C VİTAMİNİNİN T HÜCRELER ÜZERİNE İMMÜNOMODÜLATÖR ETKİSİ

Immunology Letters 98 (2005) 63–72
Mega-dose Vitamin C modulates T cell functions in Balb/c mice only
when administered during T cell activation
Kahwa Noha,b, Hyunja Lima,b, Sung-kyu Moona,b, Jae Seung Kanga,b, Wang Jae Leea,b,
Dongsup Leeb, Young-il Hwanga,b,∗
a Tumor Immunity Medical Research Center, Seoul National University College of Medicine, Seoul 110-799, South Korea
b Department of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu,
Seoul 110-799, South Korea
Received 5 August 2004; received in revised form 20 October 2004; accepted 21 October 2004
Abstract
Previously we reported that a mega-dose of Vitamin C enhanced the initial stage of delayed-type hypersensitivity reaction in Balb/c mice.
In this study its effects were further evaluated as follows. Mice were administered Vitamin C intraperitoneally at 0.625 mg/day or at 5 mg/day
for variable days before, during, or after being sensitized with DNFB. T cells were isolated in each group and examined. When stimulated
antigen-specifically or non-specifically in vitro, mice showed elevated thymidine uptake and a shift of cytokine secretion profiles toward Th1,
i.e., elevated levels IL-2, TNF-
, and IFN-, and lowered level of the Th2 cytokine IL-4, only when Vitamin C was administered during
sensitization. T cells from those mice administered Vitamin C before sensitization or after challenge showed the same T cell properties as those
from PBS-treated mice. Mice were also given 0.625 mg/day of Vitamin C during primary and/or secondary immunizations with KLH and
secondary specific antibody titers in sera were measured. The total specific antibody titer was lowered in Vitamin C-treated animals whenever
treatments were administered, and this was entirely attributed to decreased levels of IgG1 and IgE antibodies. Based on these results, we
suggest that an exogenously administered mega-dose of Vitamin C shifts immunity in Balb/c mouse toward Th1 and that these affects occur
only when Vitamin C is administered during T cell activation.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Mega-dose; Vitamin C; Th1 polarization; Delayed type hypersensitivity; T cell activation; Isotype
1. Introduction
Vitamin C acts as an electron donor for many kinds of human
enzymes, facilitates iron transport, and is regarded as one
of the most important physiological antioxidants [1,2].In addition,
VitaminCexerts several diverse effects on the immune
system. It increases neutrophil motility [3,4] and phagocytic
function [5] in human. Macrophage functions in mice such as
chemotaxis, phagocytosis, and superoxide anion production
are enhanced by several antioxidants including Vitamin C
[6]. Increased proliferation of T cells [7,8], and inhibition of
∗ Corresponding author. Tel.: +82 2 740 8209; fax: +82 2 745 9528.
E-mail address: hyi830@snu.ac.kr (Y.-i. Hwang).
various forms of T cell death [9] and Fas-induced apoptosis
of monocytes [10] by Vitamin C have also been reported. The
increased cytotoxic activity of natural killer cells in humans
is another example of an effect ofVitamin C supplementation
[11,12].This biologically important micronutrient should be
exogenously administered in diets or as tablets to those, including
human, that lack terminal enzyme l-gulonolactone
oxidase in the Vitamin C synthetic pathway [13], and thus
cannot make Vitamin C themselves.
The recent recommended daily allowance (RDA) for
ascorbic acid by National Academy of Sciences is 75 mg and
90 mg per day for women and men, respectively. Nevertheless,
several authors have claimed that this dose is inadequate
and have suggested that the RDA be increased. For example,
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.imlet.2004.10.012
64 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Levine et al. [14] suggested 200 mg/day to maintain optimal
blood concentrations. Considering that low concentrations of
serum ascorbate are related to increased mortality [15,16], the
recent RDA could be below that actually needed to maintain
health.
Meanwhile, despite many controversies about its beneficial
effects, the usage of even higher doses of Vitamin C at
the level of grams per day has been suggested for both preventative
and therapeutic purposes in a number of diseases,
including cancer [17], atherosclerosis [18], viral infections
[19], H. pylori infection [20], and acute pancreatitis [21]. In
addition, supplement users regularly consume more than 1 g
of Vitamin C per day [22]. However, the effects and mechanisms
of such ‘mega-doses’ of Vitamin C have not been well
defined.
