Dr. Mitra Basu Chhillar, M.D., M.B.A., F.A.M.

Medical Director,
SOMA Wellness Clinic

Introduction

Ozone therapy, the controlled administration of medical-grade ozone (O₃), has been explored for over a century, initially for water disinfection and wound treatment, and now for a broad range of medical conditions, including chronic inflammatory diseases, infections, circulatory disorders, and antiaging applications. Despite its historical use, ozone therapy remains controversial due to limited large-scale clinical trials and regulatory skepticism, notably from the U.S. Food and Drug Administration (FDA), which prohibits its medical use citing insufficient evidence of safety and efficacy [1]. However, countries like Germany, Italy, Russia, and Cuba integrate it into healthcare systems under strict guidelines, supported by emerging research on its biochemical mechanisms.

This lecture, designed for medical doctors, provides a detailed examination of ozone therapy’s biochemical basis, mechanisms of action, dosing protocols, clinical applications, and potential in antiaging, with a focus on the biochemical pathways involved. It addresses the user’s request to ensure all 46 references cited in the text are listed, correcting the previous discrepancy where only 25 were provided. By the end, clinicians will have a balanced perspective to evaluate ozone therapy’s role in practice responsibly.

Biochemical Basis of Ozone Therapy

The therapeutic potential of ozone therapy hinges on its ability to induce controlled, mild oxidative stress that activates protective biochemical pathways. Below, we explore the key mechanisms and pathways involved.

Formation of Ozone Peroxides

At low doses, ozone selectively reacts with mono-unsaturated fatty acids (e.g., oleic acid) in cell membranes, forming ozonides and hydroperoxides, collectively termed “ozone peroxides.” These intermediates decompose into secondary products like aldehydes, ketones, and hydrogen peroxide (H₂O₂), which act as signaling molecules. Unlike reactions with polyunsaturated fatty acids, which can lead to harmful lipid peroxidation, this selective interaction minimizes cellular damage [2, 3]. The chemical reaction can be represented as:

R-CH=CH-R’ + O₃ → R-CH(O₃)-CH-R’ → Ozonides + Hydroperoxides

Glutathione Interaction

Ozone peroxides are rapidly reduced by glutathione (GSH), the body’s primary antioxidant, generating reactive oxygen species (ROS) and lipid ozonation products (LOPs), such as 4-hydroxynonenal (4-HNE). The GSH/GSSG (glutathione/oxidized glutathione) balance is critical, serving as a limiting factor for safe ozone dosing. Therapeutic doses induce oxidative eustress, promoting cellular protection, while excessive ozone can deplete GSH, leading to oxidative distress [4]. This balance ensures ozone acts as a bioregulator rather than a toxic agent.

Key Biochemical Pathways

Ozone therapy activates several nuclear transcriptional factors, each contributing to its therapeutic effects:

• Nrf2/ARE Pathway: Mild oxidative stress activates nuclear factor erythroid 2-related factor 2 (Nrf2), which binds to antioxidant response elements (AREs). This upregulates antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO-1), and heat shock proteins (HSP). These enzymes mitigate oxidative stress, a key driver of aging and chronic diseases [5, 6]. The pathway can be summarized as:

• NFκB Pathway: Ozone suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), reducing pro-inflammatory cytokines (e.g., IL-1, IL-6, TNF-α). This anti-inflammatory effect is crucial for managing chronic inflammatory conditions like rheumatoid arthritis [7].
• HIF-1α Pathway: Hypoxia-inducible factor 1-alpha (HIF-1α) is activated, promoting genes such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO). This enhances blood flow and oxygen delivery, supporting tissue repair and regeneration [8].
• NFAT and AP-1 Pathways: These pathways regulate cytokine production (e.g., IL-2, IFNγ), supporting immune function and potentially counteracting age-related immune decline [9].
• Mitochondrial Function: Ozone enhances mitochondrial energy production by improving oxygen utilization, activating the Krebs cycle, reducing NADH, and oxidizing cytochrome C. This supports cellular energy needs, particularly in aging tissues [10].

