Redox Signaling, mitochondrial metabolism, epigenetics and redox active phytochemicals Mini-review

Abstract

Biological redox signaling plays an important role in many diseases. Redox signaling involves reductive and oxidative mechanisms. Oxidative stress occurs when reductive mechanism overwhelms oxidative challenges. Cellular oxidative stress occurs when reactive oxygen/nitrogen species (RO/NS) exceed the cellular reductive/antioxidant capacity. Endogenously produced RO/NS from mitochondrial metabolic citric-acid-cycle coupled with electron-transport-chain or exogenous stimuli trigger cellular signaling events leading to homeostatic response or pathological damage. Recent evidence suggests that RO/NS also modulate epigenetic machinery driving gene expression. RO/NS affect DNA methylation/ demethylation, histone acetylation/deacetylation or histone methylation/demethylation. Many health beneficial phytochemicals possess redox capability that counteract RO/NS either by directly scavenging the radicals or via inductive mechanism of cellular defense antioxidant/reductive enzymes. Amazingly, these phytochemicals also possess epigenetic modifying ability. This review summarizes the latest advances on the interactions between redox signaling, mitochondrial metabolism, epigenetics and redox active phytochemicals and the future challenges of integrating these events in human health.

1. Background

Cellular reduction/oxidation (redox) state regulates diverse cellular functions ranging from homeostasis, differentiation, proliferation to apoptosis. The term and concept of “redox” for electron activity in redox reaction was first introduced by WM Clark (1922) as highlighted in DC Thorstenson’s “The concept of electron activity and its relation to redox potentials in aqueous geochemical systems” in 1984 (1). Redox is a type of basic chemical reaction in which the oxidation states of atoms are altered. Redox reactions are characterized by the transfer of electrons between chemical species, most often with one specie undergoing oxidation (loss of electrons) while another specie undergoes reduction (gain of electrons).

Following the discovery of redox for basic chemical reaction, in biological systems, oxidative stress was first introduced in 1985 by H. Sies (2). Oxidative stress occurs when the reductive mechanism is overwhelmed by the oxidative challenge. Cellular redox oxidative stress occurs when reactive oxygen/nitrogen species (RO/NS) including superoxide (O⁻ ₂ •), hydroxyl ((OH•), hydrogen peroxide (H₂O₂), phenoxyl, nitric oxide (NO), nitrogen dioxide (NO⁻), peroxynitrite (OONO⁻), dinitrogen trioxide (N₂O₃), and nitrous acid (HNO₂) radicals and others, exceed the cellular reductive/antioxidant capacity like glutathione (GSH), catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX), thioredoxin, peroxiredoxin, among others (we will use RO/NS from here on to represent either ROS or RNS, unless stated otherwise). Importantly RO/NS are also secondary messengers triggering cellular signaling pathways in maintaining intracellular redox state of balance leading to normal physiologic homeostatic responses (reviewed in (3)).

Endogenous RO/NS are produced through everyday respiration in the mitochondrial citric acid cycle (CAC), also known as the TCA cycle (tricarboxylic acid cycle) or the Krebs cycle, which was first identified/discovered by Fisher and Krebs back in 1955 (4) coupled to the electron transport chain (ETC) (5) (note that we will use CAC from here on). The CAC is a series of chemical reactions used by all aerobic organisms for cellular energy adenosine triphosphate (ATP) production through the oxidation of acetyl-coenzyme A (CoA) derived from glucose, fatty acids and amino acids. The CAC cycle produces a huge flux of free radicals by being coupled to the ETC (6) with vast majority of the free radicals such as superoxide (O⁻ ₂ •), and hydrogen peroxide (H₂O₂) being neutralized by cellular antioxidant enzymes like SOD, GPX and catalase with potentially 0.1% leakage (5). In addition to mitochondria, endogenous production of RO/NS occurs in other cellular compartments including peroxisomal β-oxidation of fatty acids, respiratory burst by phagocytic cells and cytochrome P450 metabolic reactions (3, 5). Exogenous sources of RO/NS include UV light, tobacco smoke, and other environmental factors including air pollution, contaminated drinking water and certain “unhealthy high-fat” dietary food, among others.