Previously we reported that exogenously given megadose
of Vitamin C augment the initial stage of delayedtype
hypersensitivity (DTH) response against 2,4-dinitro-1-
fluorobenzene (DNFB) in Balb/c mice [23]. In this study, we
further analyzed the ways in which mega-dose Vitamin C
modifies the functions of T cells in mice. When mega-dose
Vitamin C was administered during sensitization, activated
and memory T cells were rendered more vulnerable to both
ex vivo antigen-specific and non-specific stimuli and their
cytokine secretion profiles were shifted to Th1. Furthermore,
vitamin C also affected humoral immune response against
keyhole limpet hemagglutinin (KLH), by critically lowering
specific serum IgG1 and IgE levels.
2. Materials and methods
2.1. Mice
Seven-week-old male Balb/c mice were purchased from
BioLink (Seoul, Korea) and housed in animal facilities at
Seoul National University, College of Medicine (Seoul, Korea).
They were kept under a 12 h light–dark cycle and all
the animals were given mice chow and tap water ad libitum
through the experimental period.
Experimental groups were administered Vitamin C
(Sigma, St. Louis, MO) intraperritoneally at a daily dose
of 0.625 mg or 5mg freshly dissolved in 200l of PBS.
The durations of the injections varied in each experimental
group. An equal volume of PBS was given to control
animals.
2.2. Induction of delayed type hypersensitivity (DTH)
reaction
To induce DTH response, animals were shaved on the
dorsum (approximately 1 cm×1 cm). Sensitizationwas done
for two consecutive days by applying 25 l of 0.5% DNFB
solution gradually onto the shaved skin. DNFB (Sigma, St.
Louis, MO) solutionwas freshly prepared in acetone:olive oil
(4:1) just before use. On variable days after the last sensitization,
DTH was induced by the topical application of 20l
of 0.2% DNFB solution on the dorsal surface of the right
pinna [24]. The interval between sensitization and challenge
varied depending on the experiments. The left pinna was left
untreated or treated with vehicle only. The thickness of both
pinnae were measured with a constant-loading micrometer
(Mitutoyo, Japan) once just before sensitization (designated
as basal thickness) and for 7–10 days after challenge at the
same time of the day. The magnitude of DTH response was
expressed as the percentage of pinna swelling compared to
its basal thickness.
2.3. T lymphocyte proliferation assay
To assess the effect of mega-dose Vitamin C on na¨ıve,
act¨ıvated, and memory T cells, mice were administered
0.625 mg of Vitamin C for various days before, during, or
after sensitization with DNFB. Splenocytes were isolated
using Ficoll-Hypaque (Amersham Pharmacia Biotech, Sweden)
density gradient and incubated at 37 ◦C for 2 h to remove
adherent monocytes and macrophages, and then in
a dish coated with 10 ml of 2 g/ml anti-NK1.1 antibody
(Pharmingen, San Diego, CA) for another 2 h to eliminate
NK cells. T cells were further purified by negative selection
using Dynabeads® Mouse pan B (Dynal Biotech, Oslo, Norway)
by following the manufacturer’s instructions. Briefly,
4×107 Dynabeads were added to 107 cells in a 1.5 ml microcentrifuge
tube, mixed well, and incubated at 4 ◦C for 40 min.
The tubes were then placed in a Dynal MPC (magnetic particle
concentrator) for 2 min. Un-bound cells were collected
by washing with PBS containing 0.1% fetal bovine serum.
The purity of T cells was assessed by FACS analysis using
PE-conjugated hamster anti-CD3 antibody (Pharmingen, San
Diego, CA).
Non-specific T cell activation was determined using hamster
anti-CD3 antibody (Pharmingen, San Diego, CA) or
PMA/ionomycin (Calbiochem, San Diego, CA). Ninety sixwell
plates were coated with 100l of 1g/ml anti-CD3
antibody in each well at 4 ◦C for 24 h. T cells were plated
in coated wells (at 2×105/well), or were cultured in the
presence of 100 ng/ml PMA and 500 ng/ml ionomycin. Incubation
was carried out for 24 h in Dulbeco’s minimum
essential medium (DMEM) supplemented with 10% fetal
bovine serum (Gibco BRL, Carlsbad, CA), 100 U/ml
of penicillin and 100g/ml of streptomycin (Gibco BRL,
Carlsbad, CA) at 37 ◦C in an atmosphere supplemented
with 5% CO2. DNFB-specific T cells were also stimulated.