Antimicrobial Effects

Ozone’s strong oxidative properties disrupt the cell membranes and nucleic acids of bacteria, viruses, fungi, and protozoa, making it effective against pathogens. This is particularly valuable in topical applications for wound infections (e.g., diabetic foot ulcers) and systemic treatments for conditions like hepatitis B and C [11]. At concentrations of 3.5%–5%, ozone exhibits a potent germicidal effect, inactivating pathogens like Epstein-Barr, herpes, and hepatitis viruses [12].

Redox Signaling

Ozone mimics H₂O₂ in redox signaling, restoring balance in conditions of oxidative stress. This controlled oxidative stress is the cornerstone of its bioregulatory effects, distinguishing therapeutic ozone from toxic high-dose exposure [13]. By acting as a second messenger, ozone peroxides facilitate signal transduction, enhancing cellular resilience and adaptability.

Mechanisms of Action in the Body

Ozone therapy exerts both systemic and local effects, tailored to the administration method, which influences its therapeutic outcomes.

Systemic Effects

• Bioregulation: Ozone peroxides, reduced by GSH, generate signaling molecules that activate Nrf2 and modulate NFκB, enhancing antioxidant defenses and immune regulation. This bioregulatory role is critical in chronic inflammatory diseases characterized by high oxidative stress [14].
• Oxygen Delivery: Ozone increases 2,3-diphosphoglycerate (2,3-DPG) levels in red blood cells, facilitating oxygen release to tissues. This is particularly beneficial in ischemic conditions like peripheral artery disease, where improved oxygenation supports tissue health [15].
• Immunomodulation: Ozone downregulates pro-inflammatory cytokines in chronic inflammation while enhancing immunocompetent cell activity in immune-suppressed states, offering a dual role in immune modulation [16].

Local Effects

• Germicidal Action: Ozone’s oxidative properties inactivate pathogens through radical reactions, making it effective for wound healing, such as in diabetic foot ulcers or burns. This direct antimicrobial action reduces infection rates and promotes tissue repair [17].
• Anti-inflammatory Effects: Topical ozone reduces local cytokine levels, alleviating inflammation and supporting healing processes in conditions like chronic wounds and dermatological disorders [18].

Indirect Effects

• Cytokine Modulation: Systemic ozone therapy reduces pro-inflammatory cytokines, improving outcomes in conditions like rheumatoid arthritis by restoring immune balance [19].
• Tissue Oxygenation: Enhanced red blood cell function and blood flow support tissue regeneration and repair, particularly in circulatory disorders [20].

These mechanisms highlight ozone’s multifaceted role in modulating oxidative stress, inflammation, and immune responses, making it a potential tool for various medical conditions.

Dosage and Administration Methods

Precise dosing is paramount in ozone therapy to balance efficacy and safety. The therapeutic window is narrow—concentrations below 10 µg/mL are often ineffective due to neutralization by serum antioxidants, while those above 50 µg/mL can be toxic, causing hemolysis or tissue damage [21]. Below are the key dosing protocols for common administration methods:

Method	Concentration (µg/mL)	Dosage (µg)	Volume	Frequency
Direct Intravenous ozone(DIV) or Major Autohemotherapy (MAH)	10–40 (toxic >50)	500–4000	50 mL gas	Acute RA: daily; Non-acute: 1x/week
Rectal Insufflation (RI)	Adults: 10–25; Children: 10–20	3750–9000 (adults)	150–300 mL (adults); 10–30 mL (children)	Daily, then 1–2x/week
Local (e.g., Ulcerative Colitis)	70–100 (initial), 20–30 (maintenance)	Varies	50 mL	As needed post-hemorrhage

For specific conditions like rheumatoid arthritis:

• DIV/ MAH: 20–30 µg/mL (max 40 µg/mL), dosage 1000–1500 µg (max 2000 µg).
• RI: 25–30 µg/mL, volume 150–300 mL (total dose 3750–9000 µg) [22].

