, as reported in several studies[21,31,55] may lessen clinical severity or duration. Thus therapeutic benefits have been noted in hepatitis, acute and chronic, and herpes.[55] In chronic viral infections Cytomegalic, Epstein-Barr and Retroviridae (AIDS) among others blood ozonation performed in viremic cycles or in periods of clinical exacerbation may, through direct action, through the production of cofactors inhibitory to viral replication, or through modification of immune function, be used in inducing viral quiescence. Ozone is reported to be an immuno-stimulant in low doses and immuno-inhibitory at higher levels.[15,26,27]
It is not inconceivable, in view of the possibilities given to ozone's antiviral properties that new generations of machines may be developed to test the therapeutic potential of the extra-corporeal treatment of circulating blood.
Ozone Treatment in Cancer
The logic sustaining the use of oxygen-ozone application to the treatment of carcinomas rests on the strategy of capitalizing on the disturbed metabolism of cancer cells. Since the first bio-chemical hypothesis of cancer was proposed by Warburg[56] in 1925; that all tumors have higher rates of glycolysis under aerobic conditions than do nontumor cells, efforts have been made to find the variations which could best affect treatment strategy. Although his statement has subsequently been amended considerably, there is a massive and evolving body of research centering on biochemical differences between normal and malignant cells.[57]
Some tumors have high rates of glucose use and lactic acid production in the presence of oxygen, a reflection of a number of possible mechanisms, from membrane transport differences to variations in ATP regulation. Cancer cell mitochondrial ribosomes have altered J structure and function which could diminish their oxidative energy producing abilities thus accounting for their limited aerobic potential.[57]
Some authors[5,26] report a peroxide intolerance in tumor cells. Possessing insufficient catalase and peroxidase, they are incapable of effective peroxide inactivation. Such cells exposed to ozone are said to show a significant decrease in lactate content, indicating that ozone may induce metabolic inhibition in some carcinomas.
In one study,[58] cultured cells of different carcinoma types were compared with non-cancerous human lung fibroblasts on exposure to ozonated air (0.3, 0.5, and 0.8 ppm of O3 for 8 days). Alveolar (lung) adenocarcinoma, breast adenocarcinoma, uterine carcinosarcoma and endometrial carcinoma showed 40% cell growth inhibition at 0.3 ppm and 60% at 0.5 ppm. The non-cancerous lung cells were unaffected at these levels. In 0.8 ppm exposure, cancer cell growth inhibition was 90%. Interestingly, it was at this level that the control cell group started to manifest anabolic slowdown (50%). The authors postulate that cancer cells are less able to compensate for the oxidative challenge of ozone than normal cells, possibly by way of a less functional glutathione system.
There are many clinical and anecdotal reports,[21,25,27,59] of ozone major or minor autotherapy, at times prescribed on a daily basis for several weeks applied to the treatment of various carcinomatous conditions but with a paucity of controlled data. Several researchers have focused their efforts on using ozone as an adjunct to radiation or chemotherapy.[23]
Summary and Future Directions
Ozone, an allotropic form of oxygen, possesses unique properties which are being defined and applied to biological systems as well as to clinical practice. As a molecule containing a large excess of energy, through incompletely understood mechanisms, it manifests bactericidal, virucidal and fungicidal action which may make it a treatment of choice in certain conditions and an adjunct to treatment in others. Although ozone's medicinal effects were discovered in the 19th century and clinically applied during World War I, equipment capable of purity and reliability of delivery of oxygen-ozone mixtures were not available until the late 1950s. Since then, experience has accumulated for the administration of ozone to humans and animals via a variety of routes, in doses that are both nontoxic and relevant to clinical problems, externally in gaseous form (or in solution) and systemically in blood ozonation.
A review of a large body of literature is presented which describes a spectrum of therapeutic indications. Of these, ozone application for superficial infection, burns, dental and intestinal conditions, and possibly circulatory problems seem to be the most promising. As regards blood ozonation, further research is indicated to delineate the nature of its dynamics and the extent of its effectiveness in (1) the identification of the galaxy of compounds formed in this process which, in view of doses administered, by all evidence, have metabolic, immunological, endocrine and possibly neurological effects; (2) the purification of blood or blood components for transfusion purposes; (3) the inhibition of carcinomas with reference to the types which may be the most susceptible and to its use as an adjunct to radiation or chemotherapy; and (4) the inactivation or the repression of viral diseases with special attention to chronic conditions of the Herpes or Retroviridae (HIV) families.References
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Wenzel D, Morgan D: Interactions of ozone and antineoplastic drugs on rat fibroblasts and Walker rat carcinoma cells. Res Commun Chem Patho Pharmacol 1983;40(2):279-288.16
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Tıbbın açan gülü,