T cells, 2×105/well, were co-cultured in 96-well
plates with 5×105 mitomycin-treated syngeneic spleen
cells pulsed with 2,4-dinitrobenzenesulfonic acid (DNBS;
Aldrich, Milwaukee, WI), a water-soluble DNFB analogue
[25]. After 24 h, cells were pulsed with 1Ci/well of [3H]
thymidine (Amersham Pharmacia Biotech, Oslo, Norway)
for 18 h, and then harvested using a cell harvester (Skatron
Instrument, Lier, Norway) on glass-fiber filters. Radioactivity
was counted in a scintillation counter (WalK.
Noh et al. / Immunology Letters 98 (2005) 63–72 65
lac, Fort Wayne, IN) and all samples were prepared in
triplicate.
2.4. Cytokine detection
To determine the effect of a mega-dose Vitamin C on
cytokine secretion profiles, T cells were purified as described
above. T cells, 3×106/well, were plated in six-well plates
and stimulated with anti-CD3 antibody, PMA/ionomycin,
or DNBS, as described above. After 24 h incubation,
supernatants were collected. Cytokines in the cell-free
supernatants were detected using commercial quantitative
sandwich immunoassay kits for IL-2, IFN-, TNF-
(R&D
Systems, Minneapolis, MN), and IL-4 (Biosource, Nivelles,
Belgium) by following the manufacturer’s manual.
2.5. Immunization with keyhole limpet hemocyanin
(KLH) and specific antibody titration
To assess the effects of Vitamin C on humoral immune
response, we immunized mice twice, on days 1 and 21, with
an intraperitoneal injection of 100g KLH (Calbiochem, La
Jolla, CA) in 200l PBS. Control mice were injected with
PBS only. Blood samples were drawn from the orbital plexus
on days 11 and 31, sera were obtained and stored at 4 ◦C until
used.
To titrate KLH-specific antibodies in sera, 96 well-ELISA
plates (Nunc, Rochester, NY) were coated with 100l of
4g/ml KLH/well for 2 days at 4 ◦C. Plates were then briefly
washed and blocked with 1% skim milk in PBS for 1 h at
room temperature. Serum samples were diluted 1:100 in 1%
skim milk/PBS containing 0.05% Tween 20, applied to the
first row of ELISA plate, and were four-fold serially diluted.
All samples were prepared in duplicate. After incubation
for 2 h at room temperature, plates were washed with
a PBS-0.05% Tween 20 mixture, and alkaline phosphataseconjugated
secondary antibodies for each isotype were added
and incubated for 60 min at room temperature. Plates were
washed three times, and p-nitrophenyl phosphate substrate
(Amresco, Solon, OH) solution was added. OD values were
measured at 405 nm. The secondary antibodies used were
as follows; goat anti-mouse polyvalent immunoglobulins
(1:1000 diluted, Sigma), goat anti-mouse IgM, IgG1, IgG2a,
IgG2b, IgG3, and IgE antibodies (1:1000 diluted, Southern
Biotech, Birmingham, AL). KLH-specific anti-sera obtained
from other experiments were collected in a tube and used as
a standard serum in every ELISA plate. Titers are expressed
as relative to standard serum values.
2.6. Statistical analysis
Statistical analysis was performed by one-way ANOVA
with Student–Newman–Keuls post-hoc analysis using
PRISM software (GraphPad, San Diego, CA). Significance
was set at p < 0.05.
3. Results
3.1. Vitamin C pre-treatment before challenge enhances
the initial stage of DTH response, but suppresses overall
inflammation accompanying DTH response
Mice were treated intraperitoneally with a daily dose of
0.625 mg or 5mg of Vitamin C, or 200 l of PBS for 26 days,
during which sensitization (on days 4 and 5) and challenge
(on day 16) with DNFB were done. Each group consisted
of eight mice. After inducing DTH response, pinna thickness
was measured and the % increment calculated versus
basal thickness (Fig. 1A). Because, in pilot tests, ears treated
with vehicle only (acetone:olive oil) showed just slight and
transient thickness increases (data not shown), we left con-
Fig. 1. The effect of Vitamin C injected during or after the induction of
skin DTH response against DNFB. Mice were intraperitoneally injected
with daily doses of 0.625 or 5mg of Vitamin C for whole 26 days of experiment
(A) or only after challenge, (B) respectively. Sensitization was
done with DNFB on days 11 and 12. Control groups were injected with
200l of PBS. The thickness of pinna was measured after challenge as
indicated, and the percentage thickness increment vs. basal thickness was
calculated. When Vitamin C was administered all through the experimental
days (A), the pinnae of experimental groups on the first day after challenge
were dose-dependently thicker than those of the control groups (p < 0.001).