RO/NS in general can trigger cellular signal transduction events to maintain cellular redox homeostasis. In prokaryotic bacteria system, OxyR redox sensing protein which is typically in a reducing environment such as E. coli, is highly responsive to perturbation of RO/NS. When the cells are challenged by H₂O_2, for instance, OxyR undergoes transcriptional activation by thiol-disulfide bond formation of cysteines (Cys) 199 and 208, resulting in induction of glutaredoxin (GrxA/Grx1) and glutathione reductase (GR) to counteract H₂O₂ (7). The biological specificity and activity of H₂O₂ may result from the diverse thiol sensing molecules/proteins in the different cellular compartments, with different reactivity of the respective sulfhydryl groups towards H₂O₂, activated by varying concentrations and times of exposure (8). This reactivity/sensitivity or dose response such as H₂O₂, may range from the most sensitive/responsive with catalase, perR, peroxiredoxin 2/5, GAPDH, CDC2B, Keap1, PTP25B, thioredoxin to GSH depending on the RO/NS, triggering respective signaling responsive pathways accordingly, in a concentration-dependent fashion (8). In mammalian cells, H₂O₂ activates signaling molecules including AP-1, CREB, HSF1, HIF-1, NF-κB, NOTCH, NRF2-KEAP1, SP1, TP53 and SCREB-1, typically through thiol-sulfhydryl modification (8, 9), in many in vitro or cell culture settings. For instance, many redox active electrophilic compounds activate NRF2-KEAP1 anti-oxidative stress pathway via thiol-sulfhydryl modification leading to induction of cellular defense and antioxidant enzymes GST, HO-1 and NQO1 (10). Redox regulation of signaling protein kinases by phosphorylation/dephosphorylation, could be governed by the redox sensitivity of protein tyrosine phosphatases (PTPs). Oxidation by ROS via thiol-disulfide bonding would inactivate PTPs resulting in increased steady-state protein phosphorylation. Examples include CDC25 phosphatases, PTEN (phosphatase and tensin homolog) and PTP1B (protein tyrosine phosphatase 1B) (11). In this context, redox active electrophilic compounds could modulate I-κB kinase (IKK) /I-κB/NF-κB pathway via protein phosphatase 2A (PP2A) that modulates IKK (12–14).

2. Role of redox in the modulation of epigenetic machinery

Cellular metabolism plays an important role in chromatin remodeling and epigenetic reprogramming (reviewed in (15–17)). Several epigenetic marks, including DNA methylation, histone acetylation, histone methylation, long non-coding microRNA and microRNA, and ADP-ribosylation are linked to central metabolism via redox intermediates.

DNA Methylation/demethylation:

DNA methylation, catalyzed by DNA methyltransferases (DNMT1, DNMT3A, DNMT3B), is a highly dynamic process and it profoundly impacts gene transcription, development, differentiation, and cellular responsiveness to environmental cues. Among all the epigenetic mechanisms, DNA CpG methylation is probably the most studied (reviewed in (18)). DNA methylation entails the addition of methyl groups onto C5 position of cytosine (C) residue usually occur in CpG dinucleotide sequences, dispersed across the genome, leading to the formation of 5-methylcytosine (5-mC) (19). In the context of redox signaling, RO/NS can epigenetically affect DNA methylation through DNMTs activity or gene expression. After H₂O₂ treatment, oxidative DNA damage induces the formation of a large complex(es) containing DNMTs and members of the Polycomb Repressive Complex 4, resulting in the alteration of the methylation status of CpG regions (20). In a redox oxidizing chromatin microenvironment and in the absence of S-adenosyl methionine (SAM), DNMT3A and DNMT3B may catalyze the direct conversion of 5-mC or 5-hydroxymethylcytosine (5-hmC) to an unmethylated cysteine (21). ROS-induced DNA damage of O⁶-methylguanine may inhibit binding of DNMT leading to hypomethylation and inhibit methylation of adjacent cytosine nucleotides (22). Incorporation of 8-hydroxy-deoxyglunine (8-OHdG) and the oxidation by-product of 5-mC in the Methyl-CpG Binding Protein (MBP) recognition sequence may result in the significant inhibition of the binding affinity of MBP (23). Since DNA methylation is a highly active process, and it can be oxidized to 5-hmC by DNA demethylases ten-eleven translocation enzymes (TETs) (24). In general 5-mC suppresses gene transcription, however, 5-hmC appears to activate gene transcription (25). ROS may regulate DNA methylation via expression and/or activity of TETs. Knockdown of TET1 significantly increase H₂O₂-induced apoptosis of cerebellar granule cells, and in Tet1 knockout mice, the cerebellar granule cells are more sensitive to oxidative stress (26).