Dosing must be individualized based on patient factors such as age, antioxidant status, and disease severity. For example, children require lower volumes and concentrations to avoid adverse effects, while adults with chronic conditions may tolerate higher doses within the therapeutic range [23]. Specific examples include:

• Intratumoral Ozone: 40 µg/mL, 5 mL (total 200 µg) monthly for cancer [24].
• Intra-articular Injection: 20 µg/mL × 20 mL, once weekly for 4 weeks for knee osteoarthritis [25].
• Inhalation: 8 mg/L, 60 mL/min for 10 min/day for 10 days for tinnitus [26].

Medical Applications

Ozone therapy has been investigated for a variety of conditions, particularly those involving oxidative stress, inflammation, or impaired oxygenation. Below are key applications supported by research:

Chronic Inflammatory Diseases

• Rheumatoid Arthritis (RA): A randomized trial of 60 RA patients demonstrated that ozone therapy (25–40 µg/mL, 20 rectal insufflations over 4 weeks) improved antioxidant markers (SOD, CAT, GSH), reduced oxidative stress (NO, MDA), and lowered cytokines (IL-1, IL-6, TNF-α). Clinical outcomes, including DAS-28 scores and pain, also improved significantly [27]. Additionally, ozone protected liver function in RA patients by reducing γ-GT levels [28].
• Osteoarthritis: Intra-articular ozone injections (10–20 µg/mL) reduced pain and improved joint function in randomized controlled studies, offering a non-invasive alternative for joint pain management [29].

Infectious Diseases

Ozone’s antimicrobial properties are effective against bacteria, viruses, fungi, and protozoa. It is used topically for wound infections and systemically for hepatitis B and C, where it may reduce viral load and improve liver function [30]. In dentistry, ozone treats dental caries and periodontal disease by eliminating pathogens and promoting tissue healing [31]. Studies also suggest potential in HIV treatment, though well-designed trials show no significant benefit for living patients [32].

Circulatory Disorders

Ozone improves blood flow in peripheral artery disease, with studies showing increased walking distance and reduced pain in patients with peripheral obstructive arterial disease (POAD). This is attributed to enhanced oxygen delivery and reduced oxidative stress [33].

Complementary Oncology

Ozone may enhance immune function and reduce side effects of conventional cancer treatments. Preliminary studies suggest direct antitumor effects by restoring normoxia in tumor microenvironments, though robust clinical trials are needed [34].

Wound Healing

Ozone accelerates tissue repair in chronic wounds like diabetic foot ulcers by increasing oxygen supply and reducing bacterial load. A Cuban trial showed improved glycemic control, reduced ulcer size, and fewer amputations in diabetic patients treated with ozone compared to antibiotics [35].

Dermatological Conditions

Topical ozone reduces lesion size and severity in psoriasis and eczema, leveraging its anti-inflammatory and antimicrobial effects. It also improves skin microbiomes, supporting its use in dermatology [36].

Chronic Pyelonephritis

Ozone therapy has shown promise in managing chronic pyelonephritis by reducing oxidative stress and inflammation, potentially improving renal function in affected patients [37].

Neurodegenerative Diseases

Preliminary studies suggest potential benefits in Alzheimer’s and Parkinson’s through antioxidative and anti-inflammatory effects, though more research is needed [38].

Other Conditions

Ozone has been explored for low back pain, tinnitus, COVID-19, postherpetic neuralgia, herpes zoster, sudden deafness, thromboangiitis obliterans, liver ischemia/reperfusion injury, and chronic viral hepatitis, with varying levels of evidence [39-46].

Ozone Therapy for Antiaging

Aging is driven by oxidative stress, chronic inflammation, declining mitochondrial function, and immune senescence. Ozone therapy’s ability to modulate these processes positions it as a potential antiaging intervention, though direct evidence remains limited.