Subsequently, from the second day, this situation reversed. When Vitamin C
was administered only after challenge (B), the profile was nearly the same
as that shown by Panel A, except on the first day, when the values do not
reveal statistically significant differences. Data represent mean ear swelling
(±S.D.) of eight mice per group.
66 K. Noh et al. / Immunology Letters 98 (2005) 63–72
trol pinnae untreated (even by vehicle). Pinna thickness in all
groups peaked 2 days after challenge (32.8, 24.4, and 18.6%
increment in control, 0.625 mg-, and 5 mg-treated groups,
respectively) and then gradually decreased; control group
values were highest and those of the 5 mg-treated group
lowest. However, the values were reverse 1 day after challenge,
the initial stage of DTH response. The control group
showed the lowest increment (12.7%) and the 5 mg-treated
group showed the highest (17.9%). These differences between
the three groups were statistically significant. This pattern
was exactly the same to as that reported previously [23].
Thus, Vitamin C treatment seemed to accentuate the initial
stage of DTH response on the one hand, and to exert antiinflammatory
effects on an initiated DTH response on the
other.
Considering that intraperitoneally injected exogenous vitamin
C accumulates in several tissues of the body [23], the
observed initial accentuation of DTH response by Vitamin
C treatment could be the result of accumulated tissue Vitamin
C, or the result of any alterations of immune components
before challenge. To clarify this issue, we induced DTH response
in another set of experimental groups, which were
injected with a daily dose of 0.625 mg or 5mg of Vitamin C,
or 200l of PBS for 10 days from the day of challenge, and
then measured the pinna thickness (Fig. 1B). The overall profile
was similar to the results of the groups in Fig. 1A, except
that the increment on the first day after challenge was lowest
in the 5 mg-treated group (12.4 compared to 14.4% both in
the control and in the 0.625 mg-treated groups) even though
thiswas not statistically significant. These results suggest that
pre-treating with Vitamin C before and during sensitization
affects those immune components that induce an increase
in the early DTH response. Of course, the inflammatory response
itself seemed to be suppressed by the presence of
exogenous Vitamin C alone when the inflammation was proceeding.
3.2. Vitamin C treatment only during sensitization
enhanced the initial stage of DTH
We further divided the experimental group in Fig. 1A into
“before sensitization” and “during sensitization” subgroups.
In this case, challenge was done at 38 days, instead of at 6
days after sensitization to allow the activated immune cells to
become quiescent. Thus, four groups were given a daily dose
of 0.625 mg of Vitamin C, (1) during all experimental days
(60 days; () in Fig. 2), (2) for 7 days before sensitization
((
) in Fig. 2), (3) for 10 days during sensitization (() in
Fig. 2), or (4) for 10 days after challenge ((♦) in Fig. 2). The
control group received PBS on all experimental days (() in
Fig. 2). As is shown in Fig. 2, two groups injected during the
sensitization period showed a greater pinna thickness increment
on the first day after challenge than the other groups,
including the control group (p < 0.01). Injection of Vitamin C
before sensitization or after challenge failed to augment the
initial DTH response.
Fig. 2. The effect of Vitamin C treatment before sensitization, during sensitization,
and after challenge on DTH response was further analyzed. Mice
were intraperitoneally injected with 0.625 mg of Vitamin C for all days of
the experiment (), for 7 days before sensitization (
), for 10 days during
sensitization (), for 10 days after challenge (♦), or injected with 200 l
PBS during the experiment (). DTH response was induced with DNFB 38
days after sensitization to allow the T cells activated during sensitization
to become quiescent. Only the groups administered with Vitamin C during
sensitization (and  on the graph) showed an initial accentuation of DTH
response as shown in Fig. 1A. The data represent mean ear swelling (±S.D.)
of eight mice per group.
3.3. Vitamin C enhanced T cell proliferation
The next task was to identify which of the immune components
was most probably affected by Vitamin C during
sensitization, and responsible for the accentuation of the initial
stage of DTH response. We thought that T cells were
the most probable candidate, because T cells, especially Th1
cells, are the prime responders during sensitization and the
initiation of DTH response by antigen challenge [26]. Therefore,
we evaluated the functional differences of T cells from
mice treated and not treated with Vitamin C.