Histone deacetylation:

RO/NS affects histone acetylation. To date, eighteen histone deacetylases (HDACs) have been identified in humans and classified into zinc/iron-dependent deacetylases class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 7, and 9), class IIB (HDACs 6 and 10), and class IV (HDAC11) (27) while class III HDACs (sirtuins 1–7) have an absolute requirement for NAD+ and are not zinc/iron-dependent for their activity (28). Several HDACs have been shown to be redox regulated. Redox-regulating protein thioredoxin 1 (TRX1) facilitates the reduction of oxidized signaling molecules and transcription factors by Cys’s thiol-disulfide exchange (29) including the nucleocytoplasmic shuttling of class II HDACs through a redox-dependent mechanism (30). TRX1 induces DnaJb5, a heat shock protein 40, and forms a multiple-protein complex with DnaJb5 and class II HDACs (30). In response to ROS/ H₂O₂-generating stimulus, both Cys-274/Cys-276 in DnaJb5 and Cys-667/Cys-669 in HDAC4 could be oxidized and form intramolecular disulfide bonds and both proteins could be reduced by TRX1 (30). Reduction of Cys-274/Cys-276 in DnaJb5 is essential for interaction between DnaJb5 and HDAC4, whereas reduction of Cys-667/Cys-669 in HDAC4 inhibits its nuclear export (30). Interestingly, several other Cys’s sulfhydryl (-SH) groups in HDACs appear to be sensitive to H₂O₂ in cardiomyocytes (30). Antioxidant protein peroxiredoxins (PRDX) serve not only as cellular defense against oxidative stress but also may act as redox relay for specific H₂O₂ signaling in cells (31). PRDX I and II are specific targets of HDAC6 and deacetylation of PRDXs by HDAC6 would decrease H₂O₂ reductive activity (32). Class I HDACs −1, −2 and −3 were postulated to be redox-sensitive with a putative redox-switch using chemical probes and covalent modification of HDAC1 at two conserved Cys residues, Cys-261 and Cys-273, coincided with attenuation of HDAC activity, changes in histone H3 and H4 acetylation patterns, de-repression of a LEF1/β-catenin model system, and transcription of HDAC-repressed genes, such as HO-1, Gadd45, and HSP70 (33). The catalytic activity of HDAC5 was found to suppress mitochondrial ROS generation and subsequent induction of NRF2-dependent antioxidant gene expression in cardiomyocytes (34). Cys-102 and Cys-153 of HDAC8, have been identified to be an integral components of the redox-switch in HDAC8, and disulfide bond formation under oxidizing conditions could be associated with a transition into a more stable protein resulting in a complete but reversible loss of enzyme activity (35). Collectively, there is strong evidence that redox RO/NS are capable of modulating chromatin accessibility by affecting HDAC/histone acetylation state in particular via multiple modifications of HDAC activity and expression. More studies would be needed to elucidate HDACs response to redox signaling.

Histone acetylation:

Histone acetylation is carried out by a group of histone acetyltransferases (HATs) including P300/CBP (CREB-binding protein) (36), enzymes that acetylate conserved lysine (K) residues on histone tails by transferring an acetyl group from acetyl-CoA to form ε-N-acetyl-lysine (37), resulting in decrease binding of histones to DNA, opening up the chromatin, and enhancing gene transcription. In E. coli, increasing the ratio of NADH to NAD+, inhibits pyruvate dehydrogenase (PDH) activity, and blocks formation of acetyl-CoA (38), since conversion of pyruvate to acetyl-CoA is catalyzed by PDH (39). In an in vitro study, increased ratio of NADH/NAD+ appears to be associated with the inhibition of PDH activity in pea (40). In maize seedlings, heat stress promotes accumulation of O₂−, and induces histone hyperacetylation due to the elevated expression of genes GCN5 (General Control Nondepressible 5) and HAT-B (41). In mammalian cells, H₂O₂ increases the activity of HATs and increases acetylation of H3/H4 histones (42, 43).

