During the initial stage of an inflammatory process, immune cells activated in response to invading pathogens or agents release various inflammatory mediators, aiding in increased vascular permeability and leukocyte migration towards the tissue injury site (12). ROS regulates various intracellular adhesion molecules, including ICAM-1, VCAM-1 and selectin expression, ensuring their interaction with leukocytes and transendothelial migration. On the contrary, enhanced expression of superoxide dismutase reduces leukocyte binding to endothelium by decreasing the expression of adhesion molecules (13). Additionally, a gradient increase in H₂O₂ at the tissue injury site is an early factor in leukocyte recruitment (14). Cytokines, including tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), and NOX stimulate cell migration and adhesion (15).

Metabolic reprogramming, which includes changes in the activity of fatty acid oxidation (FAO), tricarboxylic acid (TCA) cycle, glycolysis levels, involvement of pentose phosphate pathway and mitochondrial respiration mediates the phenotypical and functional role of myeloid cells.

Over the past two decades, immune cell functions, namely proliferation and differentiation, have been linked to various metabolic pathways; thus, immunometabolism is inseparable from redox reactions (11). Thus, bioenergetic and biosynthetic demands for T and B cell response, macrophages and dendritic cells are regulated by metabolic pathway and their resulting breakdown products (16). In aerobic organisms, during oxidative phosphorylation in mitochondria, the electron transport chain shuttles electrons to molecular oxygen (O₂), producing free oxygen radicals. ROS are well known for their role in inflammation through respiratory bursts, wherein macrophages and neutrophils phagocytize pathogens and cellular debris. The process activates the NOX assembly and activation, which triggers further ROS production (17). NOXs play a vital role in inflammatory response through respiratory burst and neutrophil extracellular trap formation. NOX-derived ROS are reportedly involved in angiogenesis, a process of the growth of new vessels and a key event in the proliferative phase of inflammation. It has been reported that NOX remains localized at the leading edge of migrating cells and is linked to actin and IQGAP1 protein (Ras GTPase-activating-like protein), and any disruption at this binding site results in impaired migration of endothelial cells (18).

Nevertheless, enhanced production of reactive species has to be regulated to avoid oxidation of biomolecules leading to cellular toxicity and cell death. Alterations in redox balance are often associated with chronic immune activation, such as autoimmune disorders and neurodegenerative diseases. Therefore, the redox state is an intrinsic cellular and systemic homeostasis indicator.

The release of local damage-associated molecular patterns (DAMPs) in response to tissue damage elicits enhanced cytokine production via sterile inflammation. Cytokines belonging to the interleukin-1(IL-1) family have been proposed to be essential drivers of sterile inflammation, which recruits neutrophils and macrophages to the damaged site. During sterile inflammation, host-derived DAMPs released by stressed cells contribute to macrophage polarization that aids in resolving sterile inflammation and restores homeostasis (19). In endothelial cells, under hyperglycemic conditions, an excessive glucose load triggers ROS formation in mitochondria, impairing its functions and causing cellular damage through interaction with various cellular constituents, including DNA, proteins and lipids (20). These ROS trigger the activation of pro-inflammatory transcription factors, namely NFκB and activating protein-1(AP-1), resulting in enhanced inflammatory cytokines/chemokines expression. In addition, activated endothelial cells attract monocytes that promote inflammation and macrovascular and microvascular injury (21).

Macrophages, especially adipose tissue macrophages (ATM), drive diabetic pathology. In healthy individuals, adipocytes secrete adiponectin, which induces M2-like polarization of ATM and suppresses ROS and its related pathway genes (22). On the contrary, in obesity conditions, a reduction in adiponectin levels induces M1-like polarized macrophages, thereby enhancing glucose consumption through GLUT1 (glucose transporter 1). Thus, an interplay between obesity and hyperglycemia promotes ROS formation, glycolytic metabolism, and the release of pro-inflammatory cytokine mediators by macrophages (23). Hence, focusing more on metabolic reprogramming during macrophage polarization events is essential to identify potential targets for treating inflammation and metabolic disorders. A mitochondrial reactive oxidative stress increase was reported in mice subjected to ventilator-induced lung injury, activating NLRP3 to produce IL-1β and lung inflammation in combination with TLR4 signaling (24).