Tamamlayıcı ve palyatif uygulamalar. Dünyada hızlı bir gelişme gösteren tamamlayıcı tıp uygulamaları ve palyatif uygulamalar Türkiye'de yeterli seviyeye bir türlü gelememiştir. Ancak son yıllarda tıpta ve iletişim araçlarındaki gelişmeler bu uygulamaların da yaygınlaşmasına ve tercih edilmeye başlanıyor.

Sağlık Ne Demek?
Günümüzde sağlığın tanımı yapılırken sanırım yanlış bazı şeyler yapıyoruz. Sağlık hastalıksızlık hali midir? yoksa hastalık varlığında dahi kaliteli yaşamak mıdır?

OZON YAYIN

Tumor Hypoxia and Ozone Therapy
Author: Kornel Kusznieruk
Affiliation: University of Western Ontario
Date Published: Tuesday October 24th, 2006 @ 23:30:29 EST

Abstract
This paper describes the importance of increasing oxygen content in tumors and provides some possible ways of doing this.
Introduction

Tumors are a rapidly expanding mass originating from one transformed cell. This rapid
expansion does not allow for proper vascularization to occur. At the size of 2-3 mm3 the
tumor would hypothetically stop growing because of its need for a blood supply to
provide oxygen and nutrients (1). Unfortunately, the tumor can coerce the host
endothelial cells to enter the growing mass and provide life support. This is
accomplished, in part, by release of chemoattractant compounds from the growing tumor
or from immune system cells which have entered the growing tumor. These compounds
activate neighboring endothelial cells to migrate to the tumor, to cut through host tissue so that they can arrive at the tumor, and to start proliferating and forming new blood vessels for the tumor. This process is called angiogenesis (2).

Although the host-derived endothelium allows the tumor to grow, its vascular structure is
much different from that of normal tissue. The tumor has no intratumor lymphatics, this
does not permit fluid draining from tumour tissue and as a result high interstitial fluid
pressure develops (3). The high interstitial pressure inhibits drugs from penetrating the
whole tumor tissue, as well as forcing death of some tumor cells. This alteration of
interstitial pressure combined with the rapid rate of tumor cell proliferation ends up
forming a situation where the growing tumor is vascularized, but only to the limited
extend that it needs for it’s own survival. Tumor blood vessels do not contain smooth
muscle lining, are resistant to control by the nervous system, and grow in a disorganized manner compared to vasculature in non-tumor tissue (3). An example of the difference between tumor and non-tumor vasculature is that the former relies on tumor secretion of vascular endothelial growth factor (VEGF) for its survival, whereas the former is insensitive to withdrawl of VEGF (4).

Tumors contain areas of hypoxia (4a). The cause of this is multifactorial and includes poor tumor perfusion by the blood (5), clotting of tumor blood vessels due to activated clotting factors on tumor endothelium (6), and the rapid rate of tumor growth. Hypoxia,and poor perfusion have been shown to negatively correlate with prognosis (7, 8).
Cancer cells under hypoxic environments secrete matrix metalloproteases, which allow
them to metastasize (9). In addition, hypoxia programs cancer cells and macrophages to
secrete VEGF, a protein that stimulates angiogenesis as well as immune suppression (10).
Hypoxia activates hypoxia inducible factor (HIF-1) a nuclear transcription factor which is important in promotion of angiogenesis (11). Besides local hypoxia, late stage cancer patients have lower systemic hemoglobin levels compared to healthy controls, this is due in part to lower renal production of erythropoietin (11a). Lower hemoglobin implies less oxygen transport and therefore reduced tumor oxygenation. Studies aimed at increasing hemoglobin levels by administration of erythropoietin have shown increased efficacy of radiotherapy and chemotherapy (11b).


