Back Clinic Oxidative Stress Chiropractic and Functional Medicine Team. Oxidative stress is defined as a disturbance in the balance between the production of reactive oxygen (free radicals) and antioxidant defenses. In other words, it is an imbalance between the production of free radicals and the body’s ability to counteract or detoxify the harmful effects through neutralization by antioxidants. Oxidative stress leads to many pathophysiological conditions in the body. These include neurodegenerative diseases, i.e., Parkinson’s disease, Alzheimer’s disease, gene mutations, cancers, chronic fatigue syndrome, fragile X syndrome, heart and blood vessel disorders, atherosclerosis, heart failure, heart attack, and inflammatory diseases. Oxidation happens under a number of circumstances:
the cells use glucose to make energy
the immune system is fighting off bacteria and creating inflammation
the bodies detoxify pollutants, pesticides, and cigarette smoke
There are millions of processes taking place in our bodies at any given time that can result in oxidation. Here are a few symptoms:
Fatigue
Memory loss and or brain fog
Muscle and or joint pain
Wrinkles along with grey hair
Decreased eyesight
Headaches and sensitivity to noise
Susceptibility to infections
Choosing organic foods and avoiding toxins in your environment makes a big difference. This, along with reducing stress, can be beneficial in decreasing oxidation.
Neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, affect millions of individuals worldwide. A variety of treatment options are available to treat the symptoms of several neurodegenerative diseases although the results are often limited. Research studies have found that oxidative stress caused by both internal and external factors can be a cause for the development of neurodegenerative diseases. The transcription factor, Nrf2, has been determined to function as a major defense mechanism against oxidative stress. The purpose of the article below is to show the effects of Nrf2 on neurodegenerative diseases.
Modulation of Proteostasis by Transcription Factor NRF2
Neurodegenerative diseases are linked to the accumulation of specific protein aggregates, suggesting an intimate connection between injured brain and loss of proteostasis. Proteostasis refers to all the processes by which cells control the abundance and folding of the proteome thanks to a wide network that integrates the regulation of signaling pathways, gene expression and protein degradation systems. This review attempts to summarize the most relevant findings about the transcriptional modulation of proteostasis exerted by the transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2). NRF2 has been classically considered as the master regulator of the antioxidant cell response, although it is currently emerging as a key component of the transduction machinery to maintain proteostasis. As we will discuss, NRF2 could be envisioned as a hub that compiles emergency signals derived from misfolded protein accumulation in order to build a coordinated and perdurable transcriptional response. This is achieved by functions of NRF2 related to the control of genes involved in the maintenance of the endoplasmic reticulum physiology, the proteasome and autophagy.
Keywords:Neurodegenerative diseases, Unfolded protein response, Proteasome, Ubiquitin, Autophagy, Oxidative stress
Nuclear Factor (erythroid-derived 2)-like 2 (NRF2) is a basic-leucine-zipper protein considered nowadays as a master regulator of cellular homeostasis. It controls the basal and stress-inducible expression of over 250 genes that share in common a cis-acting enhancer termed the antioxidant response element (ARE) [1], [2], [3], [4], [5]. These genes participate in phase I, II and III detoxification reactions, glutathione and peroxiredoxin/thioredoxin metabolism, NADPH production through the pentose phosphate pathway and malic enzyme, fatty acid oxidation, iron metabolism, and proteostasis [6]. Given these wide cytoprotective functions, it is possible that a single pharmacological hit in NRF2 might mitigate the effect of the main culprits of chronic diseases, including oxidative, inflammatory and proteotoxic stress. The role of NRF2 in the modulation of the antioxidant defense and resolution of inflammation have been addressed in numerous studies (reviewed in [7]). Here, we will focus on its role in proteostasis, i.e., the homeostatic control of protein synthesis, folding, trafficking and degradation. Examples will be provided in the context of neurodegenerative diseases.
Loss of Proteostasis Influences NRF2 Activity in Neurodegenerative Diseases
A general hallmark of neurodegenerative diseases is the occurrence of aberrant aggregation of some proteins. Thus, misfolded protein aggregates of ?-synuclein (?-SYN) are found in Parkinson’s disease (PD), ?-amyloid (A?) plaques and hyper-phosphorylated TAU neurofibrillary tangles in Alzheimer’s disease (AD), huntingtin (Htt) in Huntington’s disease (HD), superoxide dismutase 1 (SOD1) and TAR DNA binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS), prion protein (PrP) in spongiform encephalopathies, etc. Protein aggregates can have an impact on several cellular pathways, which in turn may affect NRF2 levels and activity.
Different Layers of Regulation Tightly Control NRF2 Activity
Under physiological conditions, cells exhibit low NRF2 protein levels because of its rapid turnover. In response to different stimuli, NRF2 protein is accumulated, enters the nucleus and increases the transcription of ARE-containing genes. Therefore, management of NRF2 protein levels is a key point that should integrate positive and negative input signals. As we will discuss further, NRF2 is activated by diverse overlapping mechanisms to orchestrate a rapid and efficient response but on the other hand NRF2 could be inhibited, probably in a second phase, in order to switch off its response.
From the classic point of view, activation of NRF2 has been considered as a consequence of the cellular response to oxidant or electrophilic compounds. In this regard, the ubiquitin E3 ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1) plays a crucial role. Molecular details will be further addressed in Section 4.1. In brief, KEAP1 acts as a redox sensor due to critical cysteine residues leading to NRF2 ubiquitination and proteasomal degradation. In addition to this classic modulation, NRF2 is profoundly regulated by signaling events. Indeed, different kinases have been shown to phosphorylate and regulate NRF2. For instance, NRF2 can be phosphorylated by mitogen activated protein kinases (MAPKs), although its contribution to NRF2 activity remains unclear [8], [9], [10], [11]. PKA kinase as well as some PKC isozymes [12], CK2 [13] or Fyn [14] phosphorylate NRF2 modifying its stability. Previous work from our group reported that glycogen synthase kinse-3? (GSK-3?) inhibits NRF2 by nuclear exclusion and proteasomal degradation [15], [25], [26], [27], [28], [29], [30]. The molecular details will be discussed in the Section 4.1. Furthermore, NRF2 is submitted to other types of regulation. For instance, NRF2 acetylation by CBP/p300 increases its activity [17], while it is inhibited by miR153, miR27a, miR142-5p, and miR144 [16], or by methylation of cytosine-guanine (CG) islands within the NRF2 promoter [18].
Impact of Protein Aggregates on NRF2 Regulatory Mechanisms
In this section we will focus in how accumulation of misfolded protein could impact NRF2 activity providing some of the pathways mentioned above as illustrative examples. Firstly, we need to consider that protein accumulation has been tightly linked with oxidative damage. Indeed, misfolded protein accumulation and aggregation induce abnormal production of reactive oxygen species (ROS) from mitochondria and other sources [19]. As mentioned above, ROS will modify redox-sensitive cysteines of KEAP1 leading to the release, stabilization and nuclear localization of NRF2.
Regarding proteinopathies, an example of dysregulated signaling events that may affect NRF2 is provided by the hyperactivation of GSK-3? in AD. GSK-3?, also known as TAU kinase, participates in the phosphorylation of this microtubule-associated protein, resulting in its aggregation, formation of neurofibrillary tangles and interruption of axonal transport (reviewed in [20]). On the other hand, GSK-3? dramatically reduces NRF2 levels and activity as mentioned above. Although not widely accepted, the amyloid cascade proposes that toxic A? oligomers increase GSK-3? activity together with TAU hyper-phosphorylation and neuron death [21], [22]. There are different models to explain how A? favors GSK3-? activity. For instance, A? binds to the insulin receptor and inhibits PI3K and AKT signaling pathways, which are crucial to maintain GSK-3? inactivated by phosphorylation at its N-terminal Ser9 residue [23]. On the other hand, extracellular A? interacts with Frizzled receptors, blocking WNT signaling [24] and again resulting in release of active GSK-3?. In summary, A? accumulation leads to abnormal hyperactivation of GSK-3?, thus impairing an appropriate NRF2 response.
As discussed in the following section, misfolded proteins lead to activation of PERK and MAPKs, which in turn up-regulate NRF2 [31], [8], [9], [10], [11]. Moreover, dysregulated CBP/p300 activity has been reported in several proteinopathies [32] and a global decrease in DNA methylation in AD brains was also shown [33], therefore providing grounds to explore the relevance of these findings in NRF2 regulation.
We and others have observed in necropsies of PD and AD patients an increase in NRF2 protein levels and some of its targets, such as heme oxygenase 1 (HMOX1), NADPH quinone oxidase 1 (NQO1), p62, etc., both by immunoblot and by immunohistochemistry [34], [35], [36], [37], [38], [39]. The up-regulation of NRF2 in these diseases is interpreted as an unsuccessful attempt of the diseased brain to recover homeostatic values. However, another study indicated that NRF2 is predominantly localized in the cytoplasm of AD hippocampal neurons, suggesting reduced NRF2 transcriptional activity in the brain [40]. It is conceivable that the disparity of these observations is related to changes in the factors that control NRF2 along the progressive stages of neurodegeneration.
Three major systems contribute to proteostasis, namely the unfolded protein response (UPR), the ubiquitin proteasome system (UPS) and autophagy. Next, we present evidence to envision NRF2 as a hub connecting emergency signals activated by protein aggregates with the protein derivative machinery.
NRF2 Participates in the Unfolded Protein Response (UPR)
NRF2 Activation in Response to the UPR
Oxidative protein folding in the ER is driven by a number of distinct pathways, the most conserved of which involves the protein disulfide-isomerase (PDI) and the sulfhydryl oxidase endoplasmic oxidoreductin 1 (ERO1? and ERO1? in mammals) as disulfide donor. Briefly, PDI catalyzes the formation and breakage of disulfide bonds between cysteine residues within proteins, as they fold, due to the reduction and oxidation of its own cysteine aminoacids. PDI is recycled by the action of the housekeeping enzyme ERO1, which reintroduces disulfide bonds into PDI [41]. Molecular oxygen is the terminal electron acceptor of ERO1, which generates stoichiometric amounts of hydrogen peroxide for every disulfide bond produced [42]. Peroxidases (PRX4) and glutathione peroxidases (GPX7 and GPX8) are key enzymes to reduce hydrogen peroxide in the ER. When this oxido-reductive system does not work properly, abnormal accumulation of misfolded proteins occurs in the ER and a set of signals named the unfolded protein response (UPR) is transmitted to the cytoplasm and nucleus to reestablish the ER homeostasis [43]. Three membrane-associated proteins have been identified for sensing ER stress in eukaryotes: activating transcription factor 6 (ATF6), pancreatic ER eIF2? kinase (PERK, also double-stranded RNA-activated protein kinase-like ER kinase), and inositol-requiring kinase1 (IRE1). The luminal domain of each sensor is bound to a 78 kDa chaperone termed glucose-regulated protein (GRP78/BIP). BIP dissociates upon ER stress to bind unfolded proteins, leading to the activation of the three sensors [44].
NRF2 and its homologue NRF1, also related to the antioxidant response, participate in the transduction of the UPR to the nucleus. In the case of NRF1, this protein is located at the ER membrane and undergoes nuclear translocation upon deglycosylation or cleavage. Then, UPR activation leads to the processing of NRF1 and nuclear accumulation of the resulting fragment in the nuclear compartment. However, the ability to transactivate ARE-containing genes of this NRF1 fragment is still under discussion [45].
Glover-Cutter and co-workers showed activation of the NRF2 orthologue of C. elegans, SKN-1, with different ER stressors. Increased SKN-1 expression was dependent on different UPR mediators, including IRE1 or PERK worm orthologues [46]. In PERK-deficient cells, impaired protein synthesis leads to accumulation of endogenous peroxides and subsequent apoptosis [47]. The effector used by PERK to protect the ER from these peroxides might be NRF2, since it has been reported that PERK phosphorylates NRF2 at Ser40, thus preventing its degradation by KEAP1 [31]. The induction of ASK1 is also likely to play a role in this route through the TRAF2-mediated kinase action of IRE1 [48]. Although the role of MAPKs in the regulation of NRF2 is still controversial, it was recently suggested that the IRE1-TRAF2-ASK1-JNK pathway might activate NRF2 [49] (Fig. 1). Interestingly, in C. elegans and human cells, new evidence suggests that cysteine sulfenylation of IRE1 kinase at its activation loop inhibits IRE1-mediated UPR and initiates a p38 antioxidant response driven by NRF2. The data suggest that IRE1 has an ancient function as a cytoplasmic sentinel that activates p38 and NRF2 [50].
Figure 1 Regulation of NRF2 by the UPR. Accumulation of unfolded or misfolded proteins inside the endoplasmic reticulum can initiate the unfolded protein response (UPR). First, the chaperone BIP is released from the intraluminal domain of the ER sensors IRE1 and PERK to bind unfolded/misfolded proteins. This enables dimerization and trans-auto-phosphorylation of their cytosolic domains. PERK activation results in direct NRF2 phosphorylation at Ser40, leading to NRF2 translocation to the nucleus and activation of target genes. IRE1 activation induces the recruitment of TRAF2 followed by ASK1 and JNK phosphorylation and activation. As JNK has been reported to phosphorylate and activate NRF2, it is reasonable to think that IRE1 activation would lead to increased NRF2 activity.
