The usually objective Wikipedia online encyclopedia had an author that declared ozone therapy “pure quackary.” This post will explore the interaction between ozone therapy and the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) transcription factor. The Wikipedia author failed to understand fundamentals of how low levels of a toxin can be protective. As the post suggests, the real crux of this post is how ozone might react with thiols in a protein called Keap1 that targets the transcription factor Nrf2 for degradation. This post will direct the reader to some good reviews regarding ozone therapy. The take home message is that we have a very good idea of how ozone therapy might be working even if governments have so far failed to fund the research to take ‘quackery” to an established medical science.
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
The Jaramillo reveiw  has a nice cartoon in Figure 2 that makes sense to present first in illustrating how ozone might be able to sense ozone and other thiol reactive compounds like nitosating agents and H2O2 .
The UniProt database has some more interesting things to say about Keap1:
- is asubstrate-specific adapter of a BCR an E3 ubiquitin ligase complex that regulates the response to oxidative stress by targeting NRF2 for ubiquitination.
- n response to oxidative stress, different electrophile metabolites trigger non-enzymatic covalent modifications of highly reactive cysteine residues in KEAP1, leading to inactivate the ubiquitin ligase activity of the BCR(KEAP1) complex, promoting NFE2L2/NRF2 nuclear accumulation and expression of phase II detoxifying enzymes
- n response to selective autophagy, KEAP1 is sequestered in inclusion bodies following its interaction with SQSTM1/p62, leading to inactivation of the BCR(KEAP1) complex and activation of NFE2L2/NRF2
- The BCR(KEAP1) complex also mediates ubiquitination of SQSTM1/p62, increasing SQSTM1/p62 sequestering activity and degradation
- Ubiquitin ligase activity of the BCR(KEAP1) complex is inhibited by oxidative stress
- Electrophile metabolites react with reactive cysteine residues in KEAP1 and trigger non-enzymatic covalent modifications of these cysteine residues, leading to inactivate the ubiquitin ligase activity of the BCR(KEAP1) complex
- KEAP1 contains reactive cysteine residues that act as sensors for endogenously produced and exogenously encountered small molecules, which react with sulfhydryl groups and modify the cysteine sensors, leading to impair ability of the BCR(KEAP1) complex to ubiquitinate target proteins.
Keap1 ozone reactive cysteines?
2010, HeLa cell line..
No study was found on ozone modified cysteines but a French group tested these thio reactive species 
- 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. 
- hydrogen peroxide (H2O2) 200 μm
- hypochlorous acid (HOCl) 1 mM
- S-nitroso cysteine 0.5 mM
These authors transfected the HeLa human cell line with the gene for murine Keap1 and a chirmeric version of Nrf2 with an added twist. Twenty three of the 25 cysteines of Keap1 were singly mutated to the hydrophilic amino acid serine. Four were the double mutations C513S/C518S and C622S/C624S. The other 19 were single mutations. The goal was to determine which of these cysteines were prone to oxidation.  Co-expression of Keap1 with Nrf2 decreased the stability of Nrf2. The Cys226 and Cys613 pair was found to form a intra molecular disulfide bond that was non essential for function. Cys 151, on the other hand, formed an inter-molecular disulfide bond resulting in an Nrf2 sparing homo-dimer.
2016 transgenic mice
by 2015 it was well known that KEAP1C273S, KEAP1C273A, KEAP1C288S, and KEAP1C288A were unable to repress NRF2 activity. For this reason a group out of todohu University in Japan created Cys273Trp and Cys288Glu mutations in mouse Keap1 in a transgenic mouse model. CRISPR techology was used to introduce Keap1 Keap1C151S, Keap1C288E , Keap1C273W&C288E knock-in mice.  A grossly abbreviated Figure 3 is shown to avoid the lethal nature of the double Keap1 knockout.
Fig 3 . (A) A schematic presentation of KRD-Keap1C273W&C288E transgene that expresses Keap1 under the regulation of KRD is shown. (B) Growth curves for Keap1−/−, Keap1−/−::Tg-Keap1WT mice (line 34) and Keap1−/−::Tg-Keap1C273W&C288E mice (line 30).