An important consideration of tumor hypoxia is the role it plays in protection of tumorsfrom anti-tumor defense systems. For example, tumor necrosis factor alpha (TNF-α) is acytokine secreted by activated macrophages and T cells, which as the name implies has antitumor activity. Interestingly, the cytotoxicity of TNF-α to tumor cells is reduced under hypoxic conditions (12). Hypoxia also stimulates production of soluble TNF-α receptors which hypothetically may block systemic activities of this cytokine (13). This may explain the poor efficacy of systemic TNF-α therapy which was attempted in the

1980s (reviewed in 13a). If TNF-α can not kill cancer cells, then it is possible that

immune effectors which use intracellular signalling similar to TNF-α will not be able to work either. Such mediators include the Fas signalling pathway (13b). Since Fas ligand and TNF-α are used by T cells in killing of target cells, the poor efficacy of immunotherapy may be explained by hypoxia.

In accordance with the above point: lymphokine activated killer (LAK) cells are effective in killing certain types of tumor cells in vitro and in vivo but the ability to generate these cells is depressed under conditions of hypoxia. Furthermore, the ability of estabilished LAK cells to kill tumor targets is decreased under conditions of hypoxia similar to those found inside the tumor (14). Proliferation of lymphocytes in response to interleukin-2 is also inhibited during hypoxia (15).

In 1953 the impact of tumor oxygenation on efficacy of radiation therapy was described by Gray et al (16). Since then a great number of studies confirming that tumor sensitivity to radiotherapy positively correlates with tumor oxygenation (reviewed in 17). Several other interventions such as etoposide, doxorubicin, camptothecin and vincristine therapy are oxygen dependent (17a).

The Problem
Tumor hypoxia:
1. Stimulates angiogenesis
2. Suppresses immune function by:
a) Blocking TNF-α toxicity
b) Blocking systemic TNF-α activity by shed receptors
c) Blocking lymphocyte proliferation
d) Blocking LAK cell generation and activity
e) Induces production of the immune-suppressive cytokine VEGF
3. Stimulates resistance to radio- and chemotherapy.

Available Solutions

Hyperbaric Chambers

Methods of increasing tumor oxygenation have been described. One such method
involves exposing a patient to higher oxygen tension by use of a hyperbaric chamber.
This approach has shown marginal increases in oxygenation of some tumors although
clinical efficacy is a matter of debate. A randomized trial of squamous cell sarcoma
patients treated with radiotherapy in the presence of air or hyperbaric oxygen
demonstrated a significantly greater number of patients achieving clinical response in the hyperbaric oxygen group (18). Another randomized study assessing the ability of hyperbaric oxygen to increase efficacy of radiotherapy in cervical carcinoma patients demonstrated no beneficial effects (19). In addition to questionable clinical efficacy hyperbaric oxygen is a costly and sometimes dangerous procedure, which is not commonly used.

Carbogen

Inhalation of carbogen, a mixture of 95% oxygen and 5% carbon dioxide has also been
shown to increase tumor oxygenation both in animal models (20,21) and in the clinical
situation (22-24). An explanation for this effect is that carbon dioxide has vasodilatory functions in this setting which allows for better tumor perfusion of the high concentration of inhaled oxygen (25). Therapeutic benefits of carbogen therapy are mixed although some radiosensitizing effects have been observed alone (26-28), or in combination with nicotinamide in Phase I/II trials (29). Phase III trials are needed.

Proposed Solutions

Hypertonic Saline

Patients suffering from hemorragic shock often incur organ failure due to hypo-perfusion and lose of oxygen. Hypertonic solutions have originally been found useful in the treatment of traumatized patients with hemorrhagic shock. In 1980 Velasco et al demonstrated that an injection of 7.5% NaCl in hemorrhaged dogs restored arterial pressure and cardiac output while increasing survival compared to dogs receiving isotonic saline (30). In the same year, his group published human study in which 12 patients with hypovolaemic shock refractory to volume replacement, corticosteroid and dopamine infusions were administered a bolus of 100-400 ml of 7.5% NaCl. 11 of the 12 patients responded with rise in arterial pressure and the resumption of urine flow, the effects lasting for several hours (31). This treatment has been shown to increase oxygen content of organs in part by causing intratissue fluid to enter blood vessels, and increase blood vessel volume. These effects were postulated to be due to the temporary disruption of osmotic balance, as well, as erythrocytes losing their volume and being able to squeeze through areas which previously have not been perfused. Hypertonic saline therapy has not been associated with toxicity and has been used extensively in the clinical setting (33,34). The hemodynamic-stabilizing effects of saline have even been shown benefitial a hamster model of endotoxemia (35).