Many studies on the induction of the UPR have been conducted with the inhibitor of protein glycosylation tunicamycin. NRF2 appears to be essential for prevention of tunicamycin-induced apoptotic cell death [31] and its activation under these conditions is driven by the autophagic degradation of KEAP1 [51]. Accordingly, shRNA-mediated silencing of NRF2 expression in ?TC-6 cells, a murine insulinoma ?-cell line, significantly increased tunicamycin-induced cytotoxicity and led to an increase in the expression of the pro-apoptotic ER stress marker CHOP10. On the other hand, NRF2 activation by 1,2-dithiole-3-thione (D3T) reduced tunicamycin cytotoxicity and attenuated the expression of CHOP10 and PERK [52]. Interestingly, olfactory neurons submitted to systemic application of tunicamycin increased NRF2 in parallel with other UPR-members such as CHOP, BIP, XBP1 [53]. These results have been extended to in vivo studies, as lateral ventricular infusion of tunicamycin in rats induced expression of PERK and NRF2 in the hippocampus accompanied by significant cognitive deficits, increased TAU phosphorylation and A?42 deposits [54].
NRF2 Up-Regulates Key Genes for the Maintenance of the ER Physiology
The ER lumen needs an abundant supply of GSH from the cytosol in order to maintain disulfide chemistry. NRF2 modulates crucial enzymes of the GSH metabolism in the brain, such as cystine/glutamate transport, ?-glutamate cysteine synthetase (?-GS), glutamate-cysteine ligase catalytic and modulator subunits (GCLC and GCLM), glutathione reductase (GR) and glutathione peroxidase (GPX) (reviewed in [55]). The relevance of NRF2 in the maintenance of GSH in the ER is supported by the finding that pharmacological or genetic activation of NRF2 results in increased GSH synthesis via GCLC/GCLM, while inhibiting the expression of these enzymes by NRF2-knockdown caused an accumulation of damaged proteins within the ER leading to the UPR activation [56].
In C. elegans several components of the UPR target genes regulated by SKN-1, including Ire1, Xbp1 and Atf6. Although NRF2 up-regulates the expression of several peroxidase (PRX) and glutathione peroxidase (GPX) genes in mammals (reviewed in [57]), only GPX8 is a bona fide ER-localized enzyme, harboring the KDEL retrieval signal [58]. Loss of GPX8 causes UPR activation, leakage of ERO1?-derived hydrogen peroxide to the cytosol and cell death. Hydrogen peroxide derived from ERO1? activity cannot diffuse from the ER to the cytosol owing to the concerted action of GPX8 and PRX4 [59]. In this regard, an analysis of the antioxidant defense pathway-gene expression array using RNA from wild type and NRF2-null mice tissue, revealed that the expression of GPX8 was down-regulated in the absence of NRF2 [60]. In line with this, transcriptome analysis from patient samples suffering from myeloproliferative neoplasms, polycythemia or myelofibrosis, diseases also associate with oxidative stress and low-grade chronic inflammation, show lower expression levels of both NRF2 and GPX8 compared with control subjects [61]. There are not yet studies that specifically involve GPX8 in human brain protection but a transcriptome analysis in mice indicates a compensatory GPX8 increase in response to the Parkinsonian toxin MPTP [62].
Impact of NRF2 on the UPR Dysregulation in Neurodegenerative Diseases
Malfunction of PDI enzymes and chronic activation of the UPR might subsequently initiate or accelerate neurodegeneration. Disease-affected neurons, animal models of neurodegenerative disease as well as post-mortem human tissues evidenced up-regulation of several UPR-markers in most of these disorders. The alteration of PDI/UPR pathway in neurodegenerative diseases has been nicely reviewed in [63] but the following highlights from brain post-mortem samples should be considered. PDI levels are increased in tangle-bearing neurons and in Lewy Bodies of AD and PD patients, respectively [64], [65]. PDI and ERP57 are up-regulated in CSF from ALS patients and in brains from CJD subjects [66], [67], [68]. BIP, PERK, IRE1 and ATF6 are elevated in samples from patients with AD, PD or ALS [69], [70], [71], [67]. BIP, CHOP and XBP1 are elevated in post-mortem brain samples from HD [72], [73]. Moreover, up-regulation of ERP57, GRP94 and BIP was found in cortex tissues from CJD patients [74]. Altogether, this evidence reveals that the accumulation of misfolded proteins in the brain parenchyma leads to a deleterious and chronic activation of the UPR. Interestingly, there is a recent study linking activation of NRF2 by PERK in early AD. In this study, the authors analyzed whether oxidative stress mediated changes in NRF2 and the UPR may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased pSer40-NRF2 were observed in human peripheral blood mononuclear cells isolated from individuals with mild cognitive impairment. Moreover, they reported impaired ER calcium homeostasis and up-regulated ER-stress markers in these cells from individuals with mild cognitive impairment and mild AD [75].
Mutual Regulation of NRF2 and the Ubiquitin Proteasome�System (UPS)
The UPS Modulates NRF2 Protein Levels
The UPS participates in the degradation of damaged or misfolded proteins and controls the levels of key regulatory molecules in the cytosol and the nucleus. The central core of this system is a large multisubunit enzyme that contains a proteolytic active complex named 20S. The 20S core proteasome degrades unfolded proteins, but binding to different regulatory protein complexes changes its substrate specificity and activity. For instance, the addition of one or two 19S regulatory subunits to the 20S core constitutes the 26S proteasome and changes its specificity towards native folded proteins [76], [77]. Proteasomal degradation needs covalent binding of ubiquitin. Conjugation of ubiquitin proceeds via a three-step cascade mechanism. First, the ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-requiring reaction. Then, one E2 enzyme (ubiquitin-carrier protein or ubiquitin-conjugating enzyme) transfers the activated ubiquitin from E1 to the substrate that is specifically bound to a member of the ubiquitin-protein ligase family, named E3. Although the exact fate of the ubiquitinated-protein will depend on the nature of the ubiquitin chain, this process generally results in the degradation by the 26S proteasome [78].
The E3-ligase KEAP1 is the best known inhibitor of NRF2. The mechanism of KEAP1 regulation elegantly explains how NRF2 levels adjust to oxidant fluctuations. Under basal conditions, newly synthesized NRF2 is grabbed by the homodimer KEAP1, which binds one NRF2 molecule at two amino acid sequences with low (aspartate, leucine, glycine; DLG) and high (glutamate, threonine, glycine, glutamate; ETGE) affinity. The interaction with KEAP1 aids to present NRF2 to the CULLIN3/RBX1 protein complex, resulting in its ubiquitination and subsequent proteasomal degradation. However, redox modification of KEAP1 impedes presentation of NRF2 to the UPS represented by CULLIN3/RBX1. As a result, newly synthetized NRF2 escapes KEAP1-dependent degradation, accumulates in the nucleus and activates ARE-containing genes [79], [80], [81], [82].
The E3-ligase adaptor ?-TrCP is also a homodimer that participates in the signaling events related to the phosphorylation of NRF2 by GSK-3?. This kinase phosphorylates specific serine residues of NRF2 (aspartate, serine, glycine, isoleucine serine; DSGIS) to create a degradation domain that is then recognized by ?-TrCP and tagged for proteasome degradation by a CULLIN1/RBX1 complex. The identification of the specific amino acids that are phosphorylated by GSK-3? in this degron was conducted by a combination of site-directed mutagenesis of the Neh6 domain, 2D-gel electrophoresis [15], [26] and mass spectroscopy [83]. Consequently, inhibition of GSK-3? by highly selective drugs or siRNAs against GSK-3 isoforms resulted in an increase in NRF2 protein levels. Similar results were found with siRNAs against ?-TrCP isoforms 1 and 2. Stabilization of NRF2 following GSK-3? inhibition occurred in KEAP1-deficient mouse embryo fibroblasts and in an ectopically expressed NRF2 deletion mutant lacking the critical ETGE residues for high-affinity binding to KEAP1, further demonstrating a KEAP1-independent regulation.
In the context of neurodegenerative diseases, we can envision the modulation of NRF2 by the UPS in two different ways. On the one hand, the KEAP1 system would sense redox imbalance derived from misfolded protein accumulation, while GSK-3/?-TrCP axis would act as an active participant in signaling transduction altered by loss of proteostasis (Fig. 2).
Figure 2 The UPS tightly controls NRF2 levels. Under homeostatic conditions, low NRF2 levels are maintained by the action of the E3 ligases adaptors KEAP1 and ?-TrCP. Left, NRF2 binds to the Kelch domains of a KEAP1 homodimer through a low (DLG) and a high (ETGE) affinity motifs. Through its BTB domain, KEAP1 simultaneously binds to a CULLIN3/RBX1 complex, enabling NRF2 ubiquitination and degradation by the 26 S proteasome. Moreover, GSK-3? phosphorylates Ser335 and Ser338 residues of NRF2 to create a degradation domain (DpSGIpSL) that is then recognized by the ubiquitin ligase adaptor ?-TrCP and tagged for proteasome degradation by a CULLIN3/RBX1 complex. Right, Upon exposure to reactive oxygen species or electrophiles critical Cys residues in KEAP1 are modified, rendering KEAP1 unable of interacting efficiently with NRF2 or CULLIN3/RBX1 and then this transcription factor increases its half-life and transcriptional activity towards ARE-genes. Signaling pathways that result in inhibition of GSK-3?, such AKT phosphorylation at Ser9, result in NRF2 impaired degradation by the proteasome, accumulation and induction of target genes.
NRF2 Increases UPS Activity Through the Transcriptional Control of Proteasome Subunits
NRF2 up-regulates the expression of several proteasome subunits, thus protecting the cell from the accumulation of toxic proteins. Twenty proteasome- and ubiquitination-related genes appear to be regulated by NRF2, according to a wide microarray analysis from liver RNA that was set up with the NRF2 inducer D3T [84]. In a posterior study, the same authors evidenced that the expression of most subunits of the 26S proteasome were enhanced up to three-fold in livers from mice treated with D3T. Subunit protein levels and proteasome activity were coordinately increased. However, no induction was seen in mice where the transcription factor NRF2 was disrupted. Promoter activity of the PSMB5 (20S) proteasome subunit increased with either NRF2 overexpression or treatment with activators in mouse embryonic fibroblasts, and AREs were identified in the proximal promoter of PSMB5 [85]. Pharmacological activation of NRF2 resulted in elevated expression levels of representative proteasome subunits (PSMA3, PSMA6, PSMB1 and PSMB5) only in non-senescent human fibroblasts containing functional NRF2 [86]. NRF2 activation during adaptation to oxidative stress results in high expression of the PSMB1 (20S) and PA28? subunits (or S11, proteasome regulator) [87]. Moreover, results from human embryonic stem cells revealed that NRF2 controls the expression of the proteasome maturation protein (POMP), a proteasome chaperone, which in turn modulates the proliferation of self-renewing human embryonic stem cells, three germ layer differentiation and cellular reprogramming [88]. All together, these studies indicate that NRF2 up-regulates the expression of key components of the UPS and therefore actively contributes to the clearance of proteins that otherwise would be toxic.
The NRF2-UPS Axis in Neurodegenerative Diseases
The role of the UPS in neurodegenerative diseases is a field of intensive debate. Initial studies reported decreased proteasome activity in human necropsies of patients affected from several neurodegenerative diseases. However, other studies employing in vitro and in vivo approaches found unchanged or even increased proteasome activity (reviewed in [89]). One possible explanation for this discrepancy is that the levels of the UPS components might change during disease progression and in different brain regions as is has been suggested for NRF2-targets.
Despite this controversy, it should be noted that up-regulation of ARE-containing proteasome genes will reinforce the UPS by increasing the clearance of toxic proteins in the brain. Indeed, ablation of NRF1, also modulator of the antioxidant response, in neuronal cells leads to impaired proteasome activity and neurodegeneration. Chromatin immunoprecipitation experiments and transcriptional analysis demonstrated that PSMB6 is regulated by NRF1. In addition, gene expression profiling led to the identification of NRF1 as a key transcriptional regulator of proteasome genes in neurons, suggesting that perturbations in NRF1 may contribute to the pathogenesis of neurodegenerative diseases [90]. Interestingly, NRF1 and its long isoform called TCF11 were shown to up-regulate ARE-containing proteasome genes upon proteasome inhibition in a feedback loop to compensate for reduced proteolytic activity [91], [92].