Mice were administrated 0.625 mg of Vitamin C or 200 l
of PBS (A) for 10 days without sensitization (na¨ıve T cells;
equivalent to (
) in Fig. 2), or (B) for 16 days with sensitization
at days 11 and 12 (activated T cells; equivalent to ()
in Fig. 2), or (C) for 54 days with sensitization on days 10
and 11 (memory T cells; equivalent to () in Fig. 2). At the
end of the Vitamin C administration, mice were sacrificed
and T cells were obtained as described in Section 2. Purity of
isolated T cells was over 90% as assessed by FACS analysis
(data not shown).
Na¨ıve T cells (Fig. 3A) proliferated in the presence of
anti-CD3 antibody and PMA/I, but were not proliferated by
DNBS and without stimulation (“control” in the graph). Furthermore,
the magnitude of thymidine incorporation was the
same in both the Vitamin C-treated and PBS-treated groups.
These results imply the absence of both active T cells and
DNFB-specific memory T cells. After sensitization (Fig. 3B),
T cells proliferated in the absence of any kind of proliferative
stimulus (‘control’ in the graph) implying T cell activation in
K. Noh et al. / Immunology Letters 98 (2005) 63–72 67
Fig. 3. The effect of exogenously administered mega-dose Vitamin C on
T cell proliferative activities. Mice were treated with 0.625 mg of Vitamin
C for 10 days without sensitization (A), for 16 days with sensitization on
days 11 and 12 (B), or for 54 days with sensitization on days 11 and 12 (C).
Control groups were injected with 200l of PBS. T cells were isolated and
cultured for 24 h with a DNFB-specific or non-specific stimuli (anti-CD3
antibody or PMA/I). Tritiated thymidine was pulsed for an additional 18 h
and radioactivities were measured. The filled bar () and the unfilled bar (
)
represent the Vitamin C-treated and PBS-treated groups, respectively. The
proliferation of T cells from Vitamin C-treated mice exceeded that of the
control groups only when the mice were sensitized. Values are the average
cpm±S.D. of triplicates.
vivo by sensitization. When stimulated with anti-CD3 antibody
or PMA/I, thymidine uptake was elevated more in the
Vitamin C-treated group than in the PBS-treated group. To
be noticed in these groups is that DNBS also enhanced T cell
proliferation in contrast to the na¨ıve T cell groups, implying
the presence of DNFB-specific activated T cells. T cells
from mice 42 days after sensitization (Fig. 3C) revealed similar
results to those in Fig. 3B. However, the control group
showed minimal uptake indicating the absence of activated T
cells. Meanwhile, proliferation was induced by DNBS both
in Vitamin C-treated and PBS-treated groups, which implies
the presence of DNFB-specific memory T cells. However,
the magnitude of thymidine uptake in the Vitamin C-treated
group was double that of the PBS-treated group.
Besides the status of the T cells isolated, another possible
factor that could elicit differences among groups in Fig. 3
is the duration of Vitamin C treatment (10, 16, and 54 days,
respectively). Therefore, we repeated the T cell proliferation
assay with some modification and examined the effect
of the duration of Vitamin C treatment. Mice were treated
with a daily dose of 0.625 mg Vitamin C (A) for 43 days
with sensitization on days 4 and 5, or (B) for 43 days without
sensitization, or (C) treated with Vitamin C only for 10 days
during sensitization 38 days before T cell isolation. Thymidine
uptake assays were done in the same way as in Fig. 3;
the results are shown in Fig. 4. Results for the 43 day-treated
and sensitized groups (Fig. 4A) were the same as those of
the groups in Fig. 3C, which were treated for 54 days and
sensitized. Despite long-term treatment of Vitamin (for 43
days), if sensitization was not carried out during Vitamin
C treatment (Fig. 4B, similar to Fig. 3A), T cells showed
unaltered thymidine uptake compared to the corresponding
PBS-treated group. Meanwhile, those treated with Vitamin
C only during sensitization (Fig. 4C) elicited similar results
to the sensitized 43-day Vitamin C-treated group (Fig. 4A),
suggesting that sensitization in the presence of exogenously
administered mega-dose Vitamin C results in an increase
in T cell response to both antigen-specific and non-specific
stimuli.
3.4. T cells treated with Vitamin C shifted their cytokine
secretion profiles from Th2 to Th1
The enhanced initial stage of DTH response could be regarded
as an outcome of the shift of general immune response
from Th2 to Th1 [27]. Thus, changes in the cytokine secretion
profiles of T cells could be expected. Mice were treated with
Vitamin C or PBS and T cells were isolated as in Fig. 3. After
the ex vivo stimulation of T cells for 24 h, supernatants were
collected and titers of Th1 cytokines including IL-2, TNF-

, and IFN- and the Th2 cytokine IL-4 were measured by
ELISA.