Histone methylation/demethylation:

Histone methylation is a highly complex process, which can lead to closed or open chromatin with increased or decreased transcription of genes (44) and is maintained by two classes of enzymes: histone methyltransferases (HMTs) and histone demethylases (HDMs) (45, 46). H₂O₂ increases several histone methylation marks including H3K4me3 and H3K27me3 while decreases histone acetylation marks including H3K9ac and H4K8ac (47). In diabetic-induced oxidative stress retinopathy model, H3K4me1, which enhances transcriptional activation, is increased at the promoter of KEAP1, and this is accompanied by increased HMT enzyme SET7/9 (SETD7) (48). Consequently, KEAP1 expression is increased, attenuating NRF2-mediated antioxidant enzyme expression (48). In diabetic nephropathy model, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which promotes oxidative stress, increases protein levels of SET7, promotes its nuclear translocation and enrichment at profibrotic gene promoters and SET7 (SETD7) gene silencing inhibited 12(S)-HETE-induced profibrotic gene expression (49). Under artery occlusion, H3K4 methyltransferase activity was decreased in astrocytes, with decreased H3K4me3 levels (50). H₂O₂ can oxidize C residues of protein arginine methyltransferases (PRMTs), decreases PRMT activity and methylation of histone arginine dose-dependently (51). RO/NS can affect HDMs, Lysine-specific demethylase (KDM) LSD1, generates H₂O₂ as a byproduct of its chromatin remodeling activity during the initial DNA damage response (52) and base excision repair proteins participate in epigenetic reprogramming and modulation of gene expression possibly via alteration of DNA methylation or histone modification (reviewed in (53)).

Overall, more studies would be needed on how different redox agents, dose response, acute versus chronic dosing, in vitro cell culture versus in vivo animal models, cell/tissue specificity and different pathological conditions can affect DNA methylation/demethylation, histone acetylation/methylation and functional gene expression.

3. Mitochondria metabolism, cellular metabolites and epigenetics

Cellular metabolism especially the mitochondrial CAC (Acetyl-CoA, NAD+/NADH, α-ketoglutarate, SAM) is tightly linked to DNA/histone modifications, chromatin remodeling (15–17) and modulating phenotypic gene expression, as shown in Figure 1. RO/NS, mitochondrial metabolites and epigenetics are highly interconnected, whether directly or indirectly. Many of the metabolites are cofactors utilized by the epigenetic enzymes that catalyze the post-translational epigenetic modification including SAM, the methyl donor for all epigenetic methylation reactions; acetyl-CoA, the acetyl (-COH) donor for histone acetylation; NAD+, the cofactor for class III HDACs sirtuins; α-ketoglutarate, the cofactor along with Fe(II) for chromatin/histones demethylases; 2-hydroxyglutarate (R-2HG), inhibitor of demethylases, among others (17), participate in the biological function of the basic epigenetic machinery. We will briefly summarize some of these metabolic co-factors in the context of oxidative stress.

Figure 1:

Interaction of mitochondrial & cellular metabolism with the epigenetic machinery. Acetyl-CoA, acetyl coenzyme A; CoA-SH, Coenzyme A with sulfhydryl (SH) functional group; NAD+/NADH, nicotinamide adenine dinucleotide oxidized (+)/reduced (H) form; JMJCs, Jumonji-C domain-containing histone lysine demethylases; SAM, S-adenosyl methionine; BOHB, β-hydroxyl-butyrate.

SAM (S-Adenosylmethionine):

As discussed above, DNMTs and HMTs add methyl group to DNA or lysine/arginine residues of histones, by transferring the methyl group of SAM to the substrate with the formation of S-Adenosyl homocysteine (SAH). SAM is synthesized from methionine catalyzed by methionine adenosyltransferase (MAT) with ATP. RO/NS reduces the level of SAM (54), and decreases the activity of DNMTs and HMTs. H₂O₂, via generation of hydroxy radical modifies Cys-121 of MAT resulting in inactivation of MAT’s activity and in SAM biosynthesis (55). In A549 cells, H₂O₂ decreased SAM levels resulting in hypomethylation of the long interspersed nuclear element-1 (LINE-1) (56). Depletion of GSH resulted in decrease of methionine and DNA hypomethylation (57). However, more work needs to be done on how different redox signals, dose response, acute versus chronic exposure of RO/NS on SAM and DNA/histone modifications.