Regarding NRF2, there is a correlation among reduction of NRF2, RPT6 (19 S) and PSMB5 (20 S) levels in the midbrain of DJ-1-deficient mice treated with the neurotoxin paraquat [93]. Moreover, the naturally-occurring compound sulforaphane (SFN) gives a more robust image of NRF2 as a crucial modulator of the UPS. In vitro experiments with murine neuroblastoma Neuro2A cells evidenced an enhanced expression of the catalytic subunits of the proteasome, as well as its peptidase activities in response to SFN. This drug protected cells from hydrogen peroxide-mediated cytotoxicity and protein oxidation in a manner dependent on proteasome function [94]. In addition, Liu and co-workers employed a reporter mouse to monitor the UPS activity in response to SFN in the brain. These mice ubiquitously express the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation by the UPS (GFPu). In cerebral cortex, SFN reduced the level of GFPu with a parallel increase in chymotrypsin-like (PSMB5), caspase-like (PSMB2), and trypsin-like (PSMB1) activities of the 20 S proteasome. In addition, treatment of Huntington-derived cells with SFN revealed that NRF2 activation enhanced mHtt degradation and reduced mHtt cytotoxicity [95]. The major mechanism of SFN action is through induction of NRF2 [96]. The specific contribution of NRF2 should be addressed employing NRF2-null systems in further studies.
Functional Connection Between NRF2 and Macroautophagy
NRF2 Protein Levels are Modulated by the Adaptor Protein P62
Autophagy refers to the degradation of cytosolic components inside lysosomes. This process is used for the clearance of long-lived and misfolded proteins as well as damaged organelles. A direct link between NRF2 and autophagy was first observed in connection with the adaptor protein p62, also termed SQSTM1 [97], [98], [99], [100], [101]. This protein shuttles ubiquitinated proteins to the proteasomal and lysosomal degradation machineries and sequesters damaged proteins into aggregates prior to their degradation. P62 presents an ubiquitin-associated (UBA) domain, for binding to ubiquitinated proteins, and a LC3-interacting region (LIR) for integration with the autophagosomal membrane through the autophagy receptor LC3.
Although the p62-mediated induction of NRF2 and its target genes was first reported in 2007 [102], the molecular mechanism was not fully understood until the discovery of its interaction with KEAP1 [103], [98], [99], [100], [101]. Komatsu and coworkers identified a KEAP1 interacting region (KIR) in p62 that bound KEAP1 in the same basic surface pocket as NRF2 and with a binding affinity similar to the ETGE motif in NRF2, suggesting competition between p62 and NRF2. The phosphorylation of Ser351 in the KIR motif in p62 (349-DPSTGE-354) was shown to increase its affinity for KEAP1, competing with NRF2 binding and allowing its accumulation and transcriptional activation of its target genes [98], [99]. In fact, p62 overexpression led to reduced NRF2 ubiquitination and consequent stabilization as well as induction of its target genes [104]. Some kinases have been suggested to participate in p62 phosphorylation. The mammalian target of rapamycin complex 1 (mTORC1) may be implicated, as treatment with the mTOR inhibitor rapamycin suppressed the phosphorylation of p62 and the down-regulation of KEAP1 upon arsenite treatment. Recently, it was demonstrated that TGF-?-activated kinase 1 (TAK1) could also phosphorylate p62, enhancing KEAP1 degradation and NRF2-up-regulation. The authors of this study suggest this is a way to regulate cellular redoxtasis under steady-state conditions, as TAK1-deficiency up-regulates ROS in the absence of any exogenous oxidant in different mouse tissues in parallel with a reduction in NRF2 protein levels [105].
A p62 construct lacking the UBA domain was still capable of binding KEAP1, implying that the interaction did not depend on ubiquitinated KEAP1 [101]. However, the p62 homologue in Drosophila melanogaster, named Ref(2), does not contain a KIR motif and does not directly interact with DmKEAP1, although it can bind to ubiquitinated DmKEAP1 through the UBA domain. Moreover, DmKEAP1 can directly interact with Atg8 (homologue to mammalian LC3). KEAP1 deficiency results in Atg8 and autophagy induction dependent on the NRF2 orthologue CncC and independent on TFEB/MITF [106]. The relationship between NRF2 and autophagy seems to be conserved though, highlighting its functional relevance.
The induction of NRF2 by p62 is the result of both the competition to bind KEAP1 and degradation of KEAP1 in the lysosome. Silencing of p62 with siRNA doubled KEAP1 half-life in parallel with a decrease in NRF2 and its target genes [101]. In agreement, ablation of p62 expression evidenced increased levels of KEAP1 compared with wild type mice. Very relevant, the increment in KEAP1 levels was not affected by proteasome inhibitors but was reduced under starvation-inducing autophagy [107]. In fact, KEAP1 is present in mammalian cells in autophagic vesicles decorated with p62 and LC3 [99], [100], [103]. All these data suggest that KEAP1 is a substrate of the macroautophagy machinery, but this issue should be analyzed with more detail because of the existence of some controversial results. KEAP1 protein levels were increased in Atg7-null mice, a key effector of macroautophagy [107], but pharmacological inhibition of macroautophagy with torin1, E64/pepstatin or bafilomycin failed to accumulate KEAP1 [107], [100]. Overall, these results suggest that increased p62 levels sequester KEAP1 into autophagic vacuoles and probably these results in KEAP1 autophagic degradation allowing NRF2 activation (Fig. 3). Two different studies reported that the sulfinic acid reductases SESTRINS play an important role in this context. SESTRIN 2 interacts with p62, KEAP1 and RBX1 and facilitates p62-dependent degradation of KEAP1 and NRF2 activation of target genes [108]. Another study showed that SESTRIN 2 interacted with ULK1 and p62, promoting phosphorylation of p62 at Ser403 which facilitated degradation of cargo proteins including KEAP1 [109].
Figure 3 NRF2 levels are regulated by the adaptor protein p62. The phosphorylation of Ser 351 in the KIR motif of p62 (349-DPSTGE-354) by mTORC1, TAK1 or other kinases results in increased affinity for binding to KEAP1 due to resemblance to the ETGE motif in NRF2. As a consequence, phosphorylated p62 displaces NRF2 and binds KEAP1. The LIR motif in p62 enables interaction with LC3 in the autophagosomal membrane, so that p62-KEAP1 complex is eventually degraded in the lysosome. As a consequence NRF2 is able to accumulate, translocate to the nucleus and increase the transcription of ARE-containing genes, including p62. This regulatory mechanism provides a perdurable NRF2 response, as KEAP1 has to be newly synthesized in order to inhibit NRF2 activity.
Modulation of Macroautophagy Genes by NRF2
NRF2 regulates the expression of relevant genes for macroautophagy as well as it does for the UPR and the UPS. The first evidence came from studies in which p62 expression was shown to be induced upon exposure to electrophiles, ROS and nitric oxide [110], [111], [112]. The mechanism of induction was described some years later with the finding that p62 contains a functional ARE in its gene promoter [99]. In a recent study, several other functional AREs were found and validated following bioinformatics analysis and ChIP assays. Moreover, mouse embryonic fibroblasts and cortical neurons from Nrf2-knockout mice exhibited reduced p62 expression, which could be rescued with an NRF2-expressing lentivirus. Similarly, NRF2 deficiency reduced p62 levels in injured neurons from mice hippocampus [36]. Therefore, it has been suggested that NRF2 activation increases p62 levels, resulting in KEAP1 degradation and favoring further NRF2 stabilization in a positive feedback loop. This non-canonical mechanism of NRF2 induction requires changes in gene expression and might be a relevant response to prolonged cellular stress.
The cargo recognition protein NDP52 was shown to be transcriptionally regulated by NRF2. NDP52 works in a similar way to p62, recognizing ubiquitinated proteins and interacting with LC3 through a LIR domain, so that cargoes are degraded in lysosomes. Five putative AREs were found in Ndp52 promoter DNA sequence. Three of them were identified with different mutant constructs and ChIP assays as indispensable for NRF2-mediated Ndp52 transcription [113]. Of note, Ndp52 mRNA levels were reduced in the hippocampus of Nrf2-knockout mice. One of these sequences was also validated in an independent study as an NRF2-regulated ARE [36].
However, the role of NRF2 in the modulation of autophagy is not limited to the induction of these two cargo-recognition proteins. In order to gain deeper insight in the role of NRF2 in the modulation of additional autophagy-related genes, our group screened the chromatin immunoprecipitation database ENCODE for two proteins, MAFK and BACH1, which bind the NRF2-regulated AREs. Using a script generated from the JASPAR’s consensus ARE sequence, we identified several putative AREs in many autophagy genes. Twelve of these sequences were validated as NRF2 regulated AREs in nine autophagy genes, whose expression was diminished in mouse embryo fibroblasts of Nrf2-knockout mice but could be restored by an NRF2-expressing lentivirus. Our study demonstrated that NRF2 activates the expression of some genes involved in different steps of the autophagic process, including autophagy initiation (ULK1), cargo recognition (p62 and NDP52), autophagosome formation (ATG4D, ATG7 and GABARAPL1), elongation (ATG2B and ATG5), and autolysosome clearance (ATG4D). Consequently, autophagy flux in response to hydrogen peroxide was impaired when NRF2 was absent [36].
Relevance of NRF2-Mediated Macroautophagy Genes Expression in Neurodegenerative Disorders
Defective autophagy has been shown to play an important role in several neurodegenerative diseases [114] and ablation of autophagy leads to neurodegeneration in mice [115], [116]. Atg7-knockout mice revealed that autophagy deficiency results in p62 accumulation in ubiquitin-positive inclusion bodies. KEAP1 was sequestered in these inclusion bodies, leading to NRF2 stabilization and induction of target genes [103]. Importantly, excessive accumulation of p62 together with ubiquitinated proteins has been identified in neurodegenerative diseases, including AD, PD and ALS [117]. In fact, neurons expressing high levels of APP or TAU of AD patients also expressed p62 and nuclear NRF2, suggesting their attempt to degrade intraneuronal aggregates through autophagy [36].
NRF2 deficiency aggravates protein aggregation in the context of AD. In fact, increased levels of phosphorylated and sarkosyl-insoluble TAU are found in Nrf2-knockout mice, although no difference in kinase or phosphatase activities could be detected comparing with the wild-type background [113]. Importantly, NDP52 was demonstrated to co-localize with TAU in murine neurons and direct interaction between phospho-TAU and NDP52 was shown by co-immunoprecipitation experiments both in mice and AD samples, pointing to its role in TAU degradation. Interestingly, silencing of NDP52, p62 or NRF2 in neurons resulted in increased phospho-TAU [113], [118]. Moreover, increased intraneuronal APP aggregates were found in the hippocampus of APP/PS1?E9 mice when NRF2 was absent. This correlated with altered autophagy markers, including increased phospho-mTOR/mTOR and phospho-p70S6k/p70S6k ratios (indicative of autophagy inhibition), augmented levels of pre-cathepsin D and a larger number of multivesicular bodies [119]. In mice co-expressing human APP (V717I) and TAU (P301L), NRF2 deficiency led to increased levels of total and phospho-TAU in the insoluble fraction and increased intraneuronal APP aggregates, together with reduced neuronal levels of p62, NDP52, ULK1, ATG5 and GABARAPL1. Co-localization between the adaptor protein p62 and APP or TAU was reduced in the absence of NRF2 [36]. Overall, these results highlight the importance of NRF2 in neuronal autophagy.
Different Transcription Factors Act Coordinately to Modulate Proteostasis
Under steady state conditions, proteostasis is controlled via protein-protein interactions and post-translational modifications obtaining a rapid response. However, cellular adaptation requires the transcriptional regulation of the UPR, UPS and autophagy genes. Considering that nerve cells are continuously submitted to low-grade toxic insults, including oxidative and proteotoxic stress, a reinforcement of proteostasis induced by transcriptional modulation might help preventing brain degeneration.
In the case of the UPR, the activation of each of the three arms will finally result in the transcriptional induction of certain genes (reviewed in [43]). For instance, an ATF6-derived fragment (ATF6f) binds to ER-stress response elements (ERSE) and induces the expression of several genes, including XBPI, BIP and CHOP. In addition, PERK signaling leads to the activation of the transcription factor ATF4, which controls the expression of multiple UPR-related genes and some others including the NRF2 target genes Hmox1 and p62. Finally, IRE1 activation results in the generation of an active transcription factor, spliced XBP1 (XBP1s), which controls the transcription of genes encoding proteins involved in protein folding.
On the other hand, NRF1 was shown to be necessary for proteasomal gene expression in the brain, as Nrf1-knockout mice exhibited reduced expression of genes encoding various subunits of the 20S core, as well the 19S regulatory complex together with impaired proteasomal function [90]. Both NRF1 and NRF2 bind to ARE sequences in the promoter regions of its target genes, which suggests they have overlapping transcriptional activities, although they differ in their regulatory mechanisms and cellular localization [120].
Transcription factors of the Forkhead box O (FOXO) family control the expression of multiple autophagy-related genes. Similar to what occurs with NRF2, there are multiple layers of regulation of the activity of FOXO members, which can be induced upon nutritional or oxidative stress [121]. Finally, the transcription factor TFEB, considered the master regulator of lysosomal biogenesis, plays a crucial role in regulation of autophagy under nutritional stress conditions. Thus, inhibition of mTORC1 leads to nuclear translocation of TFEB and induction of the expression of autophagy genes [122].
