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From the Townsend Letter
October 2015

Nrf2 Is a Master Regulator of Cytoprotective Responses Including Antioxidant, Anti-Inflammatory, Detoxification, Improved Mitochondrial Function, and Autophagy
by Martin L. Pall, PhD, and Stephen Levine, PhD
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Nrf2 (nuclear factor erythroid-2; a name derived from its role in erythropoiesis) has been known for some 15 years to be an important transcriptional activator of antioxidant genes, producing therefore important antioxidant protective responses. It has also been known for about 15 years to be activated by many (but not all) phenolic antioxidants, so that much of the antioxidant effects of these compounds are produced through this regulatory response, rather than exclusively through direct antioxidant chemistry. However, it has been shown in recent years that the cytoprotective effects of Nrf2 go far beyond antioxidant effects. They include anti-inflammatory effects, detoxification mechanisms for a wide variety of xenobiotic toxicants, improved mitochondrial function, and autophagy, a process by which both toxic protein aggregates and dysfunctional organelles can be degraded. And it has also been shown in recent years that many health-promoting factors other than phenolic antioxidants act to raise Nrf2 activity. Most of these recent findings have been reviewed in a whole series of recent reviews, and it is the role of this article to summarize the vast scope of these new findings, including the health-promoting and disease-preventing effects of Nrf2.1-22

Diseases Prevented and/or Treated by Raising Nrf2, at Least in Animal Models
There are an amazing number of diseases (Table 1) that have been shown to be prevented and/or treated by raising Nrf2. Most of these studies have been done in animal models, although there are also an increasing number of human studies being reported.

Table 1: Diseases wherein Raising Nrf2 Appears to Be Useful in Prevention and/or Treatment in Animal Models and/or Humans (pdf)
The finding that raising Nrf2 is useful in prevention and/or treatment of this list of diseases may seem surprising – almost too good to be true. However, these diseases all have both oxidative stress and inflammatory aspects to them, and many of them are also known to involve mitochondrial dysfunction. Protein aggregates have causal roles of several of them, aggregates that may be removed by Nrf2-dependent autophagy. One of us (MLP) has argued that many of these diseases are caused by the NO/ONOO(−) cycle, a vicious-cycle mechanism involving oxidative stress, inflammation, and mitochondrial dysfunction, as well as other factors. It is therefore quite plausible that because of the common factors involved in these diseases, the Nrf2 regulatory response may prevent and/or treat each of them.

Gene Activation via Nrf2
Nrf2 is most known for its role in activation of genes having antioxidant effects, acting by binding in the nucleus, along with some other proteins known as Raf to what are called antioxidant response elements (AREs) in the promoter regions of genes. However, these AREs occur not only in promoter regions of antioxidant genes but also genes involved in other functions. While over 500 genes are activated by Nrf2, there are also genes whose activity is lowered by Nrf2, some of which may be regulated by transcription factors regulated by Nrf2 and others through AREs having repressive effects.4

Nrf2-Dependent Antioxidant Effects
Among the antioxidant genes activated by Nrf2, one of the most commonly studied is the heme oxygenase 1 (HO-1) gene which converts free heme, which has prooxidant effects, into iron, carbon monoxide (CO), and biliverdin, with the last being converted into the antioxidant bilirubin via an activity also raised by Nrf2, the two biliverdin reductase genes.1,2 The iron produced by heme oxygenase is sequestered by ferritin, since Nrf2 induction of 4 ferritin genes, preventing iron-produced oxidative stress.1 This coordinate control of multiple genes producing proteins that are functionally linked in producing an important biological response has been found repeatedly in Nrf2-mediated gene regulation. There are also antioxidant responses produced by CO from its regulatory role that will not be considered here.
A second commonly studied antioxidant gene activated by Nrf2 is the quinone oxidoreductase gene (NQO1), which produces an enzyme that prevents semiquinone redox cycling and consequent oxidative stress.2 Other antioxidant genes activated by Nrf2 are two superoxide dismutase genes (SOD1 and SOD2), which lower oxidative stress by lowering superoxide and also the functionally linked catalase and two glutathione peroxidase genes, each of which lowers H2O2, produced from superoxide by the SODs. So again we see coordinate regulation of multiple antioxidant genes.
Reduced glutathione (GSH) has often been described as the most important low molecular weight antioxidant produced in the human body. Each of the three genes encoding enzymes required for the de novo synthesis of GSH is activated by Nrf2, as is the gene for glutathione reductase (the enzyme that converts oxidized glutathione [GSSG] to GSH). Genes encoding 8 enzymes that have roles in the synthesis of NADPH, the reductant needed by glutathione reductase are also activated by Nrf2. Other enzymes that have roles in using GSH for antioxidant purposes, including two glutathione peroxidase genes (discussed in the previous paragraph), and the glutaredoxin 1 gene are Nrf2 activated.