Biochemical Pathways in Antiaging

• Nrf2/ARE Pathway: Upregulation of antioxidant enzymes (SOD, CAT, GPx, HO-1, NQO-1) protects against oxidative damage, supports DNA repair, and reduces inflammation, mitigating age-related diseases like Alzheimer’s, Parkinson’s, and cardiovascular disorders [5]. Nrf2 modulation is statistically significant (p < 0.00001, OR = 1.71, 95% CI: 1.17-2.25) [6].
• HIF-1α Pathway: Improved oxygen delivery and blood flow maintain tissue vitality, potentially delaying age-related declines [8].
• NFκB Suppression: Reducing chronic inflammation prevents tissue damage associated with aging [7].
• Mitochondrial Function: Ozone enhances mitochondrial energy production by improving oxygen utilization, counteracting age-related mitochondrial decay [10].
• Immunomodulation: Mild activation of NFAT and AP-1 pathways counters immune senescence, supporting overall health in aging populations [9].

Clinical Evidence

While direct antiaging studies are sparse, ozone therapy’s effects on age-related conditions provide indirect support:

• Age-Related Macular Degeneration (ARMD): A study reported that 75% of ARMD patients showed 1–2 lines of visual acuity improvement after 15–18 treatments with ozone concentrations of 20–60 µg/mL, maintained with monthly sessions [15].
• Neurodegenerative Diseases: Anecdotal reports suggest cognitive improvements in early Alzheimer’s patients treated with ozonated autohemotherapy (O₃-AHT), though these findings are unpublished and require validation [38].
• Peripheral Obstructive Arterial Disease (POAD): Ozone improves circulation and reduces oxidative stress, supporting vascular health in aging patients [33].
• Quality of Life: Patients often report enhanced energy, euphoria, and well-being, possibly due to neuroendocrine stimulation, aligning with antiaging goals [14].
• Skin Health: Ozone therapy enhances skin microecology, boosts collagen production, and reduces wrinkles, supporting a youthful appearance [36].

Proposed Antiaging Strategy

A “bland” ozone therapy approach is proposed to delay aging:

• Low-Dose Administration: Use rectal insufflation at 10–25 µg/mL, 1–2 times weekly, to minimize risks while achieving therapeutic effects.
• Lifestyle Integration: Combine with a balanced diet rich in antioxidants, regular exercise, stress management, and adequate sleep.
• Synergistic Therapies: Integrate with other antioxidants (e.g., polyphenols, mushrooms) to enhance antiaging effects [6].
• Monitoring: Regularly assess oxidative stress markers (e.g., GSH levels, SOD activity) to tailor therapy to individual needs.

This strategy aims to enhance antioxidant defenses, improve circulation, and support immune function, addressing key aging mechanisms holistically.

Future Research in Antiaging

To establish ozone therapy’s antiaging potential, research should focus on:

• Longitudinal Studies: Assessing effects on aging biomarkers (e.g., telomere length, epigenetic markers) and longevity.
• Mechanistic Studies: Elucidating molecular pathways, such as telomere maintenance and senescent cell clearance.
• Comparative Trials: Evaluating ozone therapy against other antiaging interventions for efficacy and safety.
• Standardization: Developing protocols for antiaging applications to ensure consistency [6].

Safety and Regulatory Considerations

Ozone therapy is not without risks, particularly at high doses or with improper administration:

• Toxicity Risks: Concentrations above 50 µg/mL can cause hemolysis, methemoglobinemia, or tissue damage. Rare but severe complications, like ozone-induced encephalopathy (characterized by confusion or seizures), have been reported [11].
• Infection Risks: Non-sterile techniques may lead to infections, emphasizing the need for rigorous protocols.
• Regulatory Stance: The FDA prohibits ozone’s medical use in the U.S., citing insufficient evidence. In contrast, countries like Germany and Cuba regulate its use under strict guidelines [1].
• Clinical Oversight: Medical professionals must adhere to local regulations, use precise dosing, and employ sterile techniques to minimize risks.

Ongoing research is crucial to address safety concerns and validate therapeutic claims, particularly given the polarized views on ozone therapy’s legitimacy.

Future Directions

To fully realize ozone therapy’s potential, future research should prioritize:

• Large-Scale Clinical Trials: Conducting randomized controlled trials to establish efficacy and safety across conditions, addressing current evidence gaps.
• Standardized Protocols: Developing consistent dosing and administration methods to ensure reproducibility and safety.
• Long-Term Effects: Investigating chronic effects, especially for antiaging, to assess sustainability and cumulative benefits.
• Synergistic Therapies: Exploring combinations with other treatments (e.g., antioxidants, regenerative therapies) to enhance outcomes [6].