Acetyl-CoA:

Histone acetylation is catalyzed by HATs including P300/CBP by transferring the acetyl group from acetyl-CoA to lysine/arginine residues of histones. Acetyl-CoA can be synthesized by oxidative metabolism/decarboxylation of pyruvate during glycolysis of glucose in mitochondrial matrix, by β-oxidation of long-chain fatty acids, or by oxidative degradation of certain amino acids. Acetyl-CoA then enters the CAC for the usual energy production. Acetyl-CoA appears to diffuse freely through the nuclear pore complex, and the fluctuation of the pool of available acetyl-CoA in the cytoplasm/nucleus (nucleocytosolic) may alter histone acetylation (17). The level of cellular acetyl-CoA may fluctuate according to cellular metabolism, under certain conditions, it may be becoming limiting factor for the acetyltransferase activity in the nucleus. The oscillation of acetyl-CoA occurs, including at high glucose levels, acetyl-CoA produced through glycolysis, may promote its utilization for lipid synthesis and histone acetylation, while under fasting or survival states, acetyl-CoA may be channeled into the mitochondria for synthesis of ATP and ketone bodies and the mitochondrial amounts of acetyl-CoA increase relative to nucleocytosolic amounts (58).

NAD+:

The cofactor NAD exists in oxidized form (NAD+) and reduced form (NADH), is central to cellular metabolism involving in redox reaction by carrying electrons from one reaction to another. The oxidized NAD+ accepts electrons, becoming reduced NADH, and the reduced NADH will become reducing agent to donate electrons, which is central during glycolysis and CAC metabolism. NAD is synthesized by de novo pathway from tryptophan/aspartic acid or through salvage pathway (59). But importantly, NAD+ is involved in cellular signaling events including ADP-ribosylation, and protein deacetylation, among others (reviewed in (17, 59, 60)). NAD+ is required for ADP-ribosylation by adding one or more ADP-ribose moieties to lysine, glutamate, or aspartate residues of proteins including histones catalyzed by poly-ADP-ribose (PAR) polymerases (PARPs) (16, 60). Under conditions when NAD+ level is altered, many cellular processes involving NAD+ such as cellular metabolism, PARPs and sirtuins would be perturbed. PARPs involve in many cellular processes including cellular stress response. For instance, PARP1 is highly involved in DNA damage detection and repair (reviewed in (60)), PARP-1 is recruited to DNA double strand breaks and mediates DNA damage repair (60). PARP recruits CHD4 (chromodomain nucleosome remodeling and histone deacetylase), part of the repressive nucleosome remodeling and deacetylase (NuRD) complex, which would repress transcription and facilitate DNA repair at the break sites (61). PARP1 inhibits the binding of KDM5B to chromatin and H3K4Me3 and its demethylase activity, leading to increase levels of H3K4me3 at the promoters of PARP1-regulated genes and enhanced expression (62). PARP1 modulates HAT P300 and arginine MT CARM1 on NF-κB to support its transcriptional activity (63, 64). Under oxidative stress and energy depletion, PARP-mediated poly(ADP-ribosylation) will be compartmentalized to the mitochondria, converted from a homeostatic process to a mechanism of cell death (65). As discussed above, the class III HDACs (sirtuins 1–7) remove acetyl marks from histone tails, and have an absolute requirement for NAD+ for their activity (28).

α-Ketoglutarate:

The Jumonji C (JmjC) domain HDMs utilize α-ketoglutarate, Fe(II), and O₂ to hydroxylate histone’s methyl group with release of formaldehyde, succinate, CO₂ and demethylated histone (reviewed in (66)). In yeast, treatment with H₂O₂, Rph1, a JmJC HDM enhances the expression of stress response genes coupled with the dissociation of Rph1 from the promoters of these genes (67). In human lung BEAS-2B cells, H₂O₂ increases H3K4me3 which is blocked by preincubation with ascorbate and in a cell-free system H₂O₂ inhibited HDM activity where increased Fe(II) rescued this inhibition (47). TET enzymes also require Fe(II) and αKG as co-factors, H₂O₂ exposed cells have decreased TET protein activity (47).