This stimulates the question of whether infusions of hypertonic saline may be used in
conjunction or alone for increasing tumor oxygenation. An added benefit to this
approach is immunostimulation! It has been demonstrated in mouse and human that
hypertonic saline therapy increases T cell proliferation and IL-2 production, while
suppressing production of PGE-2 and also suppresses IL-4 production (36-40). IL-4 and
PGE-2 have both been shown to suppress antitumor immune responses (41,42).
Another interesting approach to cancer therapy, which has not been attempted is the
combination of hypertonic saline infustion with systemic interleukin-2. One of the big
problems with clinical administration of IL-2 is induction of a shock-like state called the vascular-leak syndrome (43). Along these lines, a randomized trial was conducted examining crystalloid vs colloid resuscitation. No clear benefitial effect was demonstrated (44).

Although therapy with hypertonic saline seems absurdly simple, we must not forget that cisplatin therapy would have most likely been abandoned had Cvitkovic et al not found that rapid fluid administration can spare the host of this drug’s nephrotoxic effects
(45,46).

Ozone Therapy/Stressed Cell Therapy

Introduction

There are some areas in which ideas practiced in alternative medicine may have clinical
relevance, I believe the area of ozone therapy is one of them.

Ozone therapy is administered through several routes:
1. Directly intravenously,
2. Intrarectally,
3. Exposing patient blood to ozone ex-vivo followed by subsequent re-infusion
(Autohematherapy).

The first method was described by Lacoste in 1951 who used it to treat vascular
insuffiency and gangrene (47). Intravenous administration of ozone is possible since
both ozone and oxygen are very soluble in blood (48), however this technique is
considered out-dated and is hardly used today. Rectal administration has been used with
some efficacy for AIDS associated diarrhea (49), although, by far, the safest and most
widely method of ozone therapy is the third one mention: autohematherapy.

Vasocare therapy, invented by Anthony Bolten of Vasogen, is analogous to
autohematherapy, with the exception that UV irradation and/or heat is added to the
patient cells before reintroduction. In contrast to traditional ozone therapy, the effects of Vasocare have been extensively analysized scientifically with several patents and papers available in the public domain. I will clump together Vasocare and traditional autohematherapy for purposes of this discussion.

Clinical Effects

Reports of ozone therapy are largely anectodal due to it being primarily practiced in
alternative medicine. In this field it is a panacea, irresponsibly claimed by detractors to be a cure for everything from AIDS to diabetes (50,51). Reports such has these have
severely damaged the credibility of ozone therapy, even placing it on the National Cancer
Institute’s list of questionable treatments for cancer (52).

With that said, I will examine the credible literature on this topic. Anacedotally reported, treatment of peripheral limb ischemia has been successful with autohematherapy (53,54).

Recently, Tylicki et al treated 12 patients with lower limb atherosclerotic ischemia. Of these, 11 had prolonged ability to walk on a threadmill post-treatment (55). In another pilot study symptoms of atherosclerotic vasculopathy were improved (56). Utility of Vasocare treatment was demonstrated in peripheral vascular disease in a two centered double-blind study by Baird and Belch in the United Kingdom where the treatment group reported a greater than 50% increase in walking distance compared to controls (57). Vasogen has currently received approval for a Phase III pivotal trial in peripheral arterial disease (58).

Vasogen has also demonstrated efficacy of Vasocare therapy in autoimmune diseases
both in animal models and in humans, unfortunately, these studies have not been
published in the peer reviewed literature but only as patents (59-61). More relevant to our discussion, Vasogen noticed some anti-leukemia effects in its trial on graft versus host disease, this is the basis for an ongoing trial of Vasocare in chronic lymphocytic leukemia (62).

Mechanisms

It is postulated that these effects are mediated in part by the ability of the treatment to increase tissue oxygenation, while at the same time having an anti-inflammatory effect(reviewed in 63). Erythrocytes in patients receiving autohematherapy are postulated to be better oxygen carriers than controls due to an increase in 2,3 diphosphoglycerate (64). A randomized trial comparing autohematherapy to hyperbaric oxygen demonstrated that only autohematherapy decreases blood viscosity while increasing erythrocyte filterability
(65). Both of these changes would hypothetically increase tumor oxygenation. Another
mechanism of ozone therapy may be induction of nitric oxide (NO). NO possesses many
effects, but one of the well known ones is vasodilation (66). In Vasogen mentions in two of their patents, that Vasocare blocks platelet aggregation and induces NO (67,68).
Once again, these effects could hypothetically increase tumor oxygenation.
Ozone therapy may also have direct effects on cancer cells. In the 1920s Warburg
postulated cancer cells preferentially utilize anaerobic metabolism (69). Others after him have claimed that high concentrations of oxygen can specifically kill tumor cells, although this was for the most part proven wrong. Interestingly, ozone can preferentially inhibit various primary human tumor cells while sparing normal tissue, in vitro (70).
Similar results were obtained in leukemic cell lines (71).