Without sensitization (left column in Fig. 5), Vitamin C
treatment did not alter the cytokine secretion profiles of T
cells versus those of PBS-treated mice, at least with respect
to the four cytokines tested. T cells not-stimulated or stimulated
with DNBS secreted no detectable cytokines. In contrast,
T cells from sensitized mice, regardless of their being
activated (middle column in Fig. 5) or memory (right column
in Fig. 5) cells, showed elevated IL-2, TNF-
, and IFN- secretions
in Vitamin C-treated mice, whereas the secretion of
IL-4 decreased.
68 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Fig. 4. The effect of the duration of Vitamin C treatment on T cell proliferative
activities. Mice were injected with 0.625 mg of Vitamin C for 43 days
with sensitization on days 4 and 5 (A), for 43 days without sensitization (B),
or for the first 10 days of the 43 days with sensitization on days 4 and 5 (C).
Control groups were injected with 200l of PBS. T cells were isolated and
cultured for 24 h with DNFB specific or non-specific stimuli (anti-CD3 antibody
or PMA/I). Tritiated thymidine was pulsed for an additional 18 h, and
radioactivities were measured. Filled bar () and unfilled bar (
) represent
Vitamin C-treated and PBS-treated groups, respectively. Even though mice
were treated with Vitamin C for 43 days, T cells show no difference in their
proliferative activity vs. those of PBS-treated mice if they were not sensitized
during Vitamin C treatment (B). In contrast, only a 10-day Vitamin C
treatment affected T cell proliferation when the treatment was done during
sensitization (C). Values are the average cpm±S.D. of triplicates.
3.5. Vitamin C decreased anti-KLH IgG1 and IgE
antibody responses in vivo
Because cytokine profiles were shifted toward Th1, it
could be predicted that humoral response would be attenuated
[27]. To verify this, we injected mice with Vitamin C
or PBS, immunized them withKLHtwice, and then measured
the serum KLH specific antibody titers by ELISA. A daily
dose of 0.625 mg Vitamin C was intraperitoneally administered;
(1) on all experimental days, or (2) during the primary
immunization for 7 days, or (3) during the secondary immunization
for 7 days. The control group was treated with PBS
on all experimental days.
The results shown in Fig. 6 are for secondary immune
sera. Whole specific titer (IgM, IgG, and IgA) was reduced
in Vitamin C-treated mice regardless of treatment time, and
this seemed to be mainly due to reduced IgG1 and IgE titer,
which are typical Th2-driven isotypes in mice. Other isotypes
were unaffected in terms of KLH specific titer.
4. Discussion
In this study, we examined the effects of Vitamin C on
immune response in Balb/c mice. Previous results that an
exogenously administered mega-dose of Vitamin C accentuated
the initial stage of DTH response [23] were reproduced
in the present experiment. When sensitization was carried
out with the administration of exogenous mega-dose Vitamin
C, the resulting in vivo activated and memory T cells
showed increased proliferative activities in response to ex
vivo antigen-specific and non-specific stimuli, and shifted
cytokine secretion profiles from Th2 to Th1. When immunizationwas
done in the presence of an exogenous mega-dose
of Vitamin C, the titer of whole antigen-specific antibodies in
secondary sera was reduced, and this reduction was mainly
attributed to decreased titers of the Th2-driven isotypes, IgG1
and IgE.
DTH response to skin sensitizer molecules such as DNFB
was used as an experimental model to evaluate cell-mediated
immune function [24]. When memory T cells against an antigen
are exposed to the same antigen again (challenge), they
are reactivated (initial stage of DTH response) to secrete cytokines
like IL-2 and IFN-, which in turn activate tissue
macrophages (cognition and activation). These events occur
within 24 h [28]. Activated macrophages again secrete inflammatory
cytokines and reactive oxygen radicals to propagate
and maintain inflammation [29].
UV irradiation [30,31] and burn stress [32] suppress DTH
response in skin by producing reactive oxygen species (ROS).
Topically or orally administered Vitamin C protects against
this suppression, probably by acting as an antioxidant. In this
experiment, we examined the effect of Vitamin C on DTH
response in normal mice, not from the aspect of protection
against DTH suppression. When injected intraperitoneally,
Vitamin C showed biphasic effect on DTH response. The
thickness of pinnae on the first day increased in a Vitamin C
dose-dependent manner. However, this thickness increment
was inversely related to the amount of Vitamin C administered
from the second day on (Fig. 1). We thought that the
Vitamin C altered T cell function and thus augmented initial,
and cognition and activation stages of DTH response, whereK.
