Nrf2 and Oxidative Stress

3.1.

Oxidative stress is due to the accumulation of reactive oxygen species (ROS) (O2−, H2O2 and •OH), that are generated as by-products of either physiological or exogenous stress factors (such as, e.g., the ionizing radiations). Although ROS have been recognized in the last years as functionally significant signalling molecules for the modulation of the immune system, they have long been seen as harmful factors with detrimental effect on cell homeostasis. For example, ROS are strong inducers of DNA damage [96] that irreversibly compromises cell functions and might stimulate neoplastic transformation [97]. Nrf2 prevents oxidative stress through the transcription of antioxidant enzymes, such as the catalytic and modulatory subunits of glutamate cysteine ligase (GCL), glutathione peroxidases (GPX2 and GPX4), glutathione reductase (GSR), peroxiredoxins (PRDX1 and PRDX6), thioredoxin 1 and thioredoxin reductase 1 (TXN1 and TXNRD1), HMOX1, and biliverdin reductase (BVR) [87,98,99,100]. Accordingly, we demonstrated that mild ozonisation induces modulation of genes involved in the cell response to stress (HMOX1; excision repair cross-complementation group 4, ERCC4; cyclin-dependent kinase inhibitor 1A, CDKN1A) and in the transcription machinery (CTD small phosphatase 1, CTDSP1) [101].
Oxidative stress is one of the major drivers of protein misfolding as it induces protein oxidation.
Misfolded or unfolded proteins accumulate as insoluble inclusions and aggregates in the cytoplasm and the cell nucleus, and are the hallmark of multiple aging-related neurodegenerative and metabolic disorders
3.3. Nrf2 and the Mitochondrial Function

The classic view of mitochondria as semi-independent organelles has recently been integrated by the evidence that, in response to environmental changes, they may coordinate inter-organelle signalling pathways (mitochondrial retrograde response) that ultimately instruct nuclear gene expression [109,110,111]. Maintenance of an efficient mitochondrial function is crucial to preserve cell homeostasis, as proven by the evidence that mitochondrial stimulation by mild-stress treatments (mitohormesis) [112] underlies the beneficial effects of life-extending interventions such as dietary restriction [113,114]. In contrast, mitochondrial dysfunction is ontologically linked (as either cause, con-cause or consequence) to aging and aging-related diseases [115,116]. Nrf2 has been shown to stimulate mitochondrial biogenesis through the activation of nuclear respiratory factor-1 (NRF-1) in cardiomyocytes [117,118]. Cellular respiration and ATP synthesis are impaired in conditions of Nrf2 deficiency and increased upon Keap1 loss of function [119,120]. Nrf2 is functionally linked through a positive feedback loop to Sqstm1/p62, which localizes to the mitochondria and enhances the mitochondrial transcription factor A (TFAM), a master regulator of mitochondrial biogenesis [121,122]. In addition, Nrf2 has been identified as a crucial mediator of the mitochondrial biogenesis induced by acetyl-carnitine (ALCAR), nitric oxide (NO) and resveratrol [123,124]. Accordingly, mild ozonisation was found to affect mitochondria by increasing the length of the mitochondrial cristae and the content of mitochondrial heat-shock protein 70 [125], while O3 treatment was proven to reduce mitochondrial damage in a rat heart following ischemia-reperfusion [73] as well as in a rat brain and cochlea following noise-induced hearing loss [126].
3.6. Nrf2 and Cancer

Unlike the vast majority of diseases that have an often unique pathogenic cause, cancer can be defined as multitude of possible pathologic states sharing the capability to subvert and redirect the regeneration and differentiation potential of cells and tissues toward abnormal limitless proliferation and growth. The Nrf2-mediated regulation of the biological processes described above has proven to exert beneficial effects in preventing, ameliorating or curing a multitude of diseases, but might turn detrimental in a cancer-related context. Nrf2 preserves cells from DNA damage, and therefore might help preventing the primary trigger of neoplastic transformation. However, hyperactivation of Nrf2 has been shown to support tumour progression by multiple ways [145,146]: for example, it may help incipient tumour cells to overcome oxidative stress that represents a barrier against neoplastic transformation and cancer initiation [147]. Also, Nrf2 hyperactivation supports aberrant cell proliferation by both inducing the metabolic switch towards anabolic pathways [148] and modulating mRNA translation [149]. Moreover, Nrf2 may promote tumour angiogenesis [150]. Finally, the potent cytoprotective effect of Nrf2 activation may confer drug resistance to cancer cells [151]. Thus, the possible tumour-promoting effect of Nrf2 hyperactivation still remains a crucial issue, since oncological patients are frequently administered O3 therapy due to its efficacy in reducing some adverse side-effects of the anti-cancer treatments [46,152,153].
As a whole, the direct and indirect molecular targets of Nrf2 delineates a complex network of biological processes that preserve cell homeostasis and promote cell reparative programs following chemical, physical, or biological stress [154]. The complexity of the Nrf2 functional network does not allow drawing an exhaustive molecular model that might explain the beneficial effects of O3 in preventing or ameliorating diseases. Nrf2 activation exerts positive effects especially on diseases that have oxidative stress and inflammation as primary etiopathological events [155,156].
Therefore, it may be hypothesized that the therapeutic potential of Nrf2 activation as a consequence of mild ozonisation relies on the capability of Nrf2 to maintain redox homeostasis: this would prevent DNA damage, preserve proteostasis, and improve mitochondrial function while suppressing acute and chronic inflammation.