Five genes involved in thioredoxin-related antioxidant responses are activated by Nrf2, including peroxiredoxin-1 and -6 which destroy peroxynitrite, an extremely reactive oxidant responsible for nitrosative stress.1 In summary, it can be seen from the above that there are 23 genes involved in antioxidant protection, each of which is activated by Nrf2. There are in addition still other genes activated by Nrf2 that help remove toxic products of lipid peroxidation and still others similarly regulated that help remove products of oxidative DNA damage in the process of DNA repair.

Anti-Inflammatory Effects of Nrf2
Nrf2 activation produces a wide variety of anti-inflammatory effects, including lowered NF-kB and lowered activity of a series of inflammatory mediators, including cytokines, chemokines, adhesion molecules, COX-2, MMP-9, and iNOS.15,16 Many of these changes may be indirect effects produced by raising antioxidant responses, but there are also direct anti-inflammatory effects such as raising the anti-inflammatory cytokine IL-10.5

Detoxification Genes Activated by Nrf2
Hayes and Dinkova-Kostova list a total of 25 different genes activated by Nrf2, each of which has a role in detoxification of various toxic xenobiotics.1 Nrf2 has a wide range of detoxification effects, producing increased resistance to toxic xenobiotics.

Mitochondrial Biogenesis and Autophagy
The diseases listed in Table 1 are also characterized by energy metabolism and mitochondrial dysfunction, such that one of the mechanisms that may be included as cytoprotective may be increased mitochondrial biogenesis. Such increased mitochondrial biogenesis has been shown to be produced by Nrf2 activation, acting in part by activating a related gene, Nrf1.20 A large number of other genes involved in energy metabolism are also activated by Nrf2.1
It is also the case that a number of health-promoting nutrients that stimulate Nrf2 also act to increase the process of autophagy by which damaged organelles and also damaging protein aggregates can be degraded proteolytically, with such autophagy occurring in part via a Nrf2-dependent process. This stimulation of autophagy, useful in removing damaged mitochondria, is also useful in removing protein aggregates that have roles in neurodegenerative and other diseases and has antioxidant roles are well. However, it should be noted that autophagy is inhibited by very high levels of Nrf2. It follows from this that Nrf2-dependent autophagy may be useful as a cytoprotective response in multiple ways, one of which has roles in improving mitochondrial function.

Nrf2 Activity Is Raised by Many Health-Promoting Nutrients and Other Factors
The amazing list of health-promoting factors that have been shown to act, at least in part, by raising Nrf2 are shown in Table 2.

Table 2: Health-Promoting Factors That Act to Raise Nrf2 Activity (pdf)
Each of the nine factors listed in Table 2 has an extensive literature on its health-promoting effects. Although all nine have been shown to raise Nrf2 activity, several of these can clearly act in other ways not involving Nrf2 to promote health.
For example, four of the nutritional factors are well established to act independently of Nrf2 as follows:

  • Phenolics can act as chain-breaking antioxidants.
  • Carotenoids can act as scavengers of singlet oxygen and peroxynitrite.
  • Fish oil has anti-inflammatory properties by acting as precursors of eicosanoids.
  • Exercise can act in ways independent of Nrf2.

