Hepatitis C and Ozone Therapy
by Gérard V. Sunnen, M.D.
February 2001
Abstract
Hepatitis C (HCV) is a global disease with an expanding incidence and prevalence
base. Of massive public health importance, hepatitis C presents supremely
challenging problems in view of its adaptability and its pathogenic capacity.
The unique strategies that HCV utilizes to parasitize its host make it a
formidable enemy and therapeutic interventions need considerable honing to
counter its progress. Ozone, because of its special biological properties, has
theoretical and practical attributes to make it a potent HCV inactivator.
History of the virus A form of hepatitis became recognized in the 1970's that
resembled hepatitis B, serum hepatitis, and to a lesser extent hepatitis A,
infectious hepatitis. It had, however, novel features, amongst them, a
distinctive serological profile. In 1989, the genome of hepatitis C (HCV) was
deciphered.
It is possible, by means of extrapolation from the genetic evolution of a virus,
to approximate its age. Sequence genetic analysis points to the diversification
of different HCV genotypes 200 to 400 years ago. Ancestors to these genotypes
probably date back 100,000 or so years when viruses co-evolved with modern
humans. Further analysis of genetic viral trees and Old and New World primates
take the primordial forms of these viruses to primate speciation periods some 35
million years ago.
Today, in the context of human population growth, migration, and global travel,
the hepatitis C virus has expanded its territories, geographically, and
demographically. There is every indication that the evolution of this virus, in
all its forms, is currently manifesting an accelerated phase.
Virion architecture and molecular biology The HCV particle is composed of a
nucleocapsid containing its genome, an RNA single strand composed of
approximately 9600 nucleotides, and its protein coating. The nucleocapsid is
surrounded by an envelope which allows attachment and penetration into host
cells. The genome encodes structural proteins designated as core (C), envelope 1
(E1), envelope 2 (E2), and P7 (unknown function), providing for virion
architecture, and nonstructural proteins, mainly enzymes essential to the
virion's life cycle, designated as NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
Proteases release structural and nonstructural proteins. Helicases unwind viral
nucleic acid. Polymerases replicate RNA. Within this genome is located a
hypervariable region implying an area of intensive genetic fluidity and
mutational potential. HCV displays great genotypic flexibility which makes for
sophisticated evasiveness to host defenses.
The nucleocapsid is surrounded by an envelope, a lipid bilayer associated with a
union of carbohydrates and proteins, glycoproteins. Up to 60% of the lipid
component of the envelope is phospholipid and the remainder is mostly
cholesterol. It possesses projections called peplomers which facilitate
attachment to host cells. One protein on peplomers of the HCV particle which is
thought to be instrumental in the attachment process is designated CD-81.
The sequence of nucleotides within the HCV genome shows significant variations.
Strains obtained from different parts of the world, for example, may differ
substantially in their structural and nonstructural protein compositions. This
has lead to a system of classification of the HCV family into 6 genotypes (1 to
6), and approximately 100 subtypes (designated a, b, c, ect.). Genotypes vary
from each other by a factor of 30% over the entire genome. Subtypes vary by
about 20%. Genotypes 1 to 3 have global distribution, while genotype 4 and 5 are
found mainly in Africa, and 6 is distributed in Asia. Importantly, genotype and
subtype differences have shown varying susceptibility to antiviral therapy.
Within any one afflicted individual, HCV particles do not show a homogeneous
population. Instead, they function as a pool of genetically variant strains
known as quasispecies. This is due to the high replication error inherent in the
function of the polymerase enzymes. Herein lies one of the important armaments
of HCV. Continuously generated genetic diversity gives it great advantage in
negotiating and conquering immune defense and therapeutic strategies.
Furthermore, the antigenic differences between genotypes may have implications
regarding the proper evaluation and the therapeutic regimen of patients.
Viral life cycle A freely circulating virion enters a host cell by binding to a
cell surface receptor. In the case of HCV the host cell is a hepatocyte.
However, bone marrow, kidney cells, macrophages, lymphocytes, and granulocytes
may also be trespassed.
Once cell entry is achieved, the virion sheds its envelope to commence its
replication. It binds to cellular ribosomes and released viral polymerase begins
the RNA replication cycle. Newly formed nucleocapsids continue their assembly
with the acquisition of new envelopes by means of budding through membranes of
the cell's endoplamic reticulum. Newly formed virions may number in the range of
10 billion daily. The average life span of virions is in the order of a few
hours.