These authors performed many experiments in primary cell lines derived from these mice that this post will not attempt to describe.
Diethylmaleate (DEM), dimethyl formamide (DMF), 4-Hydroxy-nonenal (4-HNE) and (±)-S-nitroso-N-acetylpenicillamine (SNAP), l-Sulforaphane (SFN), tert-butyl hydroquinone (tBHQ)
15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2),
sodium metaarsenite (NaAsO2), 9-nitro-octadec-9-enoic acid (9-OA-NO2) , 4-Hydroxynonenal
cadmium chloride (CdCl2), zinc chloride (ZnCl2), dexamethasone 21-mesylate (Dex-Mes), and hydrogen peroxide (H2O2), prostaglandin A2 (PGA2),
What redox enzymes does Nrf2 induce transcription of?
Supplemental Table 1 of the Ma review 
|Antioxidant defense||regeneration of redox factor||synthesis of reducing factor||Redox Transport|
|Cu/Zn superoxide dismutase 3 SOD3||Glutathione reductase GSR1 ||Glutamate-cysteine ligase GCLC (catalytic) GCLM (regulatory)||Cystine/glutamate transporter SLC7A11|
|glutathione peroxidase GPx2||Thioredoxin reductase TrxR1||Glucose-6-phosphate dehydrogenase G6PDH (m) ARE (3)|
|Peroxiredoxin, Prx1||Sulfiredoxin Srx1||Phosphogluconate dehydrogenase 6PGD|
|Thioredoxin Trx (h) ARE|
|Thioredoxin interacting protein TXNIP|
Two ozone reviews
An overview of ozone in medicine 
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. 
- 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
This is a very nice review that did not seem to really get into potential side effects Another paper looked at what some might consider side effects as part of the therapy.
Ozone and lipid peroxidation product signalling…
The Cenci review was written from the perspective of using ozone to treat SARS-Cov2. Some topics covered in this review include
- A Mechanistic Insight mostly concerning ozone’s reaction with poly unsaturated fatty acids. The reader is invited to consult the Wikipedia page on the teneration of 4-hydroxy nonenol from poly unsaturated fatty acids.
- O3-Derived H2O2 and Cellular Redox Signaling.. This section describes singlet oxygen water oxidation that leads to hydrogen peroxide..
- O3-Derived LOPs Biological Activity This section describes activation of phodpholipases C and A2 and down stream signaling.
- Antioxidant Effect of O3 Nrf2 is introduced in this section
- Anti-inflammatory and Immunomodulating Effects of O3 in Cells and Tissues discusses inhibition of the NLRP3 subunit of the inflammasome.
- Effect Ozone on Viruses: Action on SARS-CoV-2 and cytokines…
- Ozone and the microbiota
- Environmental Ozone and SARS-CoV-2
One neglected aspect of the extremely thorough Cenci is that 4HNE and some prostaglandins target thiols in Keap1 
To establish ozone as a drug, one needs to have a pretty good idea of the main target. The main target seems to be Keap1, the repressor of the anti-oxidant transcription factor Nrf2. All drugs have hopefully minor side targets. Some of the ozone side targets also feed forward on to keap1 and its target nrf2. These attributes make ozone extremely exciting as a drug. However the clinician must have some way of determining when the therapeutic window has been reached. To use the 1993/2001 attacks on the World Trade Center, how much tissue damage is to be considered tolerable with ozone therapy?
- Jaramillo MC, Zhang DD. The emerging role of the Nrf2-Keap1 signaling pathway in cancer. Genes Dev. 2013 Oct 15;27(20):2179-91. PMC free articlehttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814639/
- 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
- 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
- Ma Q. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. 2013;53:401-26. Supplemental Table
- 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
- Cenci A, Macchia I, La Sorsa V, Sbarigia C, Di Donna V, Pietraforte D. Mechanisms of Action of Ozone Therapy in Emerging Viral Diseases: Immunomodulatory Effects and Therapeutic Advantages With Reference to SARS-CoV-2. Front Microbiol. 2022 Apr 21;13:871645. PMC free article