Butyrate and β-Hydroxybutyrate:

Butyrate and β-hydroxybutyrate (BOHB) are short chain fatty acid metabolites found in mammals. Butyrate produced via bacterial fermentation in the colon is present at millimolar concentrations and is a well-known HDAC inhibitor (68). BOHB, a ketone body, produced by the liver during fasting, calorie restriction, or ketogenic diets can reach millimolar concentrations in the blood circulation, inhibits HDACs and modify histone acetylation (69, 70). Butyrate enhances mitochondrial function during oxidative stress (71) and BOHB may protect from oxidative stress (69). Recent study shows that butyrate is a better HDAC inhibitor than BOHB (68). Further studies would be needed to better understand the interaction of redox and butyrate/BOHB and epigenetic reprogramming.

From the above discussion, clearly, cellular metabolism especially mitochondrial CAC is tightly integrated with the epigenetic machinery DNA and histone modifications, chromatin remodeling, and modulating gene expression. Question remains how different redox signals, dose response, acute versus chronic exposure, in vitro cell culture system versus in vivo animal models, and pathological conditions, would affect cellular/mitochondrial metabolism and epigenetic reprogramming.

4. Redox active phytochemicals and epigenetics

Many health beneficial dietary phytochemicals found abundantly in fruits and vegetables are redox active and also possess epigenetic effects (72). These redox active phytochemicals modify cellular redox status, interact with CAC and cellular metabolism and the epigenetic machinery (Figure 2). We will briefly summarize the most abundant classes of these redox active phytochemicals possessing epigenetic modifying capability.

Figure 2:

Interaction of Redox Active Phytochemicals with Redox Status, CAC and Epigenetics. NRF2-ARE, Nuclear factor erythroid 2-related factor 2-antioxidant response element; GSH, glutathione.

4.1. Ally sulfides

Garlic and other plants pertaining to the allium specie have valuable influence on lipid/ carbohydrate dysmetabolism (73). Diallyl sulfide, and diallyl disulfide (DADS), and diallyl trisulfide (DATS) are oil-soluble organosulfur compounds of garlic and possess redox properties including activation of NRF2-mediating pathways (74). DADS has been shown to exert anti-proliferative effects through epigenetic regulation including suppression of HDAC activity and hyperacetylation of histone H3K14, H4K12, and H4K16 in colon cancer cell lines and in rat colonocytes (75, 76). Allyl mercaptan (AM) is a metabolite derived from DADS and is reported as the most effective HDAC inhibitor among garlic-derived organosulfur compounds and their metabolites (77). At the same concentration, AM shows higher inhibition of HDAC activity than DADS in Caco-2 cells (78). AM decreases HDAC activity in HT29 cells, with increased global histone acetylation and localized hyperacetylation of histone H3 on the P21^WAF1 promoter (79).

4.2. Flavonoids

Flavonoids are a very large class of phytochemicals and possess diverse biological activities including redox antioxidant activity, anti-inflammatory activity, and others.

Epigallocatechin-3-gallate (EGCG), found abundantly in green tea, possesses redox activities (80) and regulates NRF2-mediated antioxidant gene expression (81). EGCG inhibits HAT activities including P300, CBP, and PCAF (82). Combination of SFN and EGCG remodels chromatin structure via histone modification and alter methylation patterns in the ERα promoter (83). EGCG can also compact chromatin and silence cancer-related genes by influencing Polycomb-group (PcG) proteins via modulation of histone methylation and acetylation (84).

Genistein is the primary isoflavone found in soy and it activates Nrf2-mediated antioxidant response (85). In addition, genistein mediates promoter hypomethylation or histone hyperacetylation, re-activating different tumor suppressor-associated genes, p16, RARβ, and MGMT (86). However, genistein is known to bind to estrogen receptors (ER), triggering conformational changes leading to transcriptional activations of ER-regulated genes (87) and genistein has been shown to epigenetically activate BRCA1 In Vivo and triple negative breast cancer cells via antagonism toward aryl hydrocarbon receptor (AHR) (88). Further study would be needed the functional role of NRF2-redox-mediated, ER-mediated, AHR-mediated, or other signaling pathways-mediated epigenetic modifying effects of genistein.