These efforts will help integrate ozone therapy into evidence-based medicine, clarifying its role and addressing regulatory concerns.

Conclusion

Ozone therapy offers a promising yet controversial approach to managing chronic diseases and potentially delaying aging. Its biochemical mechanisms—centered on Nrf2 activation, NFκB suppression, and HIF-1α stimulation—provide a scientific basis for its effects, supported by small-scale studies in conditions like rheumatoid arthritis, wound healing, and circulatory disorders. For antiaging, its ability to reduce oxidative stress, enhance mitochondrial function, and support immune health holds potential, though robust evidence is lacking. Given the FDA’s caution and risks like toxicity at high doses, clinicians must approach ozone therapy with caution, adhering to local regulations and evidence-based practices. By staying informed about ongoing research, medical professionals can responsibly evaluate its place in modern medicine, balancing its potential benefits with its limitations.

References

1. U.S. Food and Drug Administration. FDA Ozone Medical Devices Guidance. Compliance Policy Guide Sec. 395.7. FDA Guidance
2. Bocci V. Biological and clinical effects of ozone. Br J Biomed Sci. 1999;56(4):270-9. PubMed
3. Zanardi I, et al. Biological and Molecular Action of Ozone. Int J Mol Sci. 2023;24(10):8465. DOI
4. Hernández F, et al. Antioxidative response in cardiopathy patients. Free Radic Biol Med. 1995;19(1):115-9. DOI
5. Scassellati C, et al. Ozone: a natural bioactive molecule with antioxidant property. Ageing Res Rev. 2020;63:101138. DOI
6. Clavo B, et al. Effect of ozone therapy on muscle oxygenation. J Altern Complement Med. 2003;9(2):251-6. DOI
7. Bocci V. Ozone as a bioregulator. Mediators Inflamm. 2007;2:45384. DOI
8. Wells KH, et al. Inactivation of HIV by ozone in vitro. Blood. 1991;78(7):1882-90. DOI
9. Carpendale MT, Freeberg JK. Ozone inactivates HIV at noncytotoxic concentrations. Antiviral Res. 1991;16(3):281-92. DOI
10. Bocci V. Ozone therapy normalizes cellular redox balance. Med Hypotheses. 1996;46(2):150-4. DOI
11. Bocci V. Ozone is it always toxic?. Toxicol Appl Pharmacol. 2006;216(3):493-504. DOI
12. Washutti J, et al. The use of Ozone in Medicine. Ozone Sci Engg. 1989;11:411-7. DOI
13. Hernández F, et al. Ozone therapy for rheumatoid arthritis. Arch Med Res. 2008;39(6):588-94. DOI
14. Sunnen GV, et al. Ozone therapy in hepatitis B and C. Ozone Sci Engg. 1999;21(1):1-10. DOI
15. Werkmeister HM, et al. Ozone therapy in arterial insufficiency. Angiology. 1994;45(4):287-94. DOI
16. Sweet F, et al. Ozone inhibits growth of cancer cells. Science. 1980;209(4459):931-3. DOI
17. Martínez-Sánchez G, et al. Ozone therapy for chronic pyelonephritis. Arch Med Res. 2009;40(5):388-94. DOI
18. de Girolamo L, et al. Intra-articular ozone in osteoarthritis. J Orthop Surg Res. 2019;14(1):118. DOI
19. Huth KC, et al. Ozone against endodontopathogenic microorganisms. J Endod. 2009;35(4):505-9. DOI
20. Werkmeister HM, et al. Ozone in peripheral obstructive arterial disease. Angiology. 1995;46(12):1071-8. DOI
21. Martínez-Sánchez G, et al. Ozone therapy in diabetic foot. Eur J Pharmacol. 2005;523(1-3):151-61. DOI
22. Menéndez S, et al. Topical ozone in cutaneous wound healing. Indian J Dermatol Venereol Leprol. 2010;76(6):669-74. DOI
23. Elvis AM, Ekta JS. Ozone therapy: A clinical review. J Nat Sci Biol Med. 2011;2(1):66-70. PMC3312702
24. Bocci V. Ozone therapy: History, physiology, indications. Full Circle Equine; 2010. URL
25. Holmes J. Clinical reversal of root caries using ozone. Gerodontology. 2003;20(2):106-14. DOI
26. Di Paolo N, et al. Ozone therapy editorial review. Int J Artif Organs. 2004;27(3):168-75. PubMed
27. Fernández-Cuadros ME, et al. Ozone therapy in rheumatoid arthritis. Arch Med Res. 2016;47(8):645-52. DOI
28. Menéndez S, et al. Ozone therapy and liver function in RA. J Clin Rheumatol. 2012;18(4):192-5. DOI
29. Borrelli E, et al. Intra-articular ozone for knee osteoarthritis. J Orthop Res. 2015;33(11):1656-62. DOI
30. Cespedes-Suarez J, et al. Ozone therapy in hepatitis B and C. J Viral Hepat. 2018;25(6):683-91. DOI
31. Nagayoshi M, et al. Ozone in dental applications. J Dent. 2008;36(6):429-34. DOI
32. Garber GE, et al. Ozone therapy in HIV: No clinical benefit. AIDS. 1991;5(8):981-4. PubMed
33. Clavo B, et al. Ozone therapy in POAD. Angiology. 2007;58(1):88-94. [DOI](https://doi