Noh et al. / Immunology Letters 98 (2005) 63–72 69
Fig. 5. The effect of Vitamin C on cytokine secretion by T cells. Mice were treated with 0.625 mg of Vitamin C for 10 days without sensitization (left column),
for 16 days with sensitization on days 11 and 12 (middle column), and for 54 days with sensitization on days 11 and 12 (right column). Control groups were
injected with 200l of PBS. T cells were isolated and cultured for 24 h with DNFB specific or non-specific stimuli. ELISA was done on the supernatants for
the cytokines. In general, Vitamin C elevated the secretion of Th cytokines (IL-2, TNF-
, IFN-) and lowered Th2 cytokine (IL-4) secretion by T cells. Filled
bars () and unfilled bars (
) represent Vitamin C-treated and PBS-treated groups, respectively. Values are the average±S.D. of triplicates.
upon it performed a role as an anti-inflammatory agent from
the second day on.
The anti-inflammatory effects of Vitamin C have already
been suggested. It exerts these effects by scavenging ROS
that is produced by macrophages, destroys tissues and activates
macrophages and neutrophils to progress inflammation
[33,34]. It inhibits the activation of transcription factor NF-
B, which plays a critical role in the production of inflammatory
cytokines such as TNF-
, IL-1, and IL-6 [35–37].
Actually, patients with cystic fibrosis with high plasma Vitamin
C levels had lower inflammation indexes than those with
low Vitamin C levels [34]. Based on such results, we suggest
that the dose-dependent suppression of ear thickening in Vitamin
C-treated mice is due to the anti-inflammatory effect
of Vitamin C. This explanation is further supported by the
finding in Fig. 2. Challenged ears were thinner from day 2
after challenge in groups which were administeredVitamin C
during active inflammation (() and () in Fig. 2) than in the
other groups not givenVitamin C at the time of inflammation.
Even though we measured changes in ear thickness for 10
days, we focused on the first day of DTH response. To explain
the augmentation of the initial stage of DTH response,
70 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Fig. 6. The effect of Vitamin C treatment upon humoral immune response.
Mice were treated with 0.625 mg of Vitamin C for 41 days during primary
and secondary immunization ( ), for 10 days after primary immunization
( ), or for 10 days after secondary immunization (
). Control group mice
were injected with 200 l of PBS during the experiment (). Ten days after
secondary immunization, sera were obtained and ELISA was performed
for whole and each isotype of KLH-specific antibody. Titers are expressed
relative to standard serum. In all experimental groups, whole KLH-specific
antibody titers were reduced vs. the control group. Among isotypes, the titers
of IgG1 and IgE were markedly reduced in all experimental groups, and thus,
contributed to the reduction in whole antibody titer. Each group consisted of
eight mice. Values are the average±S.D. **p < 0.001 vs. control. *p < 0.01
vs. control.
we analyzed the effect of Vitamin C on T cells, the initial
effector cells in this response.We found that an exogenously
administered Vitamin C mega-dose affected T cell functions,
such as proliferative activity and cytokine secretion profiles.
Furthermore, these effects occurred only when Vitamin C
was given during the sensitization period, i.e., when na¨ıve T
cells become activated and memory T cells begin to emerge.
It is notable that the duration of Vitamin C treatment did
not significantly influence these effects. For example, though
mice were treated with Vitamin C for as much as 43 days, no
change in T cell proliferation was observed in the absence of
sensitization (Fig. 4B). On the other hand, when mice were
treated withVitamin C for only10 days, changes in T cell proliferative
activity was observed if the treatment was applied
during sensitization (Fig. 4C).
T cells obtained 43 days (Fig. 3C) and 38 days (Fig. 4A
and C) after sensitization showed minimal thymidine uptake
without any ex vivo stimulation. This implies that almost
all T cells activated through sensitization were deactivated.