Virions are then released into the general blood and lymphatic circulation,
ready to infect new cells, re-infect already diseased cells, or a new host,
mainly through bodily fluid transmission pathways. HCV RNA, as measured by
polymerase chain reaction (PCR) may show 10 million or more virions per ml. As
little as 0.0001 ml of blood may be sufficient to impart infection. The
evolution of hepatitis C is characterized by phases of accentuated viremia
punctuated by periods of relative quiescence. The presence and timely detection
of these viremic waves may offer novel therapeutic considerations.
Clinical and laboratory manifestations Hepatitis, from anyone of the several
viruses capable of inducing liver inflammation, produce a spectrum of clinical
and laboratory manifestations. Hepatitis C distinguishes itself by the low
incidence of acute phases and by the high incidence of progression to
chronicity. Acute hepatitis C progresses from exposure, to incubation, to
pre-icteric, icteric, and convalescent phases. With an incubation period of
about 6 weeks, the first and sometimes only symptoms include weakness, fatigue,
indolence, headache, nausea, poor appetite, and vague abdominal pain. The
pre-icteric period extends from the onset of symptoms to the appearance of
jaundice, ranging usually from 2 to 12 days. The icteric phase corresponds to
the declaration of jaundice and darkened urine. The convalescent phase is marked
by the gradual disappearance of symptoms.
Chronic hepatitis C is characterized by the presence of HCV RNA and the
elevation of liver enzymes for 6 months or longer. Patients may be asymptomatic,
or at times suffer an acute exacerbation with a return of symptoms.
Approximately 75% of acutely ill patients continue into a chronic phase
evidenced by parameters of viral presence.
Hepatitis C can only be distinguished from other viral hepatic conditions by
serological and virological determinations. Liver enzymes characteristically
affected by HCV infection include serum alanine transfesferase (ALT), aspartate
aminotransferase (AST), gamma- glutamyl transpeptidase (GGTP), and alkaline
phosphatase; in addition, there may be abnormalities in bilirubin, serum
albumin, prothrombin time, and platelet density.
Cirrhosis, a diffuse disruption of liver tissue architecture with regenerative
nodules surrounded by fibrosis, is an important sequel to hepatitis C. Within 20
years post HCV infection 20 to 25% of patients will develop cirrhosis. Hepatic
decompensation ensues with ascites as the salient marker.
Hepatocellular carcinoma, another notable outcome of HCV infection is present in
approximately 5% of patients post infection. The presence of cirrhosis is
central to its genesis. Although the mechanisms by which cirrhosis ushers
carcinoma are unknown, it is likely that chronic inflammation and the sustained
pressure of cellular regeneration play important roles.
Up to 10% of patients appear to have fully conquered the disease. HCV antibodies
are undetectable, as is HCV RNA. Liver enzymes are fully normalized, but liver
biopsy may show lingering areas of stagnant inflammation and spotty necrosis. It
is thus possible for host immunocompetence to vanquish HCV infection and
therapeutic strategies aim to assist the host immune system to achieve this
goal.
Immunological response to the virus HCV particles are detected early in the
infection, usually 1 to 2 weeks following exposure. Antibodies to HCV core,
nonstructural, and envelope elements appear about 6 weeks after exposure. A
broad range of cytokines are mobilized. Cellular immunity is activated with
broad recruitment of neutrophils, natural killer (NK), macrophages, and CD4 and
CD8 T helper cells.
Current and experimental treatment strategies As of this date the main treatment
strategies for hepatitis C include interferon and ribavirin. Interferons are
natural cellular products which activate macrophages, neutrophils and natural
killer cells. There is controversy as to interferon's biological effects, be
they mostly immunoregulatory or directly antiviral. Ribavirin is a guanosine
analog that represses messenger RNA formation thus inhibiting the replication of
many DNA and RNA viruses. It is, however, mutagenic to mammalian cells.
Ribavirin and interferon have significant medical and psychiatric side effects.
Treatment response is defined as undetectable viral load 6 months following
therapy. Contemporary detection methods of quantitative HCV RNA determinations
are capable of detecting approximately 1000 viral copies per serum ml.
Resistance to antiviral therapies is a particularly vexing problem in anti HCV
treatment. Novel and experimental antiviral compounds include inhibitors of
protease, polymerase and helicase.
Vaccine development needs to take into account HCV's antigenic rainbow and its
high mutability. High mutation rates in this condition implies a dauntingly
diverse and variable array of viral antigenic components. It is estimated, for
example, that HCV mutates significantly in its own host approximately a thousand
times a year. This implies that within any one afflicted individual there exists
an awesomely large array of viral quasispecies, which in turn creates
commensurate difficulties in the creation of effective vaccines.
