Ozone Therapy

This post explores the interaction between ozone therapy and the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) transcription factor. Ozone might react with thiols in a protein called Keap1 that targets the transcription factor Nrf2 for degradation. Nrf2 is responsible for transcription of genes whose proteins code for enzymes that protect against oxidative stress.
PEMF therapy has been performed in conjunction with ozone therapy. Magnetic and electric fields have been used in conjugation with ozone for the purpose of chemical degradation of dyes in waste water. What could possibly be the connection? Ozone has zero unpaired electrons and is considered to be diamagnetic. Libre Chemist was consulted to answer this question.

Ozone and EMF [1]

Is ozone and PEMF being used to treat any condition after this first was first completed? Yes a group out of Vercruz Mexico treated three females with fibromyagia as follows:

• PEMF Therapy: The PEMF device (EIMA, Magnetic Inductive Stabilizer) was applied systemically. During each session, the patient remained seated with the forearm placed inside the applicator for a duration of 45 min. Using a frequency of 60 Hz, a semi-sinusoidal wave form, with a duty cycle of 12% in a repetitive single-pulse mode (true pulsed mode). The treatment was administered using a magnetic flux of 0.1968 Tesla (equivalent to 4.92 mT/kg), twice weekly.
• Ozone Therapy: Rectal insufflation of 100–200 mL of medical-grade ozone at a concentration of 28 μg/mL, administered twice weekly. The ozone concentration was fixed at 28 μg/mL for all patients throughout the treatment, following protocols established by the Scientific Society for Oxygen-Ozone Therapy. The volume of the gas mixture (ozone/oxygen) was individually titrated based on patient tolerability and colonic capacity, starting at 100 mL and gradually increasing to a maximum of 200 mL by the end of the treatment course, as tolerated.
• Supplementation: Daily Omega-3 fatty acids and Vitamin D3.

Following the intervention, patients experienced substantial pain relief and significant improvement in quality of life, which were sustained at the 30-day follow-up assessment. These findings suggest that the combination of PEMF and ozone therapy may induce durable physiological benefits, presenting a promising complementary strategy for managing refractory fibromyalgia.

In a rat model of fatty liver disease treated with 15 Hz and 1.6 mT PEMF. PEMF EMF may activate the CaMKKβ/AMPK/SREBP-1c and Nrf2 pathways to control hepatic lipid deposition and oxidative stress. PMC free paper.

In subsequent posts since 2023 I wrote on the concept of hormesis. This section illustrates the concept better than what I did in subsequent years.

A parallel in Human Society

Acccordng to history.com a truck bomb explored in the parking garage in the north tower of the world trade center. Only six people were killed in this initial attack while 1000 had to be evacuated due to the smoke.

Image of the 1993 attack on the north tower of the World Trade Center from history.com

CBS news reports that it took ten hours to evacuate after the 1993 attack because fire fighter’s radios were not receiving signals, stairwells had gone black, Radio repeaters were installed in the twin towers and batteries in the stairwell lights. CBS news claims that those who were evacuated immediately after both the 1993 and 2001 attacks said the second evacuation went smoother. .. In a very relative way! Many people died or were injured for life in the 1001 attack. Had there been biannual attacks on the World Trade Center killing half a dozen people, we can bet that less people would have died in the 2001 attack. The Wikipedia author who declared ozone therapy “quackery” simply had no concept of how a small attack can cause a cell or whole organism prepare for a major attack. The “devil in the details comes in how much of a small attack is needed to instate adequate defense measures.

Keap1, an adaptor protein that keeps Nrf2 turned off [1]

The Jaramillo review [1] has a nice cartoon in Figure 2 that makes sense to present first in illustrating how Keap1 might be able to sense ozone and other thiol reactive compounds like nitosating agents and H₂O₂ [2].

Keap1 ozone reactive cysteines?

Fourquet 2010, mapping Keap1 oxidant reactive thiols.[2].

No study has found on ozone modified cysteines but a French group tested these thio reactive species [2]

• nitric oxide (NO) donor spermidine NONOate 2 mM This is a slow NO releasing compound with a half life of about 2 hours. It had a long lived but modest ncrase in Nrf2 stability. [2]
• hydrogen peroxide (H₂O₂) 200 μm
• hypochlorous acid (HOCl) 1 mM
• S-nitroso cysteine 0.5 mM

1. Fig 1 says that agents that oxidize Keap1 cause some pretty major structural changes that go beyond monomers and disulfide bond cross linked dimers. This is not a really good figure for lay people
2. Fig 2 panel A is a good figure for everyone as it tells the story of just how many cysteines in Keap1 that could be oxidized and regulatory.
3. Fig 3 Keap1 forms disulfide linked homo dimers. Not a lay friendly figure.
4. Fig 4 simply shows that oxidizing Keap1 keeps Nrf2 from being degraded.
5. Fig 5 There are ways of reducing oxidized thiols on Keap1 essentially turning Nrf2 acivation off.

If ozone and/or PEMF were to activate Nrf2, this is one way to prove it.