However, each of these 9 factors, when tested in Nrf2−/− mouse knockout mutants, has been shown to have lost most of its health-promoting properties as compared with their activity in Nrf2+/+ mice.32-39 This shows, therefore, that much of their health promotion requires the presence of a functional Nrf2 gene, at least in the mouse. Other cell culture studies on these nutritional factors have also supported an important role for Nrf2 elevation in response to these factors.
Three of these classes of chemicals act via oxidation products to raise Nrf2 levels. The long-chain omega-3 fatty acids DHA and EPA act via their oxidation product 4-hydroxy hexenal to raise Nrf2.27,28 The carotenoids act, primarily and possibly entirely, via their oxidation products to raise Nrf2.25,26 And the phenolic antioxidants are thought to act via their quinone and semiquinone oxidation products in raising Nrf2.1-6 This pattern is important because it means that each of these will be most active in raising Nrf2 in tissues that are under oxidative stress and therefore in most need of raising Nrf2 to protect themselves from the consequences of oxidative stress and other related cell damage.

The Two Most Healthful Known Diets, the Traditional Mediterranean Diet and the Traditional Okinawan Diet, Are Both Rich in Nrf2 Activating Nutrients
The traditional Mediterranean diet, thought to be ideally described as the Cretan diet of the 1960s, and the traditional Okinawan diet of the same time period, are thought to be the most healthful diets known, with high overall lifespans, large numbers of centenarians, and low incidences of cancer and cardiovascular disease.40-46 Diets in both of these locations are thought to have become considerably less healthful in recent decades, but studies of these two traditional diets are still important parts of our understanding of dietary factors that may influence human health. The question being raised here is whether nutrients raising Nrf2 activity in these diets are likely to have an important role in their health-promoting properties.
The dietary factors which raise Nrf2 (Table 2) are all of plant origin except for the long-chain omega-3 fatty acids, which are best obtained from seafood. Consequently, it may be argued that the best diets for raising Nrf2 are those with regular seafood consumption but otherwise containing large amounts of foods derived from plants, particularly plants with low calorie densities which are likely to be consumed in larger quantities and therefore provide in general more phytochemicals. Both the traditional Mediterranean and Okinawan diets clearly fit this description.40-46 Furthermore, several of the nutrient categories known to raise Nrf2 listed in Table 2 are thought to be high in each of these diets (see Table 3).

Table 3: Overall Apparent Consumption of Nrf2 Raising Nutritional Components40-46 (pdf)
It can be seen from Table 3 that both of these health-promoting diets are very rich in nutritional components that raise Nrf2, including five of the six types of Nrf2-activating components listed in Table 3. In addition, the traditional Mediterranean diet is most characterized by high consumption of olives and olive oil, which are known to contain very high levels of phenolics and terpenoids, both of which have been shown to raise Nrf2. The main caloric source in the Okinawan diet is the sweet potato, often including purple sweet potatoes; all sweet potatoes are very high in carotenoids, and purple sweet potatoes are very high in anthocyanin phenolics, which are potent Nrf2 activators.40 Murakami et al. showed that a large number of specific vegetables in the traditional Okinawan diet are potent agents that lower the production of both superoxide and nitric oxide in leukocytes, suggesting agents that act in part by raising Nrf2. In some cases, they implicated both phenolics and terpenoids in producing these responses, again suggesting a possible Nrf2 effect.41 While it is unlikely that all of the phytochemicals that may produce health-promoting effects in these two diets are acting mainly or solely via Nrf2, it may be the case that Nrf2 has a major role in the health promotion in each of these two diets.
The Okinawan diet is thought to be very similar to what is often called the Paleolithic diet, the diet that our ancestors ate during much of human evolution.47 The only substantial difference is that in the Paleolithic diet, most of the omega-3 fatty acids come from wild animals and plants, both of which are quite rich in omega-3 fatty acids, rather than more substantially from fish.48 Specifically, the Okinawan diet closely resembles the Paleolithic diet in having very high levels of phenolic and carotenoid antioxidants as well as high omega-3 levels, probably also terpenoids and essentially no grain consumption, all of which will act to raise Nrf2. It may be argued that we evolved with much higher levels of Nrf2-raising nutrients in our diets and that almost all of us are currently in a dietary deficiency state for Nrf2-raising nutrients. This may in turn be responsible for much of the extraordinary predominance of chronic diseases afflicting modern populations, characterized by oxidative stress, inflammation, and mitochondrial dysfunction.

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