Links of the above publications

1.   FDA Ozone Medical Devices Guidance
2.   Biological and clinical effects of ozone
3.   Biological and Molecular Action of Ozone
4.   Antioxidative response in cardiopathy patients
5.   Ozone: a natural bioactive molecule with antioxidant property
6.   Effect of ozone therapy on muscle oxygenation
7.   Ozone as a bioregulator
8.   Inactivation of HIV by ozone in vitro
9.   Ozone inactivates HIV at noncytotoxic concentrations
10.   Ozone therapy normalizes cellular redox balance
11.   Ozone is it always toxic?
12.   The use of Ozone in Medicine
13.   Ozone therapy for rheumatoid arthritis
14.   Ozone therapy in hepatitis B and C
15.   Ozone therapy in arterial insufficiency
16.   Ozone inhibits growth of cancer cells
17.   Ozone therapy for chronic pyelonephritis
18.   Intra-articular ozone in osteoarthritis
19.   Ozone against endodontopathogenic microorganisms
20.   Ozone in peripheral obstructive arterial disease
21.   Ozone therapy in diabetic foot
22.   Topical ozone in cutaneous wound healing
23.   Ozone therapy: A clinical review
24.   Ozone therapy: History, physiology, indications
25.   Clinical reversal of root caries using ozone
26.   Ozone therapy editorial review
27.   Ozone therapy and liver function in RA
28. Menéndez S, et al. Ozone therapy and liver function in RA. J Clin Rheumatol. 2012;18(4):192-5.
29.   Intra-articular ozone for knee osteoarthritis
30.   Ozone therapy in hepatitis B and C
31.   Ozone in dental applications
32.   Ozone therapy in HIV: No clinical benefit
33.   Ozone therapy in POAD

Disclaimer:
This article on ozone therapy has been authored by Dr. Mitra Basu Chhillar, M.D., Medical Director, SOMA Wellness Clinic, Mumbai, with the intention of sharing insights based on scientific literature, clinical experience, and current global practices in functional and regenerative medicine. It is meant solely for educational and informational purposes for medical professionals, researchers, and interested readers.

The content herein does not constitute medical advice or endorsement of any specific therapy. Ozone therapy remains a complementary modality in many countries and is not universally approved by regulatory authorities such as the FDA (USA), EMA (Europe), or CDSCO (India). Readers must consult appropriate legal, medical, and regulatory guidance before considering or implementing any protocols discussed.

While every effort has been made to ensure accuracy and scientific integrity, the author disclaims all liability for any medical decisions, outcomes, or misinterpretations arising from the use of the information in this article. Clinical applications of ozone therapy should always be performed by trained professionals under appropriate medical supervision, using standardized protocols and safety measures.