Overall, the existence of different transcriptional regulators of these machineries also suggests crosstalk and partially redundant mechanisms that may assure proteostasis under different circumstances. Accordingly, NRF2 may have a relevant role in tissues that support high levels of oxidative stress. For instance, oxidative stress-induced NRF2 may function under nutrient-rich conditions to transcriptionally up-regulate autophagy, similar to what has been found for TFEB under starvation conditions. Moreover, the brain functions largely under nutrient-rich conditions, posing NRF2 as a relevant mechanism to activate autophagy in neurons.
Promising�Therapeutic Potential for NRF2 in Proteinopathies
In the past few years, a great progress has been made in the knowledge of the regulatory roles of the UPR, UPS and autophagy on NRF2 activity, as well as the reciprocal NRF2-mediated transcription of components of these three systems. Therefore, new therapeutic possibilities may arise based on the exploitation of NRF2 as a crucial regulator of protein clearance in neurodegenerative diseases.
However, a key remaining question is whether it will be useful or deleterious to increase NRF2 levels in brain. Analysis of epidemiological data may provide a partial answer, as it indicates that the NFE2L2 gene is highly polymorphic and some single nucleotide polymorphisms found in its promoter regulatory region may provide a range of �physiological� variability in gene expression at the population level and some haplotypes were associated with decreased risk and/or delayed onset of AD, PD or ALS [123]. Moreover, as discussed by Hayes and colleagues [124], NRF2 effect might have an U-shaped response, meaning that too low NRF2 levels may result in a loss of cytoprotection and increased susceptibility to stressors, while too much NRF2 might disturb homeostatic balance towards a reductive scenario (reductive stress), which would favor protein misfolding and aggregation. Low NRF2 levels in the brain support the idea that a slight up-regulation may be sufficient to achieve a benefit under pathological conditions. In fact, the protective role of pharmacological NRF2-mediated activation of protein clearance has been shown in different neurodegeneration cell culture and in vivo models.
SFN is a pharmacological NRF2 activator that was demonstrated to induce proteasomal and autophagy gene expression [95], [36]. Interestingly, Jo and colleagues demonstrated that SFN reduced the levels of phosphorylated TAU and increased Beclin-1 and LC3-II, suggesting NRF2 activation may facilitate degradation of this toxic protein through autophagy [113]. Moreover, degradation of mHtt was enhanced with SFN, and this was reverted with the use of MG132, indicating proteasomal degradation of this toxic protein [95]. Autophagy-mediated degradation of phospho- and insoluble-TAU was reported with the organic flavonoid fisetin. This compound was able to induce autophagy by simultaneously promoting the activation and nuclear translocation of both TFEB and NRF2, along with some of its target genes. This response was prevented by TFEB or NRF2 silencing [125]. Bott and colleagues reported beneficial effects of a simultaneous NRF2, NRF1 and HSF1 activator on protein toxicity in spinal and bulbar muscular atrophy, a neurodegenerative disorder caused by expansion of polyglutamine-encoding CAG repeats in which protein aggregates are present [126]. The potential of NRF2 activation for the treatment of neurodegenerative disorders has been demonstrated with the approval of BG-12, the oral formulation of the NRF2 inducer dimethyl fumarate (DMF), for the treatment of multiple sclerosis [127], [128]. The success of DMF with autoimmune diseases with a strong inflammatory component suggests that neurodegenerative diseases might benefit from repositioning this drug. In a recent preclinical study of an ?-synucleinopathy model of PD, DMF was shown to be neuroprotective due, in part, to its induction of autophagy [129]. Studies reporting beneficial effects of NRF2 on neurodegeneration but not focusing on its effect on protein clearance are even more abundant (for a comprehensive review, see [7]). This is quite relevant, as it highlights the multiple damaging processes that can be simultaneously targeted by a single hit in NRF2, also including oxidative stress, neuroinflammation or mitochondrial dysfunction. However, future work will be needed to definitely determine if pharmacological activation of NRF2 may be a valid strategy to facilitate degradation of toxic proteins in the brain.
As explained before, exacerbated GSK-3? activity was reported in neurodegenerative diseases and it has been speculated that consequent NRF2 reduction can be partially responsible for the deleterious outcome. Under these pathological conditions, GSK-3 inhibitors could also cooperate to increase NRF2 levels and proteostasis. The beneficial effects of GSK-3 inhibitors have been reported in different models of neurodegeneration and, more interesting, GSK-3 repression was shown to reduce the levels of toxic proteins [130], [131], [132], [133]. Although no direct links between GSK-3 inhibition and NRF2-transcriptional regulation of genes promoting proteostasis have been observed yet, it is reasonable to speculate that down-regulation of GSK-3 activity would result in increased NRF2 levels, which eventually will result in reinforced proteostasis.
The transcriptional activity of NRF2 as well as the cellular capacity to maintain proteostasis decrease with age, the main risk factor for the development of neurodegenerative diseases. It is reasonable to think that the reinforcement of NRF2 and, consequently, proteostasis would, at least, delay the accumulation of protein aggregates and neurodegeneration. Indeed, treatment of human senescent fibroblasts with 18?-glycyrrhetinic acid (18?-GA) triterpenoid promoted NRF2 activation, leading to proteasome induction and enhanced life span. This study suggests that pharmacological activation of NRF2 is possible even in late life [86]. Moreover, a later study showed that this compound mediated SKN-1 and proteasome activation in C.elegans with beneficial effects on AD progression in relevant nematode models [134].
All things considered, NRF2-mediated induction of proteostasis-related genes seems to be beneficial in different proteinopathies.
Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More
Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.
Key sections:
00:01:14 – Cancer and mortality
00:19:04 – Aging
00:26:30 – Brain and behavior
00:38:06 – Final recap
00:40:27 – Dose
Full timeline:
00:00:34 – Introduction of sulforaphane, a major focus of the video.
00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
00:02:12 – Prostate cancer risk.
00:02:23 – Bladder cancer risk.
00:02:34 – Lung cancer in smokers risk.
00:02:48 – Breast cancer risk.
00:03:13 – Hypothetical: what if you already have cancer? (interventional)
00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
00:04:38 – Sulforaphane and cancer.
00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
00:08:32 – Inhibition of breast cancer stem cells.
00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
00:19:04 – Beginning of aging section.
00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
00:20:34 – Importance of low inflammation for longevity.
00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
00:37:19 – Sulforaphane and duchenne muscular dystrophy.
00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
00:41:01 – Anecdotes on sprouting at home.
00:43:14 – On cooking temperatures and sulforaphane activity.
00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
00:44:56 – Cooking techniques and cruciferous vegetables.
00:46:06 – Isothiocyanates as goitrogens.
The nuclear factor erythroid-derived 2 (NF-E2)-related factor 2, otherwise known as Nrf2, is a transcription factor which regulates the expression of a variety of antioxidant and detoxifying enzymes. Research studies have also demonstrated its role in controlling oxidative stress. Most neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, are characterized by oxidative stress and chronic inflammation, the common targets of Nrf2 treatment approaches. Dr. Alex Jimenez D.C., C.C.S.T. Insight
Concluding Remarks
Transcription factor NRF2 orchestrates a proteostatic response by sensing to and modulating changes in the UPR, UPS and autophagy (Fig. 4). Consequently, the lack of NRF2 has been shown to aggravate proteinopathy, suggesting that NRF2 is necessary for optimal protein clearance. All together, we can speculate that NRF2 might be an interesting therapeutic target for proteinopathies.
Figure 4 NRF2 as a hub connecting proteotoxic-derived emergency signals to a protective transcriptional response. The accumulation of unfolded/misfolded proteins will lead to the activation of the unfolded protein response (UPR) in the ER. Activation of PERK or MAPK may result in the transcriptional induction of the ER-resident Gpx8 and several enzymes regulating GSH levels, critical to ensure correct protein folding. Protein aggregates inhibit proteasome activity (UPS), probably avoiding NRF2 degradation. NRF2 has been shown to specifically modulate the transcription of Psma3, Psma6, Psmb1, Psmb5 and Pomp genes. Several other subunits were up-regulated in an NRF2-dependent manner in response to D3T, probably enlarging the list of proteasome subunits regulated by NRF2. Autophagy is the main pathway for the degradation of protein aggregates. Autophagy also regulates NRF2, connecting this degradation pathway with NRF2 transcriptional induction of p62, Ndp52, Ulk1, Atg2b, Atg4c, Atg5, Atg7 and Gabarapl1.
According to the article above, while the symptoms of neurodegenerative diseases can be treated through a variety of treatment options, research studies have demonstrated that Nrf2 activation can be a helpful treatment approach. Because Nrf2 activators target broad mechanisms of disease, all neurodegenerative diseases can benefit from the use of the Nrf2 transcription factor. The findings of Nrf2 have revolutionized the treatment of neurodegenerative diseases. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.
Additional Topic Discussion: Relieving Knee Pain without Surgery
Knee pain is a well-known symptom which can occur due to a variety of knee injuries and/or conditions, including�sports injuries. The knee is one of the most complex joints in the human body as it is made-up of the intersection of four bones, four ligaments, various tendons, two menisci, and cartilage. According to the American Academy of Family Physicians, the most common causes of knee pain include patellar subluxation, patellar tendinitis or jumper’s knee, and Osgood-Schlatter disease. Although knee pain is most likely to occur in people over 60 years old, knee pain can also occur in children and adolescents. Knee pain can be treated at home following the RICE methods, however, severe knee injuries may require immediate medical attention, including chiropractic care. �
Oxidative stress is described as cell damage caused by free radicals, or unstable molecules, which can ultimately affect healthy function. The human body creates free radicals to neutralize bacteria and viruses, however, external factors, such as oxygen, pollution, and radiation, can often also produce free radicals. Oxidative stress has been associated with numerous health issues.
Oxidative stress and other stressors turn on internal protective mechanisms which can help regulate the human body’s antioxidant response. Nrf2 is a protein which senses levels of oxidative stress and enables the cells to protect themselves from internal and external factors. Nrf2 has also been demonstrated to help regulate genes involved in the production of antioxidant enzymes and stress-response genes. The purpose of the article below is to explain the effects of Nrf2 in cancer.
Abstract
The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. Although cell signaling pathways triggered by the transcription factor Nrf2 prevent cancer initiation and progression in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth. In this graphical review, we provide an overview of the Keap1-Nrf2 pathway and its dysregulation in cancer cells. We also briefly summarize the consequences of constitutive Nrf2 activation in cancer cells and how this can be exploited in cancer gene therapy.
The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by reactive oxygen species (ROS) and electrophiles [1]. The key signaling proteins within the pathway are the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) that binds together with small Maf proteins to the antioxidant response element (ARE) in the regulatory regions of target genes, and Keap1 (Kelch ECH associating protein 1), a repressor protein that binds to Nrf2 and promotes its degradation by the ubiquitin proteasome pathway (Fig. 1). Keap1 is a very cysteine-rich protein, mouse Keap1 having a total of 25 and human 27 cysteine residues, most of which can be modified in vitro by different oxidants and electrophiles [2]. Three of these residues, C151, C273 and C288, have been shown to play a functional role by altering the conformation of Keap1 leading to nuclear translocation of Nrf2 and subsequent target gene expression [3] (Fig. 1). The exact mechanism whereby cysteine modifications in Keap1 lead to Nrf2 activation is not known, but the two prevailing but not mutually exclusive models are (1) the �hinge and latch� model, in which Keap1 modifications in thiol residues residing in the IVR of Keap1 disrupt the interaction with Nrf2 causing a misalignment of the lysine residues within Nrf2 that can no longer be polyubiquitinylated and (2) the model in which thiol modification causes dissociation of Cul3 from Keap1 [3]. In both models, the inducer-modified and Nrf2-bound Keap1 is inactivated and, consequently, newly synthesized Nrf2 proteins bypass Keap1 and translocate into the nucleus, bind to the ARE and drive the expression of Nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), glutamate-cysteine ligase (GCL) and glutathione S transferases (GSTs) (Fig. 2). In addition to modifications of Keap1 thiols resulting in Nrf2 target gene induction, proteins such as p21 and p62 can bind to Nrf2 or Keap1 thereby disrupting the interaction between Nrf2 and Keap1 [1], [3] (Fig. 3).