Curcumin, a natural compound found in turmeric, possess multi-faceted redox and epigenetic mechanisms including modifying CpG methylation, modulation of DNMTs and HDACs, inhibition of HAT activities, and miRNA modulation (reviewed in (89)). In rat lymphocytes, orally-administered curcumin suppresses lipopolysaccharide (LPS)-induced Dnmt3a and Hdac2/3/4 mRNA expression (90). Pharmacokinetics and pharmacodynamics study of orally administered curcumin in healthy human volunteers shows that curcumin decreases HDAC1, HDAC2, HDAC3, and HDAC4 mRNA expression in leukocytes (91). High concentration of curcumin increases ROS production, induces DNA damage and DNA demethylation potentially via DNA repair pathway (92). DNA methylomic and transcriptomic alterations and cancer preventive effect by curcumin was reported in the azoxymethane (AOM)-dextran sulfate sodium (DSS)-induced colon cancer C57BL/6 mice (93). Analysis of the differentially expressed and differentially methylated genes in pairwise comparisons, several signaling pathways including LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, production of NO and ROS in macrophages and IL-6 signaling were identified to be modified by curcumin (93). Among all the differentially methylated genes, tumor necrosis factor (TNF) was selected for further study and its CpG promoter was hypomethylated and mRNA expression was decreased in colon epithelial cells and colon tumors of AOM-DSS mice and these events were reversed by curcumin (93). Using ex vivo lipopolysaccharide (LPS) treated peritoneal macrophages from Nrf2+/+and Nrf2−/− mice, curcumin’s anti-inflammatory and anti-oxidant effects require Nrf2 (94). CUR can both increase and decrease the accessibility of DNA and thereby influence transcriptional responses to the ligand-activated AHR (95). Question remains on the contribution of redox-mediated, AHR-mediated, or other signaling pathway-mediated epigenetic modifying effects of curcumin.

Anthocyanidins are the sugar-free counterparts of anthocyanins. Anthocyanidins have been shown to activate Nrf2 pathway, correlating with demethylation of CpGs of the mouse Nrf2 promoter region coupled with decrease protein expression DNMT1, DNMT3a, and class I/II HDACs (96). In a high-fat diet-induced redox sensitive signaling (NF-κB and ERK1/2), anthocyanin supplementation attenuated these pathways (97). Delphinidin and pelargonidin inhibit TPA-induced transformation of mouse epidermal JB6 P+ cells coupled with promoter demethylation-mediated activation of Nrf2 (96, 98).

4.3. Isothiocyanates

Isothiocyanates, derived from glucosinolates found abundantly in crucifers, are redox active electrophiles and possess many epigenetic properties (reviewed in (99, 100)). The isothiocyanate sulforaphane was first reported to inhibit HDAC, enhanced histone acetylation, depression of P21 and BAX, and induction of cell cycle arrest/apoptosis (reviewed in (101)). Subsequently, many studies show that isothiocyanates modulate the epigenetic machinery in multiple systems (reviewed in (102)). Metanalysis of SFN shows SFN could exert its effect by restoring the ER gene expression by modulating epigenetic events in human breast cancer cell line MDA-MB-231 (103). Since the isothiocyanates are potent electrophiles, question remains on the contribution of redox electrophile-mediated, ER-mediated, or other signaling pathway-mediated epigenetic modifying effects of isothiocyanates.

4.4. Triterpenoids

Triterpenoids are structurally diverse redox active phytochemicals and possess epigenetics properties (reviewed in (104)). Ursolic acid demethylates the CpG sites in the Nrf2 promoter region, and correlates with the re-expression of Nrf2, coupled with inhibition of mRNA expression of DNMTs and HDACs, and HDAC activity (105). In rat leukocytes, orally administered ursolic acid inhibits LPS-induced DNMTs and HDACs mRNA (106) and in a UVB-induced skin carcinogenesis model, ursolic acid significantly reduced tumorigenesis coupled with modification of DNA methylome of antioxidative, anti-inflammatory, and anticancer pathways (107).

Due to space limitation, not all the redox active phytochemicals that possess capability to modify epigenetic machinery are discussed here. Some of the future challenges in integrating redox signaling, mitochondrial metabolism, epigenetics and redox active phytochemicals include dose response, in vitro cell culture versus in vivo animal models versus in human, different redox stimuli, acute versus chronic dosing and in vivo absorption, distribution, and bioavailability of phytochemicals (108).