Ozone: Physical and physiological properties Ozone (O3) is a naturally occurring
configuration of three oxygen atoms. With a molecular weight of 48, the ozone
molecule contains a large excess of energy. It has a bond angle of 127° and
resonates among several forms. At room temperature, ozone has a half life of
about one hour, reverting to oxygen. A powerful oxidant, ozone has unique
biological properties which are being investigated for applications in various
medical fields. Basic research on ozone's biological dynamics have centered upon
its effects on blood cellular elements (erythrocytes, leucocytes, and
platelets), and to its serum components (proteins, lipoproteins, lipids,
carbohydrates, electrolytes). Administrating increaing dosages of ozone to whole
blood shows that beyond a certain threshold there is a rise in the rate of
hemolysis. This threshold, depending upon various parameters, begins to be
reached at 40 to 60 micrograms per milliliter, and becomes significant when
higher levels are attained. Precise ozone dosing capacity is therefore essential
in clinical practice and research.
Leucocytes show good resistance to ozone because they have enzymes which protect
them from oxidative stress. These enzymes include superoxide dismutase,
glutathione, and catalase. Research has shown that platelets also maintain their
integrity after ozone administration. In ozone therapy, the doses applied to
blood are gauged to avoid disruption of its cellular elements. Serum components
remain viable during ozone therapy. Lipid and protein peroxides, produced in
small amounts by ozonation, have demonstrable antiviral properties.
Interestingly, ozone tends to stimulate leucocyte function and cytokine
production. Ozone increases the oxygen saturation (p02) in erythrocytes and
enhances their pliability so that capillary circulation is facilitated.
Ozone: Antiviral properties Recently, there has surged renewed interest in the
potential of ozone for viral inactivation. It has long been established that
ozone neutralizes bacteria, viruses, and fungi in aqueous media. This has
prompted the creation of water purification processing plants in many major
municipalities worldwide.
Ozone's antiviral properties may also be applied to the treatment of biological
fluids, albeit in technologically and physiologically appropriate ways. Indeed,
it is noted that ozone, administered in such dosages designed to respect the
integrity of blood's cellular and constituent elements, is capable of
inactivating a spectrum of viral families.
Some viruses are much more susceptible to ozone's action than others. It has
been found that lipid-enveloped viruses are the most sensitive. This group
includes, amongst others, HCV, Herpes 1 and 2, Cytomegalus, HIV1 and 2.
The envelopes of viruses provide for intricate cell attachment, penetration, and
cell exit strategies. Peplomers, finely tuned to adjust to changing receptors on
a variety of host cells, constantly elaborate new glycoproteins under the
direction of E1 and E2 portions of the HCV genome. Envelopes are fragile. They
can be disrupted by ozone and its by-products.
In HCV, viral load appears to be a major factor in the invasiveness and
virulence of the disease process. Preliminary research has shown that reduction
of viral load in Hepatitis C by means of ozone therapy can significantly
normalize hepatic enzymes and improve measures of global patient health.
Volunteers administered ozone therapy according to the method outlined below
achieved a viral load reduction in the order of 5 log, or 99.9%, along with a
normalization of liver enzyme levels.
Ozone: Clinical methodology Ozone may be utilized for the therapy of a spectrum
of clinical conditions. Routes of administration are varied and include external
and internal (blood interfacing) methods. In the technique of ozone major
autohemotherapy for hepatitis C, an aliquot of blood is withdrawn from a
virally-afflicted patient, anticoagulated, interfaced with an ozone/oxygen
mixture, then re-infused. This process is repeated serially until viral load
reduction is documented.
The aliquots of blood range from 50 ml. to 300 ml. Ozone dosages and treatment
frequency vary according to treatment protocols. The reason aliquots of blood
are treated and not, as one would propose, the entire blood volume, is that in
the latter case the total ozone dosage administered would exceed toxic limits.
The average adult has 4 to 6 liters of blood, accounting for about 7% of body
weight. How can the viral load reduction observed via ozone therapy be explained
in the face of a technique that treats relatively small amount of blood, albeit
serially?