Fig. 1. Structures of Nrf2 and Keap1 and the cysteine code. (A) Nrf2 consists of 589 amino acids and has six evolutionarily highly conserved domains, Neh1-6. Neh1 contains a bZip motif, a basic region � leucine zipper (L-Zip) structure, where the basic region is responsible for DNA recognition and the L-Zip mediates dimerization with small Maf proteins. Neh6 functions as a degron to mediate degradation of Nrf2 in the nucleus. Neh4 and 5 are transactivation domains. Neh2 contains ETGE and DLG motifs, which are required for the interaction with Keap1, and a hydrophilic region of lysine residues (7 K), which are indispensable for the Keap1-dependent polyubiquitination and degradation of Nrf2. (B) Keap1 consists of 624 amino acid residues and has five domains. The two protein�protein interaction motifs, the BTB domain and the Kelch domain, are separated by the intervening region (IVR). The BTB domain together with the N-terminal portion of the IVR mediates homodimerization of Keap1 and binding with Cullin3 (Cul3). The Kelch domain and the C-terminal region mediate the interaction with Neh2. (C) Nrf2 interacts with two molecules of Keap1 through its Neh2 ETGE and DLG motifs. Both ETGE and DLG bind to similar sites on the bottom surface of the Keap1 Kelch motif. (D) Keap1 is rich in cysteine residues, with 27 cysteines in human protein. Some of these cysteines are located near basic residues and are therefore excellent targets of electrophiles and oxidants. The modification pattern of the cysteine residues by electrophiles is known as the cysteine code. The cysteine code hypothesis proposes that structurally different Nrf2 activating agents affect different Keap1 cysteines. The cysteine modifications lead to conformational changes in the Keap1 disrupting the interaction between the Nrf2 DLG and Keap1 Kelch domains, thus inhibiting the polyubiquitination of Nrf2. The functional importance of Cys151, Cys273 and Cys288 has been shown, as Cys273 and Cys288 are required for suppression of Nrf2 and Cys151 for activation of Nrf2 by inducers [1], [3].
Fig. 2. The Nrf2-Keap1 signaling pathway. (A and B) in basal conditions, two Keap1 molecules bind to Nrf2 and Nrf2 is polyubiquitylated by the Cul3-based E3 ligase complex. This polyubiquitilation results in rapid Nrf2 degradation by the proteasome. A small proportion of Nrf2 escapes the inhibitory complex and accumulates in the nucleus to mediate basal ARE-dependent gene expression, thereby maintaining the cellular homeostasis. (C) Under stress conditions, inducers modify the Keap1 cysteines leading to the inhibition of Nrf2 ubiquitylation via dissociation of the inhibitory complex. (D) According to the hinge and latch model, modification of specific Keap1 cysteine residues leads to conformational changes in Keap1 resulting in the detachment of the Nrf2 DLG motif from Keap1. Ubiquitination of Nrf2 is disrupted but the binding with the ETGE motif remains. (E) In the Keap1-Cul3 dissociation model, the binding of Keap1 and Cul3 is disrupted in response to electrophiles, leading to the escape of Nrf2 from the ubiquitination system. In both of the suggested models, the inducer-modified and Nrf2-bound Keap1 is inactivated and, consequently, newly synthesized Nrf2 proteins bypass Keap1 and translocate into the nucleus, bind to the Antioxidant Response Element (ARE) and drive the expression of Nrf2 target genes such as NQO1, HMOX1, GCL and GSTs [1], [3].
Fig. 3. Mechanisms for constitutive nuclear accumulation of Nrf2 in cancer. (A) Somatic mutations in Nrf2 or Keap1 disrupt the interaction of these two proteins. In Nrf2, mutations affect ETGE and DLG motifs, but in Keap1 mutations are more evenly distributed. Furthermore, oncogene activation, such as KrasG12D[5], or disruption of tumor suppressors, such as PTEN [11] can lead to transcriptional induction of Nrf2 and an increase in nuclear Nrf2. (B) Hypermethylation of the Keap1 promoter in lung and prostate cancer leads to reduction of Keap1 mRNA expression, which increases the nuclear accumulation of Nrf2 [6], [7]. (C) In familial papillary renal carcinoma, the loss of fumarate hydratase enzyme activity leads to the accumulation of fumarate and further to succination of Keap1 cysteine residues (2SC). This post-translational modification leads to the disruption of Keap1-Nrf2 interaction and nuclear accumulation of Nrf2 [8], [9]. (D) Accumulation of disruptor proteins such as p62 and p21 can disturb Nrf2-Keap1 binding and results in an increase in nuclear Nrf2. p62 binds to Keap1 overlapping the binding pocket for Nrf2 and p21 directly interacts with the DLG and ETGE motifs of Nrf2, thereby competing with Keap1 [10].
Mechanisms of Activation and Dysregulation of Nrf2 in Cancer
Although cytoprotection provided by Nrf2 activation is important for cancer chemoprevention in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth [4]. Several mechanisms by which Nrf2 signaling pathway is constitutively activated in various cancers have been described: (1) somatic mutations in Keap1 or the Keap1 binding domain of Nrf2 disrupting their interaction; (2) epigenetic silencing of Keap1 expression leading to defective repression of Nrf2; (3) accumulation of disruptor proteins such as p62 leading to dissociation of the Keap1-Nrf2 complex; (4) transcriptional induction of Nrf2 by oncogenic K-Ras, B-Raf and c-Myc; and (5) post-translational modification of Keap1 cysteines by succinylation that occurs in familial papillary renal carcinoma due to the loss of fumarate hydratase enzyme activity [3], [4], [5], [6], [7], [8], [9], [10] (Fig. 3). Constitutively abundant Nrf2 protein causes increased expression of genes involved in drug metabolism thereby increasing the resistance to chemotherapeutic drugs and radiotherapy. In addition, high Nrf2 protein level is associated with poor prognosis in cancer [4]. Overactive Nrf2 also affects cell proliferation by directing glucose and glutamine towards anabolic pathways augmenting purine synthesis and influencing the pentose phosphate pathway to promote cell proliferation [11] (Fig. 4).
Fig. 4. The dual role of Nrf2 in tumorigenesis. Under physiological conditions, low levels of nuclear Nrf2 are sufficient for the maintenance of cellular homeostasis. Nrf2 inhibits tumor initiation and cancer metastasis by eliminating carcinogens, ROS and other DNA-damaging agents. During tumorigenesis, accumulating DNA damage leads to constitutive hyperactivity of Nrf2 which helps the autonomous malignant cells to endure high levels of endogenous ROS and to avoid apoptosis. Persistently elevated nuclear Nrf2 levels activate metabolic genes in addition to the cytoprotective genes contributing to metabolic reprogramming and enhanced cell proliferation. Cancers with high Nrf2 levels are associated with poor prognosis because of radio and chemoresistance and aggressive cancer cell proliferation. Thus, Nrf2 pathway activity is protective in the early stages of tumorigenesis, but detrimental in the later stages. Therefore, for the prevention of cancer, enhancing Nrf2 activity remains an important approach whereas for the treatment of cancer, Nrf2 inhibition is desirable [4], [11].
Given that high Nrf2 activity commonly occurs in cancer cells with adverse outcomes, there is a need for therapies to inhibit Nrf2. Unfortunately, due to structural similarity with some other bZip family members, the development of specific Nrf2 inhibitors is a challenging task and only a few studies of Nrf2 inhibition have been published to date. By screening natural products, Ren et al. [12] identified an antineoplastic compound brusatol as an Nrf2 inhibitor that enhances the chemotherapeutic efficacy of cisplatin. In addition, PI3K inhibitors [11], [13] and Nrf2 siRNA [14] have been used to inhibit Nrf2 in cancer cells. Recently, we have utilized an alternative approach, known as cancer suicide gene therapy, to target cancer cells with high Nrf2 levels. Nrf2-driven lentiviral vectors [15] containing thymidine kinase (TK) are transferred into cancer cells with high ARE activity and the cells are treated with a pro-drug, ganciclovir (GCV). GCV is metabolized to GCV-monophosphate, which is further phosphorylated by cellular kinases into a toxic triphosphate form [16] (Fig. 5). This leads to effective killing of not only TK containing tumor cells, but also the neighboring cells due to the bystander effect [17]. ARE-regulated TK/GCV gene therapy can be further enhanced via combining a cancer chemotherapeutic agent doxorubicin to the treatment [16], supporting the notion that this approach could be useful in conjuction with traditional therapies.
Fig. 5. Suicide gene therapy. Constitutive Nrf2 nuclear accumulation in cancer cells can be exploited by using Nrf2-driven viral vector for cancer suicide gene therapy [16]. In this approach, lentiviral vector (LV) expressing thymidine kinase (TK) under minimal SV40 promoter with four AREs is transduced to lung adenocarcinoma cells. High nuclear Nrf2 levels lead to robust expression of TK through Nrf2 binding. Cells are then treated with a pro-drug, ganciclovir (GCV), which is phosphorylated by TK. Triphosphorylated GCV disrupts DNA synthesis and leads to effective killing of not only TK containing tumor cells, but also the neighboring cells due to the bystander effect.
Nrf2 is a master regulator which triggers the production of powerful antioxidants in the human body which help eliminate oxidative stress. Various antioxidant enzymes, such as superoxide dismutase, or SOD, glutathione, and catalase, are also activated through the Nrf2 pathway. Furthermore, certain phytochemicals like turmeric, ashwagandha, bacopa, green tea, and milk thistle, activate Nrf2. Research studies have found that Nrf2 activation can naturally enhance cellular protection and restore balance to the human body.
Dr. Alex Jimenez D.C., C.C.S.T. Insight
Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More
Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.
Key sections:
00:01:14 – Cancer and mortality
00:19:04 – Aging
00:26:30 – Brain and behavior
00:38:06 – Final recap
00:40:27 – Dose
Full timeline:
00:00:34 – Introduction of sulforaphane, a major focus of the video.
00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
00:02:12 – Prostate cancer risk.
00:02:23 – Bladder cancer risk.
00:02:34 – Lung cancer in smokers risk.
00:02:48 – Breast cancer risk.
00:03:13 – Hypothetical: what if you already have cancer? (interventional)
00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
00:04:38 – Sulforaphane and cancer.
00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
00:08:32 – Inhibition of breast cancer stem cells.
00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
00:19:04 – Beginning of aging section.
00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
00:20:34 – Importance of low inflammation for longevity.
00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
00:37:19 – Sulforaphane and duchenne muscular dystrophy.
00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
00:41:01 – Anecdotes on sprouting at home.
00:43:14 – On cooking temperatures and sulforaphane activity.
00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
00:44:56 – Cooking techniques and cruciferous vegetables.
00:46:06 – Isothiocyanates as goitrogens.
Acknowledgments
This work was supported by the Academy of Finland, the Sigrid Juselius Foundation and the Finnish Cancer Organisations.
In conclusion, nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or Nrf2, is a protein which increases the production of antioxidants which protect the human body against oxidative stress. As described above, the stimulation of the Nrf2 pathway are being studies for the treatment of diseases caused by oxidative stress, including cancer. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.
Additional Topic Discussion: Relieving Knee Pain without Surgery
Knee pain is a well-known symptom which can occur due to a variety of knee injuries and/or conditions, including�sports injuries. The knee is one of the most complex joints in the human body as it is made-up of the intersection of four bones, four ligaments, various tendons, two menisci, and cartilage. According to the American Academy of Family Physicians, the most common causes of knee pain include patellar subluxation, patellar tendinitis or jumper’s knee, and Osgood-Schlatter disease. Although knee pain is most likely to occur in people over 60 years old, knee pain can also occur in children and adolescents. Knee pain can be treated at home following the RICE methods, however, severe knee injuries may require immediate medical attention, including chiropractic care.
DNA supports approximately 20,000 genes, each holding a program for the creation of a protein or enzyme required for a healthy lifestyle. Every one of these patterns needs to be constantly regulated by a sort of “promoter” which manages exactly how much of each substance and/or chemical is generated and under which conditions these will also develop.
By connecting to a particular kind of the switch-like promoter areas, known as the Antioxidant Response Element, or ARE, the Nrf2 factor�supports the speed of creation for hundreds of distinct genes which enable the cells to survive under stressful circumstances. These genes then generate a selection of antioxidant enzymes which develop a defense network by neutralizing oxidants and by cleaning up the toxic by-products left behind in their�production, in addition to helping restore the�damage they caused.
What is Oxidative Stress?
Several oxidants like the superoxide radical, or O2-., and hydrogen peroxide, or H2O2, have been created through the practice of burning off the substances and/or chemicals which sustain the human body. The human body�possesses antioxidant enzymes which�neutralize and detoxify reactive foods and drinks we consume. The Nrf2 modulates their production to keep equilibrium and underscores the demand for all these enzymes. This balance can be interrupted by a�couple of factors, including age.
As we age,�the human body creates less Nrf2 and this delicate equilibrium can gradually begin to�turn towards the oxidative side, a state referred to as oxidative stress. Disease may also cause the overproduction of oxidants. Infections, allergies, and autoimmune disorders can additionally trigger our immune cells to create reactive oxidants, such as O2-. , H2O2, OH and HOCl, where healthy cells become damaged and respond with inflammation. Diseases associated with aging, including heart attacks, stroke, cancer, and neurodegenerative conditions like Alzheimer’s disease, also increase the development of oxidants, generating stress and an inflammation response.
What are Nrf2 Activators?
The Nrf2 protein, also called a transcription factor due to the way it can support and control enzymes and genes, is the secret element of a sequence of biochemical reactions within the cell which reacts to modifications in cognitive equilibrium as well as oxidative balance. The sensing elements of this pathway modify and discharge Nrf2, triggering it so it might spread into the nucleus of the cell towards the DNA. The Nrf2 may alternatively turn on or switch off the genes and enzymes it supports to protect the cell.