5. Gut metabolites and epigenetics

Gut microbiota-derived metabolites are small molecules that are produced as intermediate or end products of microbial metabolism. These metabolites can be derived from bacterial metabolism of dietary phytochemicals or modification of host molecules such as bile acids. Alterations by dietary phytochemicals in the composition and function of the microbiota have been implicated in patients with inflammatory conditions of the gastrointestinal tract (109–111). These alterations are likely to cause changes to gut microbial metabolite profiles, of which the main classes include short-chain fatty acids, bile acids, and tryptophan metabolites. Many phytochemicals, together with dietary fiber, have been proposed to modulate the gut microbiota. Some of the phytochemicals that positively affect the gut microbiota include polyphenols, carotenoids, phytosterols/phytostanols, lignans, alkaloids, glucosinolates and terpenes (112). Some polyphenols may act as prebiotics, while carotenoids have been shown to alter immunoglobulin A expression, an important factor for bacteria colonization (111, 113–115). Other phytochemicals may interact with the gut mucosa, another important factor for colonization, and prevent their degradation. Some polyphenols have shown to influence bacterial communication, interacting with quorum sensing. Short chain fatty acids production depends on gut microbiota composition and diet. Short chain fatty acids, mainly butyrate and acetate, reduce inflammation but increase the anti-inflammatory response of the adaptive immunity. Butyrate has also been shown to inhibit the activity of HDAC through suppressing NF-κB activation and synthesis of interferon gamma and increasing PPAR- γ (116, 117). More study would be needed on the interaction of microbiota-derived metabolites and the epigenetic machinery in the gut as well as in other tissues/organs.

6. Conclusion and perspective

Biological redox signaling and epigenetics play important roles in many diseases’ manifestation. Increasing evidence suggests that redox signaling and cellular/mitochondrial metabolism are highly integrated with reprogramming of epigenetic machinery contributing to many human diseases. Many health beneficial phytochemicals are redox active and possess epigenetic modifying capability. Future challenges include how redox bioactive phytochemicals would interact with the basic redox signaling, cellular/mitochondrial metabolism and the epigenetic machinery contributing to the overall health beneficial effects and impacting diseases.

Highlights.

1. Cellular oxidative stress occurs when reactive oxygen/nitrogen species (RO/NS) exceed the cellular reductive/antioxidant capacity.

2. Many health beneficial phytochemicals possess redox capability that counteract RO/NS either by directly scavenging the radicals or via inductive mechanism of cellular defense antioxidant/reductive enzymes.

3. This review summarizes the latest advances on the interactions between redox signaling, mitochondrial metabolism, epigenetics and redox active phytochemicals and the future challenges of integrating these events in human health.

Abbreviations

ROS

reactive oxygen species

RNS

reactive nitrogen species

RO/NS

reactive oxygen/nitrogen species

CAC

citric acid cycle

TCA

tricarboxylic acid cycle

SOD

superoxide dismutase

MnSOD

manganese superoxide dismutase

NOS

nitric oxide synthase

GST

glutathione-S-transferase

DNMT

DNA methyltransferase

SAM

S-adenosyl methionine

TET

ten-eleven translocation enzyme

HDAC

histone deacetylase

HAT

histone acetyltransferase

HMT

histone methyltransferase

HDM

histone demethylase

CVD

cardiovascular disease

NOS

nitric oxide synthase

AD

Alzheimer’s disease

COPD

chronic obstructive pulmonary disease

NAFLD

non-alcoholic fatty liver disease

IKK

inhibitory κB kinase

DAS

diallyl sulfide

DADS

diallyl disulfide

DATS

diallyl trisulfide

GPS

glutathione peroxidase

iNOS

inducible nitric oxide synthase

AM

Allyl mercaptan

ITC

isothiocyanate

SFN

sulforaphane

AITC

allyl-ITC

BITC

benzyl-ITC

PEITC

phenethyl-ITC

UA

ursolic acid

OA

oleanolic acid

BA

betulinic acid

CDDO

cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic

DMF

dimethyl fumarate

LPS

lipopolysaccharide

IL-6

interleukin-6