Conclusion

Ozone therapy has been used for millions of treatments without toxicity (cited in 72).
Under a different form (Vasocare) ozone therapy is now entering Phase III trials. The
combination of ozone therapy with radio, chemo and immune therapies should increase
efficacy of these treatments by overcoming tumor hypoxia. Future studies in this area are
definitely warranted.
________________________________________
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Ozon Tedavisi Genel Bilgi

3 Major Effects of Ozone on the Body by Dr. Frank Shallenberger

1. Ozon beyaz kan hücreleri üretimini stimüle eder. Bu hücreler vücudu virüs, bakteri, mantar ve kansere karşı koruyan hücrelerdir. Bu hücrelerin oksijenden yoksun kalması sonucunda fonksiyonlarını tam olarak yerine getiremezler. Eğer bu mekanizma düzgün olarak çalışmaz ve tehditleri ortadan kaldıramazsa normal hücrelere karşı da reaksiyon gösterebilir ve sonuç olarak alerjik reaksiyonlar ortaya çıkar. Ozon oksijen seviyelerini normal sınırlara çekerek desentizasyonu ortadan kaldırır.

2. İnterferon bağışıklık sistemini bir orkestra gibi yöneten çok önemli bir maddedir. Bazı interferonlar virüsle enfekte olmuş hücreler tarafından üretilirler. Bu interferonlar komşu hücreleri uyarır infekte hücrelere karşı kensini savunur ve virüs replikasyonunu engeller (çoğalmasını). Diğer interferonlar, kas, bağ dokusu ve beyaz kan hücreleri tarafından salgılanır. İnterferon gamma düzeyleri ozon tedavisi ile %400-900 arasında artırılır. Bu interferon fagositik hücrelerin patojenler ve anormal hücreleri öldürmesi ve fagosite etmesindeki karışıklıkları giderir. İnterferonlar kronik hepatit B ve C, genital siğil (papilloma virüs sebepli), Hairy cell lösemi, kaposi sarkomu, relapsing-remitting multiple sclerosis ve kronik granülomatoz hastalıklarda kullanımı FDA tarafından onaylanmıştır. İnterferon güncel çalışmalarında ağız içi siğilleri (papilloma virüs kaynaklı), HIV enfeksiyonu, kronik myeloid lösemi, non-hodkin lenfoma, kolon tümörleri, böbrek tümörleri, mesane kanseri, malign melanom, bazal cell karsinoma ve leishmaniazis tedavisinde interferon kullanımını araştırmaktadır. İnterferonun dışarıdan verilmesi toksik etkiler yaparken ozon tedavisi ile vücutta güvenli bir şekilde yükseltilebilir.

     3. Tümör büyümesi esnasında vücut tümör necrosis faktörü üretir. Ozon tedavisi de TNF’nin üretimini artırır. Tümör ne kadar büyürse o kadar TNF üretilir. Tümör ne zaman metastatik hale gelirse, kanser hücreleri lenf ve kan damarları vasıtasıyla taşınırlar. Vücudun başka yerlerine ulaşır ve yerleşerek orada çoğalmaya başlar ve TNF üretilmesine sebep olur bu madde de orijinal tümörü baskılar. Ne zaman ki tümör cerrahi olarak çıkarılırsa TNF seviyesi dramatik olarak düşer ve sağlıklı dokuda yeni tömör görülür.

      4. Ozon IL-2 salgılanmasına neden olur. IL-2 bağışıklık sistemin temel taşıdır. T-Helper hücreler tarafından salınır. Bu süreç otostimülasyon olarak bilinir. IL-2 T-helperlar üzerindeki reseptörleri bir araya toplar ve bunların daha fazla IL-2 salınımı sağlamasına neden olur. Bu lenfositlerin değişimine ve hızla çoğalmasına neden olur, T-helpers, T-supressors, sitotoksik T’s, T-delayed ve T-memory hücrelerinin artışı meydana gelir.