Ozone: Possible mechanisms of anti-viral action
The viral culling effects of ozone in infected blood may recruit the following
mechanisms:
Denaturation of virions through direct contact with ozone. Ozone, via this
mechanism, disrupts viral envelope proteins, lipoproteins, lipids, and
glycoproteins. The presence of numerous double bonds in these unsaturated
molecules makes them vulnerable to the oxidizing effects of ozone which readily
donates its oxygen atom and accepts electrons in these redox reactions. Double
bonds are thus reconfigured, molecular architecture is disrupted and widespread
breakage of the envelope ensues. Deprived of an envelope, virions cannot sustain
nor replicate themselves.
Ozone proper, and the peroxide compounds it creates, may directly alter
structures on the viral envelope which are necessary for attachment to host
cells. Peplomers, the viral glycoproteins protuberances which connect to host
cell receptors are likely sites of ozone action. Alteration in peplomer
integrity impairs attachment to host cellular membranes foiling viral attachment
and penetration.
Introduction of ozone into the serum portion of whole blood induces the
formation of lipid and protein peroxides. While these peroxides are not toxic to
the host in quantities produced by ozone therapy, they nevertheless possess
oxidizing properties of their own which persist in the bloodstream for several
hours. Peroxides created by ozone administration show long-term antiviral
effects which serve to further reduce viral load. This factor may explain in
part the reason for the fact that ozonated blood in the amount processed in
usual treatment protocols is able to reduce viral load values in the total blood
volume.
Immunological effects of ozone have been documented. Cytokines are proteins
manufactured by several different types of cells which regulate the functions of
other cells. Mostly released by leucocytes, they are important in mobilizing the
immune response. It has been found that ozone induces the release of cytokines
which in turn activate a spectrum of immune cells. This is likely to constitute
a significant avenue for the reduction of circulating virions.
Ozone action on viral particles in infected blood yield several possible
outcomes. One outcome is the modification of virions so that they remain
structurally grossly intact yet sufficiently dysfunctional as to be
nonpathogenic. This attenuation of viral particle functionality through slight
modifications of the viral envelope, and possibly the viral genome itself,
modifies pathogenicity and allows the host to increase the sophistication of its
immune response. The creation of dysfunctional viruses by ozone offers unique
therapeutic possibilities. In view of the fact that so many mutational variants
exist in any one afflicted individual, the creation of an antigenic spectrum of
crippled virions could provide for a unique host-specific stimulation of the
immune system, thus designing what may be called a host-specific autovaccine.
Summary
Viruses are far from being static entities. As quintessential intracellular
parasites they have developed, through millions of years of cohabitation with
their hosts, astoundingly sophisticated structures, survival, and propagation
mechanisms. They have adapted, modified their biological strategies, and evolved
impressive genetic diversity and mutational capacity to cope with the changing
ecology of planetary life.
HCV has an extremely high rate of mutation and within any one individual there
may exist millions of antigenic quasispecies. The disease process is marked by
periods of viral quiescence alternating with viremic waves whereby billions of
virions are poured into the blood and lymphatic reservoirs. Their astounding
numbers stress the immune system relentlessly and produce an inexorable
compromise in all parameters of its functioning.
Viral load reduction by means of ozone blood treatment alleviates immune system
fatigue. Ozone-mediated viral culling may be achieved by anyone of a number of
possible mechanisms. Direct virion denaturation, peplomer alteration, lipid and
protein peroxide formation, cytokine induction, host pan-humoral activation, and
host-specific autovaccine creation are suggested mechanisms. Due to the excess
energy contained within the ozone molecule, it is theoretically likely that
ozone, unlike antiviral options available today, will show effectiveness across
the entire genotype and subtype spectrum.
Ozone embodies unique physico-chemical and biological properties which suggest
an important role in the therapy of hepatitis C, either as a monotherapy, or as
an adjunct to standard treatment regimens.