Saito 2015, more on the many thiols of Keap1 [3]

The Saito 2015 was mapping reactive thiols in Keap1, just like the Fourquet 2010 study. Fig 10 is a good lay summary to ge a feel for how scientists were thinking that there is fine tuning of thiols to oxidants.

An overview of ozone in medicine [4]

In 2019 Galiè and coauthors wrote an excellent review on the many modalities of ozone therapy and made a good case for a link to Nrf2 signalling. The following are the topics covered in the Galiè. [4]

Section 2 covers ozone activation of Nrf2 without naming any one particular thiol.

• 3.1. Nrf2 and Oxidative Stress
• 3.2. Nrf2 and Proteostasis
• 3.3. Nrf2 and the Mitochondrial Function
• 3.4. Nrf2 and Inflammation
• 3.5. Nrf2 and Adipose Biology
• 3.6. Nrf2 and Cancer

Fig 5 is a nice summary Main functional pathways depending on Nrf2 activation induced by low ozone concentrations. Arrows indicate up- (red) or down- (blue) regulation.

Ozone and magnetic fields to oxidize a yellow dye [5]

A quote from the abstract The summary figure is public access.

“At present, there is no systematic research on the synergistic enhancement mechanism of ozone activation by magnetic field and electric field applied to the treatment of high-salinity dye-containing wastewater. On this basis, this study introduced an external magnetic field and electric field into the single ozonation system, and developed a magnetoelectric synergistic activated ozonation process (E-M/O₃). This process can achieve the efficient removal of Reactive Yellow K-RN (K-RN) over a wide pH range and under high-salinity conditions. Compared with the single ozone process, electro/ozone process, and magnetic/ozone process, the E-M/O₃ process achieved a removal efficiency of 90.19% for K-RN within 15 min (pH = 9, electrolyte concentration of 300 mM and process synergistic index of 4.26). Moreover, the E-M/O3 process had the lowest energy consumption, amounting to only 3.33 kW·h·g-1. Based on the radical quenching experiments and electron spin resonance (ESR) tests, hydroxyl radicals (•OH) and singlet oxygen (¹O₂) played a dominant role in the removal of K-RN. Furthermore, by combining reactive species generation pathways, K-RN UV full-wavelength scans, and HPLC-MS analysis, the possible degradation pathways of K-RN were speculated. It was also found that Cl^– could promote the degradation of K-RN in the process, while humic acid (HA), NH4+, and NO3- exerted varying degrees of inhibitory effects on the removal of K-RN. In addition, the E-M/O₃ process exhibited good removal efficiency for various organic dyes and excellent decolorisation performance for printing and dyeing wastewater.”

The perplexing thing is that ozone is diamagnetic, no unpaired electrons. It might be repelled by a magnetic field.

Ozone in magnetic fields?

Oxygen as eight neutrons and electrons. Two of these electrons are in the 1s shell and don’t do much. The other six are in the 2s and 2p shells. This image from Wikipedia Commons does a nice job of illustrating the resonance nature of electrons in ozone. The dashed line in this image tells us that the side oxygens have only half a negative charge over any given time because an electron is resonating between the two.

Libretexts has a good piece on ozone delocalization. Much of this image was adapted from one in the given link. Bonding orbitals in the lowest energy state keep the molecule together. All three orbitals out of phase creates an anti-bonding situation encourage dissolution of the ozone molecule.

I have no clue if pulsed electromagnetic frequencies of any wave form would enhance or diminish the effect of ozone in biological systems. It would be a very interesting exeriment!

References

1. Lara-Reyes JA, Aranda-Abreu GE, Cortijo-Palacios X, Aquino-Gálvez A, Catillejos-López M, Ruiz-Moya EG, Rojas-Durán F. Case Report: Integrative management of refractory fibromyalgia with pulsed electromagnetic fields and ozone therapy: a case series. Front Med (Lausanne). 2026 Apr 15;13:1763506. PMC free paper
2. Fourquet S, Guerois R, Biard D, Toledano MB. Activation of NRF2 by nitrosative agents and H2O2 involves KEAP1 disulfide formation. J Biol Chem. 2010 Mar 12;285(11):8463-71.PMC free article
3. Saito R, Suzuki T, Hiramoto K, Asami S, Naganuma E, Suda H, Iso T, Yamamoto H, Morita M, Baird L, Furusawa Y, Negishi T, Ichinose M, Yamamoto M. Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response. Mol Cell Biol. 2015 Nov 2;36(2):271-84. PMC free article
4. Galiè M, Covi V, Tabaracci G, Malatesta M. The Role of Nrf2 in the Antioxidant Cellular Response to Medical Ozone Exposure. Int J Mol Sci. 2019 Aug 17;20(16):4009. PMC free article
5. Sun Z, Liu G, Lu H, Ji Y, Zhao H, Li X, Wang H, Zhou Z, Sun Y. Synergistic magnetic and electric activation of ozone for the efficient treatment of high-salinity dyeing wastewater over a wide pH range. Environ Technol. 2026 May;47(13):2163-2178. PubMed