Fortunately, a variety of substances which are Nrf2 activators develop through the consumption of certain plants and extracts utilized centuries ago in Chinese and Native American traditional remedies. These phytochemicals seem to be equally as powerful with fewer side-effects, as the Nrf2-activating pharmaceutical products which are being used today.
Nuclear factor erythroid 2-related factor, more commonly known as Nrf2, is a transcription factor which protects the cell by regulating genes, enzymes and antioxidant responses. Transcription factors are a type of protein which attach to DNA to promote the creation of specific substances and chemicals, including glutathione S-transferases, or GSTs. Nrf2 activation induces the production of active proteins which exhibit a powerful antioxidant capacity to help decrease oxidative stress.
Dr. Alex Jimenez D.C., C.C.S.T. Insight
The Science Behind Nrf2 Activation
Once the initial Nrf2-activating dietary supplement was created in 2004, minimal information was known concerning the function of the Nrf2 pathway. Approximately 200 newspapers in the literature on Nrf2, also known as nuclear factor-like 2 or NFE2L2, existed and researchers were only just starting to discover the antioxidant response of Nrf2 in mammals. As of 2017, however, over 9,300 academic research studies on this “master regulator,” have been printed.
In reality, Nrf2 regulates many antioxidant enzymes which don’t correlate to the genes, instead, they offer protection against a variety of stress-related circumstances which are encountered by cells, organs and ultimately organisms, under healthy and pathological conditions. Based on this new quantity of information from published academic research studies, researchers can now develop better Nrf2 dietary supplements.
As of 2007,�research studies have demonstrated the complex function of the Nrf2 pathway. Nrf2 activators have been found to mimic factors of different structures within the human body. Through these pathways, Nrf2 activators have been equipped to feel changing conditions throughout the cell in order to keep balance and respond to the evolving requirements of the genes.
Why Use Nrf2-Activating Supplements?
As Nrf2-activation abilities diminish with age in organisms, changes may begin to occur. Research studies have demonstrated that the focus of Nrf2 in cells declines with age, showing increased markers of oxidative stress. A variety of age-related diseases like atherosclerosis and cardiovascular disease, arthritis, cancer, obesity, type 2 diabetes, hypertension, cataracts, and Alzheimer’s disease as well as Parkinson’s diseases can develop due to these changes. Oxidative stress has been found with these health issues.
By stimulating the cell’s capacity to increase the production of Nrf2 activators, Nrf2 dietary supplements can help revive the human body’s own ability to counteract the effects of oxidative stress. Polyunsaturated fatty acids, or PUFAs, are one of the most readily oxidized molecules and they’re particularly vulnerable to suffer damage from free radicals. Thiobarbituric acid, or TBARS, production can increase with age, indicating heightened oxidative stress along with a drop in Nrf2-regulated pathways.
Biologically, gene induction is a really slow mechanism, generally requiring hours to transfer through a pathway. As a result,�many enzymes possess their very own on/off switches which could be triggered in minutes by different regulatory enzymes. Researchers have developed proprietary compositions of Nrf2 activators which utilize this knowledge base of activation. Nrf2 activation is composed not just of the Nrf2 transcription factor being discharged from its inhibitor and migrating to the cell nucleus, but also binding to specific DNA sequences to encourage cytoprotective gene expression, regulating the pace at that Nrf2 is taken out of the nucleus.
Understanding the elimination procedure and the activation of Nrf2 in the human body has allowed researchers to build combinations of different Nrf2 activators to accomplish the reflection of genes through its modulation. The combination of the knowledge base, together with the wide variety of other research studies has�helped produce Nrf2 activators for use as dietary supplements. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.
Curated by Dr. Alex Jimenez
Additional Topic Discussion: Relieving Knee Pain without Surgery
Knee pain is a well-known symptom which can occur due to a variety of knee injuries and/or conditions, including�sports injuries. The knee is one of the most complex joints in the human body as it is made-up of the intersection of four bones, four ligaments, various tendons, two menisci, and cartilage. According to the American Academy of Family Physicians, the most common causes of knee pain include patellar subluxation, patellar tendinitis or jumper’s knee, and Osgood-Schlatter disease. Although knee pain is most likely to occur in people over 60 years old, knee pain can also occur in children and adolescents. Knee pain can be treated at home following the RICE methods, however, severe knee injuries may require immediate medical attention, including chiropractic care.
Antioxidants are scientifically referred to as compounds which restrict the oxidation process in the human body, that if left unchecked, it can create free radicals which can develop numerous chain reactions that may cause cellular damage. Fortunately, the human body can create such built-in immune mechanisms, however, when mounting reactive oxygen species, or ROS, are unable to be neutralized, envision a tiny flame which gets out of control when infused with oxygen, harm is bound to occur.
To continue expanding on the metaphor of the flame, the final product of not having the ability to neutralize the impact of ROS, or reactive oxygen species, is damage as well as inflammation, in other words, the human body is quite literally on fire. The fantastic thing is there are antioxidants which can tremendously help fight this health issue and this antioxidant is glutathione. Though found in 1889, glutathione’s antioxidant effect has become one of the most interesting topics in modern research studies.
Master of Antioxidants: Glutathione
The powerful�substance is a tripeptide that develops from cysteine, glutamic acid, and glycine. Because of its capability to protect the human body against the creation of free radicals, glutathione can ultimately help promote a healthy immune system. Based on Scientific Reports in 2015, it was determined that glutathione’s capacity to function synergistically with peroxiredin and catalase helps guard cells against hydrogen peroxide. This synergistic formula functions against reactive oxygen species, or ROS. Glutathione, peroxidredin and catalase are essential elements in the increase of cellular homeostasis, which is an essential process of healthy cells, tissues and organs altogether.
Additionally, glutathione increases overall immune system structure and function utilizing its important effect on lymphocyte functions. According to the Department of Immunochemistry, properly supplementing levels of glutathione in the human body can greatly enhance immune reactions. By way of example, two randomized placebo-controlled trials demonstrated that the therapeutic treatment of immune-compromised patients with N-acetyl-cysteine, or NAC, resulted, in both cases, in a substantial growth in most immunological processes which included an entire rejuvenation of natural killer cell activity. N-acetyl-cysteine, or NAC, uses the sulfur from glutathione and combines it with poisonous molecules, which then become water-soluble and are discharged in the human body.
Glutathione also has the capability to revitalize lipoic acid as well as to recycle Vitamin C and E, which are necessary in order to initiate certain system processes by sending electrons to neutralize free radicals. Based on a research study from PLOS ONE, glutathione affected patients with diabetes metillus, or T2DM, and mycobacterium tuberculosis. Normally, individuals with weak immune systems have a tendency to show greater exposure to M. tb, or mycobacterium tuberculosis, disease or infection. Furthermore, individuals with Type 2 diabetes metilllus, or T2DM, are two to three times more prone to TB than people without T2DM. The research study also suggested that boosting the levels of glutathione in macrophages isolated from patients with T2DM led to improved control of M.Tb disease or infection. These results demonstrate that lower levels of glutathione in patients with T2DM contributes to a heightened chance of M. tb disease or infection. Moreover, dependent on Dietro Ghezzi in Brighton and Sussex Medical School, oxidative stress can ultimately cause poor immune system structure and function.
Fortunately, glutathione plays an essential role in strengthening and controlling immunity. By way of instance, glutathione is essential for innate and adaptive processes within the immune system, including T-lymphocyte proliferation, phagocytic activity of polymorphonuclear neutrophils, and dendritic cell functions, which can be fundamental because these are made-up of antigen-presenting cells. Cell-meditated immunity includes protein antigens which initially begin to degenerate in the endocytic vesicles of macrophages and dendritic cells, therefore, the smaller peptides are demonstrated on the surface to activate proliferation of antigen-specific T cells. In addition, glutathione helps with the creation of cytokines, and it is necessary to maintain interferon-gamma production by dendritic cells, which is important towards protecting against intracellular pathogens including mycobacteria.
N-acetyl-cysteine, or NAC, scientifically referred to as the precursor of glutathione, is also a very powerful cellular antioxidant used as a free radical scavenger antioxidant. Commonly recognized for its role in averting acetaminophen toxicity, NAC, or�N-acetyl-cysteine, has been demonstrated to possess several health and wellness benefits. According to Cell Journal, NAC helps support a healthy inflammatory response and may positively impact human term and preterm labors. The research study concluded that in women with previous preterm birth and bacterial vaginosis, 0.6 gram of NAC per day taken orally together with progesterone after week 16 of pregnancy shielded against preterm birth recurrence and improved neonatal outcome. In conclusion, NAC’s positive effects on muscle building was also detected. After three minutes of persistent contractions, there was a 15 percent enhanced output, demonstrating how NAC plays a fundamental role in improving muscle building and reducing overall fatigue during labour.
Researchers also discovered that NAC, or�N-acetyl-cysteine, may benefit those who have polycystic ovarian syndrome, or PCOS. PCOS, or�polycystic ovarian syndrome, is a common endocrine glands-related disease which impacts approximately 5 to 10 percent of reproductive-age women. In such patients, there is a greater risk of experiencing metabolic syndrome, where the use of NAC helped restore healthy insulin levels and sensitivity.
Dr. Alex Jimenez’s Insight
Glutathione has been referred to as the “master of antioxidants” due to its fundamental role in achieving and maintaining overall health and wellness. While the human body is capable of producing its own glutathione, poor nutrition, pollution, toxins, excessive use of drugs and/or medications, stress, trauma, aging, disease and radiation can all decrease our natural levels of glutathione. This can in turn make individuals more susceptible to cell damage from oxidative stress, free radicals, infections and cancer. Glutathione supplementation can therefore have tremendous benefits on the human body. Together with alternative treatment options, such as chiropractic care, glutathione levels can once again be regulated to improve well-being.
Additionally, healthcare professionals have suggested implementing the use of glutathione supplementation together with other alternative treatment options, such as chiropractic care, to further improve overall health and wellness. Antioxidants are important towards maintaining maximum well-being as well as to inhibit the chain reaction of free radicals that cause cell harm or damage. Powerful antioxidants like glutathione, as previously mentioned above, ultimately help regulate the development of these free radicals and provide a healthier immune system response. Research studies have found that chiropractic care may also play an essential role in this process, naturally boosting the activity of antioxidants in the human body. Chiropractic care is a safe and effective treatment approach which utilizes spinal adjustments and manual manipulations to correct spinal misalignments, or subluxations, in order to allow the human body to naturally heal itself without the use of drugs/medications and/or surgical interventions.
Finally, antioxidants demonstrate their biological properties through a great deal of health benefits, which might be necessary now more than ever with the every so increasing onslaught of stress, disease and pollution in our modern world, which all contribute to cell harm and/or damage. Glutathione and its precursor, NAC, or�N-acetyl-cysteine, continue to show their powerful status in the realm of antioxidants. Together with alternative treatment options, such as chiropractic care, people can take advantage of all the benefits that this powerful antioxidant has to offer. The scope of our information is limited to chiropractic as well as to spinal injuries and conditions. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.
Curated by Dr. Alex Jimenez
Additional Topics: Back Pain
Back pain is one of the most prevalent causes for disability and missed days at work worldwide. As a matter of fact, back pain has been attributed as the second most common reason for doctor office visits, outnumbered only by upper-respiratory infections. Approximately 80 percent of the population will experience some type of back pain at least once throughout their life. The spine is a complex structure made up of bones, joints, ligaments and muscles, among other soft tissues. Because of this, injuries and/or aggravated conditions, such as herniated discs, can eventually lead to symptoms of back pain. Sports injuries or automobile accident injuries are often the most frequent cause of back pain, however, sometimes the simplest of movements can have painful results. Fortunately, alternative treatment options, such as chiropractic care, can help ease back pain through the use of spinal adjustments and manual manipulations, ultimately improving pain relief.
Science based Chiropractor Dr. Alexander Jimenez takes a look at oxidative stress, what it is, how it affects the body and the antioxidant defense to remedy the situation.
Esra Birben PhD,1 Umit Murat Sahiner MD,1 Cansin Sackesen MD,1 Serpil Erzurum MD,2 and Omer Kalayci, MD1
Abstract: Reactive oxygen species (ROS) are produced by living organisms as a result of normal cellular metabolism and environ- mental factors, such as air pollutants or cigarette smoke. ROS are highly reactive molecules and can damage cell structures such as carbohydrates, nucleic acids, lipids, and proteins and alter their functions. The shift in the balance between oxidants and antioxidants in favor of oxidants is termed �oxidative stress.� Regulation of reducing and oxidizing (redox) state is critical for cell viability, activation, proliferation, and organ function. Aerobic organisms have integrated antioxidant systems, which include enzymatic and non- enzymatic antioxidants that are usually effective in blocking harmful effects of ROS. However, in pathological conditions, the antioxidant systems can be overwhelmed. Oxidative stress contributes to many pathological conditions and diseases, including cancer, neurological disorders, atherosclerosis, hypertension, ischemia/perfusion, diabetes, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and asthma. In this review, we summarize the cellular oxidant and antioxidant systems and discuss the cellular effects and mechanisms of the oxidative stress.