BIBLIOGRAPHY
a.. Bartenschlager R. Candidate targets for hepatitis C virus-specific
antiviral therapy. Intervirology 1997; 40:378-393
b.. Bocci V, Luzzi E, Corradeschi F, Paulesu, et al. Studies on the biological
effects of ozone: 5. Evaluation of immunological parameters and tolerability in
normal volunteers receiving ambulatory autohaemotherapy. Biotherapy 1994;
7:83-90
c.. Bocci V. Ozonation of blood for the therapy of viral diseases and
immunodeficiencies. A hypothesis. Medical Hypotheses 1992 Sept; 39(1):30-34
d.. Bolton DC, Zee YC, Osebold JW. The biological effects of ozone on
representative members of five groups of animal viruses. Environmental Research
1982; 27:476-48
e.. Buckley RD, Hackney JD, Clarck K, Posin C. Ozone and human blood. Archives
of Environmental Health 1975; 30:40-43
f.. Cardile V, et al. Effects of ozone on some biological activities of cells
in vitro. Cell Biology and Toxicology 1995 Feb; 11(1):11-21
g.. Carpendale MT, Freeberg JK. Ozone inactivates HIV at noncytotoxic
concentrations. Antiviral Research 1991; 16:281-292
h.. Dailey JF. Blood. Medical Consulting Group, Arlington MA, 1998
i.. Di Bisceglie AM, Bacon BR. The unmet challenge of hepatitis C. Scientific
American 1999; 281:58-63
j.. Dienstag JL. Sexual and perinatal transmission of hepatitis C. Hepatology
1997; 26: 66S-70S
k.. Dieperink E, Willenbring M, Ho SB. Neuropsychiatric symptoms associated
with hepatitis and interferon alpha: A review. Am J Psychiatry 2000 June;
157(6):867-876
l.. Evans AS, Kaslow RA (Eds). Viral Infections in Humans: Epidemiology and
Control, Fourth Edition, Plenum, New York, 1997
m.. Gonzalez-Peralta RP, Qian K, She JY, et al. Clinical implications of viral
quasispecies heterogeneity in chronic hepatitis C. J Med Virology 1996;
49:242-24
n.. Harrison TJ, Zuckerman AJ (Eds). The Molecular Medicine of Viral
Hepatitis. Molecular Medical Science Series. John Wiley & Sons, New York, 1997
o.. Konrad H. Ozone therapy for viral diseases. In: Proceedings 10th Ozone
World Congress 19-21 Mar 1991, Monaco. Zurich: International Ozone Association
1991:75-83
p.. Liang TJ, Hoofnagle JH, (Eds). Hepatitis C. Academic Press, San Diego,
2000
q.. Maggi F, Fornai C, Morrica A, et al. Divergent evolution of hepatitis C
virus in liver and peripheral blood mononuclear cells of infected patients. J
Med Virology 1999; 57:57-63
r.. Major ME, Feinstone SM. The molecular virology of hepatitis C. Hepatology
1997; 25: 1527-1538
s.. Maertens G, Stuyver L. Genotypes and genetic variation of hepatitis C
virus. In: The Molecular Medicine of Viral Hepatitis. John Wiley $ Sons Ltd.,
London, 1997: 225-227
t.. Monjardino J. Molecular Biology of Hepatitis Viruses, Imperial College
Press, London, 1998
u.. Par A, et al. Hepatitis C virus infection: pathogenesis, diagnosis and
treatment. Scandinavian Journal of Gastroenterology. 1998 Suppl; 228: 107-114
v.. Paulesu L, Luzzi L, Bocci V. Studies on the biological effects of ozone:
Induction of tumor necrosis factor (TNF-alpha) on human leucocytes. Lymphokine
Cytokine Research 1991; 5:409-412
w.. Pawlotsky J. Hepatitis C virus resistance to antiviral therapy. Hepatology
Nov. 5, 2000; 32: 889-89
x.. Roy D, Wong PK, Engelbrecht RS, Chian ES. Mechanism of enteroviral
inactivation by ozone. Applied Environmental Microbiology 1981; 41: 728-733
y.. Sarara AI. Chronic hepatitis C. South Med J. 1997; 90: 872-877
z.. Seeff LB. Natural history of hepatitis C. Hepatology 1997; 26: 21S-28S
aa.. Sunnen GV. Ozone in Medicine. Journal of Advancement in Medicine. 1988
Fall; 1(3): 159-174
ab.. Trivedi M. Newly diagnosed hepatitis C: Lack of symptoms doesn't mean
lack of progression. Postgraduate Medicine 1997; 102: 95-98
ac.. Valentine GS, Foote CS, Greenberg A, Liebman JF (Eds). Active Oxygen in
Biochemistry. Blackie Academic and Professional, London, 1995
ad.. Vaughn JM, Chen Y, Linburg K, Morales D. Inactivation of human and simian
rotaviruses by ozone. Applied Environmental Microbiology 1987; 48:2218-2221
ae.. Viebahn R. The Use of Ozone in Medicine. Haug, Heildelberg, 1994
af.. Wells KH, Latino J, Gavalchin J, Poiesz BJ. Inactivation of human
immunodeficiency virus Type 1 by ozone in vitro. Blood 1991 Oct; 78(7):1882-1890
ag.. Yu BP. Cellular defenses against damage from reactive oxygen species.
Physiological Reviews 1994 Jan; 74(1):139-162
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