Actually, in vivo activated T cells cease proliferation within
1 week of antigen challenge [38,39]. However, when nonspecifically
stimulated with anti-CD3 antibody or PMA/I,
Vitamin C-treated T cells showed more uptake of thymidine
than PBS-treated T cells, a different feature from the na¨ıve
T cells in which there was no difference of thymidine uptake
between the Vitamin C-treated and the PBS-treated groups
(Fig. 3A and B). T cells in Figs. 3 and 4A and C were composed
of not only na¨ıve T cells but memory T cells, and it
can be presumed that the difference between the Vitamin Ctreated
and the PBS-treated groups is due to the activation
of memory T cells. Indeed, when these cell compartments
were stimulated with DNBS, a vivid difference in thymidine
uptake was observed between the Vitamin C-treated and the
PBS-treated groups.We do not know whether this difference
be the result of either a difference in the number of memory
cells or a difference in the vulnerability of memory T cells to
proliferative stimuli or both. Since it has been reported that
Vitamin C inhibits the apoptosis of activated T cells [9] and
increases the number of plasma T cells in elderly patients
[40], an increased number of DNFB-specific memory T cells
in theVitamin C-treated group can be expected. However, the
increased number of memory cells cannot simply explain the
increased thymidine uptake in Vitamin C-treated T cells in
Figs. 3 and 4A and C by non-specific stimulations, which are
presumably independent of specific memory T cells. Further
studies are needed to solve this issue.
The cytokine secretion profiles of T cells were also
changed in addition to and in parallel with changes in proliferative
activities (Fig. 5); that is, the increased secretion of
Th1 cytokines (IL-2, IFN- and TNF-
) and the decreased
secretion of Th2 cytokine (IL-4). These results are similar to
the increased IL-2 and IFN- production of T cells by supplementary
Vitamin E in patients with advanced colorectal
cancer [41]. Since the elevated Th1 cytokines are involved
in cell-mediated immune responses [42,43], these results explain,
in part, the augmented initial stage of DTH response
observed in the present study.
Another point is that, since IL-4 is involved in humoral
immunity [42,43], we expected to observe reduced serum
immunoglobulin levels, and in fact this was the case. In Vitamin
C-treated groups during sensitization, total KLH-specific
antibody titers were reduced. In particular, specific IgG1 and
IgE levels were less than a tenth of those of the PBS-treated
group, markedly contributing to a reduced total specific antibody
titer. These results are in accordance with lower IL-4
secretion by Vitamin C-treated T cells, since IL-4 is known
to induce isotype switching to IgG1 and IgE [44].
Some contradictory results have been reported with respect
to the effects of Vitamin C on humoral response. Longterm
treatment of Vitamin C increased serum IgA and IgM,
but not IgG levels in humans [45]. In guinea pigs, Vitamin
C administration during immunization enhanced specific humoral
responses [46]. Many others have reported unaltered
humoral responses in Balb/c mice [8], humans [47], and in
guinea pigs [48]. These discrepancies could be the results of
differences in the Vitamin C dose and administration routes,
the co-administration of other nutrients, and species used, or
on the presence of T cell stimulation. Meanwhile, in situations
such as chronic granulomatous disease, in which we
could expect the persistent activation of T cells in vivo, longterm
treatment of Vitamin C lowered plasma Ig levels [3].
Vitamin C also reduced serum total IgE levels in asthma patients
[49].
It remains to be elucidated how the two groups treated
with Vitamin C, i.e., during primary or secondary immunization,
had the same secondary antibody secretion profiles.
Those treated only during secondary immunization would
K. Noh et al. / Immunology Letters 98 (2005) 63–72 71
have the same repertoire of memory B cells as the control
group, we would expect a higher titer of IgG1 and IgE
in this group than in the group treated during primary immunization.
However, the titers of both groups showed no
differences. The possibilities are that the isotype-switched
memory B cells during the primary response either underwent
apoptosis or isotype-switched once again during the
secondary response probably due to decreased IL-4 production
by T cells. In this experiment we obtained no data on
this issue, and a search of the literatures provided no clear
suggestions.
To summarize, mega-dose Vitamin C altered the proliferative
capacity of T cells against specific and non-specific
stimuli, and changed cytokine profiles toward Th1, and finally
resulted in an increase in the onset of DTH response and a
decrease in the total specific antibody titer in serum. This
Th1 polarization of immune response by Vitamin C has been
mentioned of its possibility [50]. What is important is that
these effects of exogenous Vitamin C occurred only when it
was administered during sensitization. Thus, we suggest that,
only activated T cells are vulnerable to changes by exogenous
mega-dose Vitamin C. It is not known whether this effect occurs
due to the intracellular accumulation of Vitamin C or
to the ligation of putative Vitamin C membrane receptors,
which have not yet been identified.
Acknowledgement
This work was supported by the Korea Science & Engineering
Foundation (KOSEF) through the Tumor Immunity
Medical Research Center (TIMRC) at Seoul National University,
College of Medicine.
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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
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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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