Reactive oxygen species (ROS) are produced by living organisms as a result of normal cellular metabolism. At low to moderate concentrations, they function in physiological cell processes, but at high concentrations, they produce adverse modifications to cell components, such as lipids, proteins, and DNA.1�6 The shift in balance between oxidant/ antioxidant in favor of oxidants is termed �oxidative stress.� Oxidative stress contributes to many pathological conditions, including cancer, neurological disorders,7�10 atherosclerosis, hypertension, ischemia/perfusion,11�14 diabetes, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease,15 and asthma.16�21 Aerobic organisms have integrated antioxidant systems,� which include enzymatic and nonenzymatic antioxidants that are usually effective in blocking harmful effects of ROS. However, in pathological conditions, the antioxidant systems can be overwhelmed. In this review, we summarize the cellular oxidant and antioxidant systems and regulation of the reducing and oxidizing (redox) state in health and disease states.
OXIDANTS
Endogenous Sources of ROS
ROS are produced from molecular oxygen as a result of normal cellular metabolism. ROS can be divided into 2 groups: free radicals and nonradicals. Molecules containing one or more unpaired electrons and thus giving reactivity to the molecule are called free radicals. When 2 free radicals share their unpaired electrons, nonradical forms are created. The 3 major ROS that are of physiological significance are superoxide anion (O22.), hydroxyl radical ( OH), and hydro- gen peroxide (H2O2). ROS are summarized in Table 1.
Superoxide anion is formed by the addition of 1 electron to the molecular oxygen.22 This process is mediated by nicotine adenine dinucleotide phosphate [NAD(P)H] oxidase or xanthine oxidase or by mitochondrial electron trans- port system. The major site for producing superoxide anion is the mitochondria, the machinery of the cell to produce adenosine triphosphate. Normally, electrons are transferred through mitochondrial electron transport chain for reduction of oxygen to water, but approximately 1 to 3% of all electrons leak from the system and produce superoxide. NAD(P)H oxidase is found in polymorphonuclear leukocytes, monocytes, and macrophages. Upon phagocytosis, these cells produce a burst of superoxide that lead to bactericidal activity. Superoxide is converted into hydrogen peroxide by the action of superoxide dismutases (SODs, EC 1.15.1.1). Hydrogen peroxide easily diffuses across the plasma membrane. Hydrogen peroxide is also produced by xanthine oxidase, amino acid oxidase, and NAD(P)H oxidase�23,24 and in peroxisomes by consumption of molecular oxygen in metabolic reactions. In a succession of reactions called Haber�Weiss and Fenton reactions,H2O2 can breakdown to OH2 in the presence of transmission metals like Fe21 or Cu21.25
Fe31 +�.O2�?Fe2 +�O2 Haber Weiss
Fe2 +�H2O2�?Fe3 +�OH�+ .OH Fenton reaction
O 2 �itself can also react with H2 O2 and generate OH�.26,27 Hydroxyl radical is the most reactive of ROS and can damage proteins, lipids, and carbohydrates and DNA. It can also start lipid peroxidation by taking an electron from polyunsaturated fatty acids.
Granulocytic enzymes further expand the reactivity of H2O2 via eosinophil peroxidase and myeloperoxidase (MPO). In activated neutrophils, H2O2 is consumed by MPO. In the presence of chloride ion, H2O2 is converted to hypochlorous acid (HOCl). HOCl is highly oxidative and plays an important role in killing of the pathogens in the airways.28 However, HOCl can also react with DNA and induce DNA�protein interactions and produce pyrimidine oxidation products and add chloride to DNA bases.29,30 Eosinophil peroxidase and MPO also contribute to the oxidative stress by modification of proteins by halogenations, nitration, and protein cross-links via tyrosyl radicals.31�33
Other oxygen-derived free radicals are the peroxyl radicals (ROO$ ). Simplest form of these radicals is hydro- peroxyl radical (HOO$ ) and has a role in fatty acid peroxidation. Free radicals can trigger lipid peroxidation chain reactions by abstracting a hydrogen atom from a side- chain methylene carbon. The lipid radical then reacts with oxygen to produce peroxyl radical. Peroxyl radical initiates a chain reaction and transforms polyunsaturated fatty acids into lipid hydroperoxides. Lipid hydroperoxides are very unstable and easily decompose to secondary products, such as aldehydes (such as 4-hydroxy-2,3-nonenal) and malondialdehydes (MDAs). Isoprostanes are another group of lipid peroxidation products that are generated via the peroxidation of arachidonic acid and have also been found to be elevated in plasma and breath condensates of asthmatics.34,35 Peroxidation of lipids disturbs the integrity of cell membranes and leads to rearrangement of membrane structure.
Hydrogen peroxide, superoxide radical, oxidized glutathione (GSSG), MDAs, isoprostanes, carbonyls, and nitrotyrosine can be easily measured from plasma, blood, or bronchoalveolar lavage samples as biomarkers of oxidation by standardized assays.
Exogenous Source of Oxidants
Cigarette Smoke
Cigarette smoke contains many oxidants and free radicals and organic compounds, such as superoxide and nitric oxide.36 In addition, inhalation of cigarette smoke into the lung also activates some endogenous mechanisms, such as accumulation of neutrophils and macrophages, which further increase the oxidant injury.
Ozone Exposure
Ozone exposure can cause lipid peroxidation and induce influx of neutrophils into the airway epithelium. Short-term exposure to ozone also causes the release of inflammatory mediators, such as MPO, eosinophil cationic proteins and also lactate dehydrogenase and albumin.37 Even in healthy subjects, ozone exposure causes a reduction in pulmonary functions.38 Cho et al39 have shown that particulate matter (mixture of solid particles and liquid droplets suspended in the air) catalyzes the reduction of oxygen.
Hyperoxia
Hyperoxia refers to conditions of higher oxygen levels than normal partial pressure of oxygen in the lungs or other body tissues. It leads to greater production of reactive oxygen and nitrogen species.40,41
Ionizing Radiation
Ionizing radiation, in the presence of O2, converts hydroxyl radical, superoxide, and organic radicals to hydrogen peroxide and organic hydroperoxides. These hydroperoxide species react with redox active metal ions, such as Fe and Cu, via Fenton reactions and thus induce oxidative stress.42,43 Narayanan et al44 showed that fibroblasts that were exposed to alpha particles had significant increases in intracellular O2 2. and H2O2 production via plasma membrane-bound NADPH oxidase.44 Signal transduction molecules, such as extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38, and transcription factors, such as activator protein-1 (AP-1), nuclear factor-kB (NF-kB), and p53, are activated, which result in the expression of radiation response�related genes.45�50 Ultraviolet A (UVA) photons trigger oxidative reactions by excitation of endogenous photosensitizers, such as porphyrins, NADPH oxidase, and riboflavins. 8-Oxo-7,8- dihydroguanine (8-oxoGua) is the main UVA-mediated DNA oxidation product formed by the oxidation of OH radical, 1-electron oxidants, and singlet oxygen that mainly reacts with guanine.51 The formation of guanine radical cation in isolated DNA has been shown to efficiently occur through the direct effect of ionizing radiation.52,53 After exposure to ionizing radiation, intracellular level of glutathione (GSH) decreases for a short term but then increases again.54
Heavy Metal Ions
Heavy metal ions, such as iron, copper, cadmium, mercury, nickel, lead, and arsenic, can induce generation of reactive radicals and cause cellular damage via depletion of enzyme activities through lipid peroxidation and reaction with nuclear proteins and DNA.55
One of the most important mechanisms of metal- mediated free radical generation is via a Fenton-type reaction. Superoxide ion and hydrogen peroxide can interact with transition metals, such as iron and copper, via the metal catalyzed Haber�Weiss/Fenton reaction to form OH radicals.
Besides the Fenton-type and Haber�Weiss-type mechanisms, certain metal ions can react directly with cellular molecules to generate free radicals, such as thiol radicals, or induce cell signaling pathways. These radicals may also react with other thiol molecules to generate O22.. O22. is converted to H2O2, which causes additional oxygen radical generation. Some metals, such as arsenite, induce ROS formation indirectly by activation of radical producing systems in cells.56
Arsenic is a highly toxic element that produces a variety of ROS, including superoxide (O2 2), singlet oxygen (1O2), peroxyl radical (ROO ), nitric oxide (NO ), hydrogen peroxide (H2O2), and dimethylarsinic peroxyl radicals [(CH3)2AsOO ].57�59 Arsenic (III) compounds can inhibit antioxidant enzymes, especially the GSH-dependent enzymes, such as glutathione-S-transferases (GSTs), glutathione peroxidase (GSH-Px), and GSH reductase, via bind- ing to their sulfhydryl (�SH) groups.60,61
Lead increases lipid peroxidation.62 Significant decreases in the activity of tissue SOD and brain GPx have been reported after lead exposure.63,64 Replacement of zinc, which serves as a cofactor for many enzymes by lead, leads to inactivation of such enzymes. Lead exposure may cause inhibition of GST by affecting tissue thiols.
ROS generated by metal-catalyzed reactions can mod- ify DNA bases. Three base substitutions, G / C, G / T, and C / T, can occur as a result of oxidative damage by metal ions, such as Fe21, Cu21, and Ni21. Reid et al65 showed that G / C was predominantly produced by Fe21 while C / T substitution was by Cu21 and Ni21.
ANTIOXIDANTS
The human body is equipped with a variety of antioxidants that serve to counterbalance the effect of oxidants. For all practical purposes, these can be divided into 2 categories: enzymatic (Table 2) and nonenzymatic (Table 3).
Enzymatic Antioxidants
The major enzymatic antioxidants of the lungs are SODs (EC 1.15.1.11), catalase (EC 1.11.1.6), and GSH-Px (EC 1.11.1.9). In addition to these major enzymes, other antioxidants, including heme oxygenase-1 (EC 1.14.99.3), and redox proteins, such as thioredoxins (TRXs, EC 1.8.4.10), peroxiredoxins (PRXs, EC 1.11.1.15), and glutaredoxins, have also been found to play crucial roles in the pulmonary antioxidant defenses.
Since superoxide is the primary ROS produced from a variety of sources, its dismutation by SOD is of primary importance for each cell. All 3 forms of SOD, that is, CuZn- SOD, Mn-SOD, and EC-SOD, are widely expressed in the human lung. Mn-SOD is localized in the mitochondria matrix. EC-SOD is primarily localized in the extracellular matrix, especially in areas containing high amounts of type I collagen fibers and around pulmonary and systemic vessels. It has also been detected in the bronchial epithelium, alveolar epithelium, and alveolar macrophages.66,67 Overall, CuZn- SOD and Mn-SOD are generally thought to act as bulk scavengers of superoxide radicals. The relatively high EC-SOD level in the lung with its specific binding to the extracellular matrix components may represent a fundamental component of lung matrix protection.68
H2O2 that is produced by the action of SODs or the action of oxidases, such as xanthine oxidase, is reduced to water by catalase and the GSH-Px. Catalase exists as a tetra- mer composed of 4 identical monomers, each of which con- tains a heme group at the active site. Degradation of H2O2 is accomplished via the conversion between 2 conformations of catalase-ferricatalase (iron coordinated to water) and com- pound I (iron complexed with an oxygen atom). Catalase also binds NADPH as a reducing equivalent to prevent oxidative inactivation of the enzyme (formation of compound II) by H2O2 as it is reduced to water.69
Enzymes in the redox cycle responsible for the reduction of H2O2 and lipid hydroperoxides (generated as a result of membrane lipid peroxidation) include the GSH-Pxs.70 The GSH-Pxs are a family of tetrameric enzymes that contain the unique amino acid selenocysteine within the active sites and use low-molecular-weight thiols, such as GSH, to reduce H2O2 and lipid peroxides to their corresponding alcohols. Four GSH- Pxs have been described, encoded by different genes: GSH- Px-1 (cellular GSH-Px) is ubiquitous and reduces H2O2 and fatty acid peroxides, but not esterified peroxyl lipids.71 Esterified lipids are reduced by membrane-bound GSH-Px-4 (phospholipid hydroperoxide GSH-Px), which can use several different low-molecular-weight thiols as reducing equivalents. GSH-Px-2 (gastrointestinal GSH-Px) is localized in gastrointestinal epithelial cells where it serves to reduce dietary peroxides.72 GSH-Px-3 (extracellular GSH-Px) is the only member of the GSH-Px family that resides in the extracellular compartment and is believed to be one of the most important extracellular antioxidant enzyme in mammals. Of these, extracellular GSH-Px is most widely investigated in the human lung.73
In addition, disposal of H2O2 is closely associated with several thiol-containing enzymes, namely, TRXs (TRX1 and TRX2), thioredoxin reductases (EC 1.8.1.9) (TRRs), PRXs (which are thioredoxin peroxidases), and glutaredoxins.74
Two TRXs and TRRs have been characterized in human cells, existing in both cytosol and mitochondria. In the lung, TRX and TRR are expressed in bronchial and alveolar epithelium and macrophages. Six different PRXs have been found in human cells, differing in their ultrastructural compartmentalization. Experimental studies have revealed the importance of PRX VI in the protection of alveolar epithelium. Human lung expresses all PRXs in bronchial epithelium, alveolar epithelium, and macrophages.75 PRX V has recently been found to function as a peroxynitrite reductase,76 which means that it may function as a potential protective compound in the development of ROS-mediated lung injury.77
Common to these antioxidants is the requirement of NADPH as a reducing equivalent. NADPH maintains catalase in the active form and is used as a cofactor by TRX and GSH reductase (EC 1.6.4.2), which converts GSSG to GSH, a co-substrate for the GSH-Pxs. Intracellular NADPH, in turn, is generated by the reduction of NADP1 by glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the pen- tose phosphate pathway, during the conversion of glucose- 6-phosphate to 6-phosphogluconolactone. By generating NADPH, glucose-6-phosphate dehydrogenase is a critical determinant of cytosolic GSH buffering capacity (GSH/ GSSG) and, therefore, can be considered an essential, regulatory antioxidant enzyme.78,79
GSTs (EC 2.5.1.18), another antioxidant enzyme family, inactivate secondary metabolites, such as unsaturated aldehydes, epoxides, and hydroperoxides. Three major families of GSTs have been described: cytosolic GST, mitochondrial GST,80,81 and membrane-associated microsomal GST that has a role in eicosanoid and GSH metabolism.82 Seven classes of cytosolic GST are identified in mammalian, designated Alpha, Mu, Pi, Sigma, Theta, Omega, and Zeta.83�86 During non-stressed conditions, class Mu and Pi GSTs interact with kinases Ask1 and JNK, respectively, and inhibit these kinases.87�89 It has been shown that GSTP1 dissociates from JNK in response to oxidative stress.89 GSTP1 also physically interacts with PRX VI and leads to recovery of PRX enzyme activity via glutathionylation of the oxidized protein.90
Nonenzymatic Antioxidants
Nonenzymatic antioxidants include low-molecular-weight compounds, such as vitamins (vitamins C and E), b-carotene, uric acid, and GSH, a tripeptide (L-g-glutamyl-L-cysteinyl-L- glycine) that comprise a thiol (sulfhydryl) group.
Vitamin C (Ascorbic Acid)
Water-soluble vitamin C (ascorbic acid) provides intracellular and extracellular aqueous-phase antioxidant capacity primarily by scavenging oxygen free radicals. It converts vitamin E free radicals back to vitamin E. Its plasma levels have been shown to decrease with age.91,92
Vitamin E (a-Tocopherol)
Lipid-soluble vitamin E is concentrated in the hydrophobic interior site of cell membrane and is the principal defense against oxidant-induced membrane injury. Vitamin E donates electron to peroxyl radical, which is produced during lipid peroxidation. a-Tocopherol is the most active form of vitamin E and the major membrane-bound antioxidant in cell. Vitamin E triggers apoptosis of cancer cells and inhibits free radical formations.93
Glutathione
GSH is highly abundant in all cell compartments and is the major soluble antioxidant. GSH/GSSG ratio is a major determinant of oxidative stress. GSH shows its antioxidant effects in several ways.94 It detoxifies hydrogen peroxide and lipid peroxides via action of GSH-Px. GSH donates its electron to H2O2 to reduce it into H2O and O2. GSSG is again reduced into GSH by GSH reductase that uses NAD(P)H as the electron donor. GSH-Pxs are also important for the pro- tection of cell membrane from lipid peroxidation. Reduced glutathione donates protons to membrane lipids and protects them from oxidant attacks.95
GSH is a cofactor for several detoxifying enzymes, such as GSH-Px and transferase. It has a role in converting vitamin C and E back to their active forms. GSH protects cells against apoptosis by interacting with proapoptotic and antiapoptotic signaling pathways.94 It also regulates and activates several transcription factors, such as AP-1, NF-kB, and Sp-1.
Carotenoids (b-Carotene)
Carotenoids are pigments found in plants. Primarily, b-carotene has been found to react with peroxyl (ROO ), hydroxyl ( OH), and superoxide (O22.) radicals.96 Carotenoids show their antioxidant effects in low oxygen partial pressure but may have pro-oxidant effects at higher oxygen concentrations.97 Both carotenoids and retinoic acids (RAs) are capable of regulating transcription factors.98 b-Carotene inhibits the oxidant-induced NF-kB activation and interleukin (IL)-6 and tumor necrosis factor-a production. Carotenoids also affect apoptosis of cells. Antiproliferative effects of RA have been shown in several studies. This effect of RA is mediated mainly by retinoic acid receptors and vary among cell types. In mammary carcinoma cells, retinoic acid receptor was shown to trigger growth inhibition by inducing cell cycle arrest, apoptosis, or both.99,100
THE EFFECT OF OXIDATIVE STRESS: GENETIC, PHYSIOLOGICAL, & BIOCHEMICAL MECHANISMS
Oxidative stress occurs when the balance between antioxidants and ROS are disrupted because of either depletion of antioxidants or accumulation of ROS. When oxidative stress occurs, cells attempt to counteract the oxidant effects and restore the redox balance by activation or silencing of genes encoding defensive enzymes, tran- scription factors, and structural proteins.101,102 Ratio between oxidized and reduced glutathione (2GSH/GSSG) is one of the important determinants of oxidative stress in the body. Higher production of ROS in body may change DNA structure, result in modification of proteins and lipids, activation of several stress-induced transcription factors, and production of pro-inflammatory and anti-inflammatory cytokines.
Effects Of Oxidative Stress On DNA
ROS can lead to DNA modifications in several ways, which involves degradation of bases, single- or double- stranded DNA breaks, purine, pyrimidine or sugar-bound modifications, mutations, deletions or translocations, and cross-linking with proteins. Most of these DNA modifications (Fig. 1) are highly relevant to carcinogenesis, aging, and neurodegenerative, cardiovascular, and autoimmune diseases. Tobacco smoke, redox metals, and nonredox metals, such as iron, cadmium, chrome, and arsenic, are also involved in carcinogenesis and aging by generating free radicals or bind- ing with thiol groups. Formation of 8-OH-G is the best- known DNA damage occurring via oxidative stress and is a potential biomarker for carcinogenesis.
Promoter regions of genes contain consensus sequences for transcription factors. These transcription factor�binding sites contain GC-rich sequences that are susceptible for oxidant attacks. Formation of 8-OH-G DNA in transcription factor binding sites can modify binding of transcription factors and thus change the expression of related genes as has been shown for AP-1 and Sp-1 target sequences.103 Besides 8-OH-G, 8,59 -cyclo-29 -deoxyadenosine (cyclo-dA) has also been shown to inhibit transcription from a reporter gene in a cell system if located in a TATA box.104 The TATA-binding protein initiates transcription by changing the bending of DNA. The binding of TATA-binding protein may be impaired by the presence of cyclo-dA.
Oxidative stress causes instability of microsatellite (short tandem repeats) regions. Redox active metal ions, hydroxyl radicals increase microsatellite instability.105 Even though single-stranded DNA breaks caused by oxidant injury can easily be tolerated by cells, double-stranded DNA breaks induced by ionizing radiation can be a significant threat for the cell survival.106
Methylation at CpG islands in DNA is an important epigenetic mechanism that may result in gene silencing. Oxidation of 5-MeCyt to 5-hydroxymethyl uracil (5-OHMeUra) can occur via deamination/oxidation reactions of thymine or 5-hydroxymethyl cytosine intermediates.107 In addition to the modulating gene expression, DNA methylation also seems to affect chromatin organization.108 Aberrant DNA methylation patterns induced by oxidative attacks also affect DNA repair activity.
Effects Of Oxidative Stress On Lipids
ROS can induce lipid peroxidation and disrupt the membrane lipid bilayer arrangement that may inactivate membrane-bound receptors and enzymes and increase tissue permeability.109 Products of lipid peroxidation, such as MDA and unsaturated aldehydes, are capable of inactivating many cellular proteins by forming protein cross-linkages.110�112 4-Hydroxy-2-nonenal causes depletion of intracellular GSH and induces of peroxide production,113,114 activates epidermal growth factor receptor,115 and induces fibronectin production.116 Lipid peroxidation products, such as isoprostanes and thiobarbituric acid reactive substances, have been used as indirect biomarkers of oxidative stress, and increased levels were shown in the exhaled breath condensate or bronchoalveolar lavage fluid or lung of chronic obstructive pulmonary disease patients or smokers.117�119
Effects Of Oxidative Stress on Proteins
ROS can cause fragmentation of the peptide chain, alteration of electrical charge of proteins, cross-linking of proteins, and oxidation of specific amino acids and therefore lead to increased susceptibility to proteolysis by degradation by specific proteases.120 Cysteine and methionine residues in proteins are particularly more susceptible to oxidation.121 Oxidation of sulfhydryl groups or methionine residues of proteins cause conformational changes, protein unfolding, and degradation.8,121�123 Enzymes that have metals on or close to their active sites are especially more sensitive to metal catalyzed oxidation. Oxidative modification of enzymes has been shown to inhibit their activities.124,125
In some cases, specific oxidation of proteins may take place. For example, methionine can be oxidized methionine sulfoxide126 and phenylalanine to o-tyrosine127; sulfhydryl groups can be oxidized to form disulfide bonds;128 and carbonyl groups may be introduced into the side chains of proteins. Gamma rays, metal-catalyzed oxidation, HOCl, and ozone can cause formation of carbonyl groups.129
Effects of Oxidative Stress on Signal Transduction
ROS can induce expression of several genes involved in signal transduction.1,130 A high ratio for GSH/GSSG is important for the protection of the cell from oxidative dam- age. Disruption of this ratio causes activation of redox sensitive transcription factors, such as NF-kB, AP-1, nuclear factor of activated T cells and hypoxia-inducible factor 1 , that are involved in the inflammatory response. Activation of transcription factors via ROS is achieved by signal transduction cascades that transmit the information from outside to the inside of cell. Tyrosine kinase receptors, most of the growth factor receptors, such as epidermal growth factor receptor, vascular endothelial growth factor receptor, and receptor for platelet-derived growth factor, protein tyrosine phosphatases, and serine/threonine kinases are targets of ROS.131�133 Extra- cellular signal-regulated kinases, JNK, and p38, which are the members of mitogen-activated protein kinase family and involved in several processes in cell including proliferation, differentiation, and apoptosis, also can be regulated by oxidants.
Under oxidative stress conditions, cysteine residues in the DNA-binding site of c-Jun, some AP-1 subunits, and inhibitory k-B kinase undergo reversible S-glutathiolation. Glutaredoxin and TRX have been reported to play an important role in regulation of redox-sensitive signaling pathways, such as NF-kB and AP-1, p38 mitogen-activated protein kinase, and JNK.134�137
NF-kB can be activated in response to oxidative stress conditions, such as ROS, free radicals, and UV irradiation.138 Phosphorylation of IkB frees NF-kB and allows it to enter the nucleus to activate gene transcription.139 A number of kinases have been reported to phosphorylate IkBs at the serine residues. These kinases are the targets of oxidative signals for activation of NF-kB.140 Reducing agents enhance NF-kB DNA binding, whereas oxidizing agents inhibit DNA binding of NF-kB. TRX may exert 2 opposite actions in regulation of NF-kB: in the cytoplasm, it blocks degradation of IkB and inhibits NF-kB activation but enhances NF-kB DNA binding in the nucleus.141 Activation of NF-kB via oxidation-related degradation of IkB results in the activation of several antioxidant defense�related genes. NF-kB regulates the expression of several genes that participate in immune response, such as IL-1b, IL-6, tumor necrosis factor-a, IL-8, and several adhesion molecules.142,143 NF-kB also regulates angiogenesis and proliferation and differentiation of cells.
AP-1 is also regulated by redox state. In the presence of H2O2, some metal ions can induce activation of AP-1. Increase in the ratio of GSH/GSSG enhances AP-1 binding while GSSG inhibits the DNA binding of AP-1.144 DNA binding of the Fos/Jun heterodimer is increased by the reduction of a single conserved cysteine in the DNA-binding domain of each of the proteins,145 while DNA binding of AP-1 can be inhibited by GSSG in many cell types, suggesting that disulphide bond formation by cysteine residues inhibits AP-1 DNA binding.146,147 Signal transduction via oxidative stress is summarized in Figure 2.
CONCLUSIONS
Oxidative stress can arise from overproduction of ROS by metabolic reactions that use oxygen and shift the balance between oxidant/antioxidant statuses in favor of the oxidants. ROS are produced by cellular metabolic activities and environmental factors, such as air pollutants or cigarette smoke. ROS are highly reactive molecules because of unpaired electrons in their structure and react with several biological macromolecules in cell, such as carbohydrates, nucleic acids, lipids, and proteins, and alter their functions. ROS also affects the expression of several genes by upregulation of redox-sensitive transcription factors and chromatin remodeling via alteration in histone acetylation/ deacetylation. Regulation of redox state is critical for cell viability, activation, proliferation, and organ function.
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