Though the primary source of ROS in vivo is through aerobic respiration, cellular events, including peroxisomal β-oxidation of fatty acids, arginine metabolism, tissue-specific cellular enzymes and phagocytosis stimulation by pathogens also contributed to ROS production (3). Based on its source, cell type and environment, ROS signaling contributes to either normal physiological processes or metabolic dysfunction through inflammatory signaling. Diseased conditions, including diabetes mellitus, atherosclerosis and stroke, are known to be associated with redox balance (25).

The NADPH oxidase (NOX) family of proteins remains the primary cytosolic source of ROS, consist of seven different isoforms and comprises membrane and cytosolic components that are actively involved in the host response to various stimuli including bacterial and viral infections, cellular signaling and regulation of gene expression. Among the isoforms, NOX2 is well characterized for its role in phagocytic functions. Both NOX and inducible nitric oxide synthase (iNOS) are vital in generating enhanced ROS levels within phagocytes via oxidative burst to kill invading pathogens (26). In comparison to mitochondria, O₂ ^•- produced by NOX is dismutated into H₂O₂ by superoxide dismutase1 (SOD1), whereas nitric oxide produced by inducible nitric oxide synthase (iNOS) reacts with O₂ ^•- resulting in peroxynitrite production (19). For example, ROS drive hypoxia-inducible factor 1α (HIF1α) mediated GLUT1 expression, hexokinase activity, and resultant glycolysis in response to low oxygen tension as part of the angiogenic response (27) The co-localization of neutrophil phosphofructokinase 2 with NOX2 leads to its activation, resulting in NADP+ production as its byproduct, facilitating an enhanced glycolytic rate. The increase in glycolysis rate and enhancement of NOX2 activity and the relation between these processes are still under study (28).

Mitochondria are considered as the redox-active compartment within the cell, accounting for nearly 90% of oxygen utilization (1). They serve as a significant contributor to ROS in the form of O₂ ^•-. Mitochondrial SOD converts O₂ ^•- into H₂O_2, which in turn gets converted into HO^• through the Fenton reaction, which in turn oxidizes biomolecules. SOD1 is constitutively expressed and regulates cytosolic O₂ ^•- levels (29). Factors like hyperoxia, oxidative stress, and inflammatory cytokines induce SOD2, whereas SOD3 are cell and tissue-specific and likely of significant importance in protection against stress factors from the extracellular environment. In addition, Grx (glutaredoxin), glutathione, and Trx (thioredoxin) systems play a predominant role in mitochondrial ROS buffering, like SOD (30).

Under stress conditions, the ER tubular network holds a unique oxidizing environment, wherein redox signaling mediators play a vital role in ROS generation and mediate protein folding. Protein folding is highly sensitive to ER redox status, and dysregulation of disulfide bond formation in response to ER stress increases luminal oxidative stress, leading to a decline in ER function. During protein folding, protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductase 1 (ERO1) introduce disulphide bonds into folded proteins, resulting in H₂O₂ formation (31). Protein disulfide isomerase introduces disulfide bonds onto protein substrates through thiol oxidation, resulting in a reduced state. However, PDI is reoxidized through ERO1, which transfers electrons from O₂ through the flavin adenine dinucleotide cofactor, forming H₂O₂ (32). In addition to the PD1/ERO1 pathway, ROS are produced through NOX4, NADPH-P450 reductase (NPR), and GSH (33). NOX4 is reported to be consistently associated with ER, and NOX4 associated with p22phox utilizes NADH or NADPH as an electron donor to produce O₂ ^•-. They are also reported to interact with PDI, whereas the absence of PDI results in cell death (34). In macrophages, the interaction between p22phox and PDI was observed (26).

Peroxisomes, like mitochondria, are vital organelles that regulate crucial processes such as α- and β-oxidation, amino acid catabolism, glyoxylate metabolism, ketogenesis, polyamine oxidation and isoprenoid and cholesterol metabolism (35). Peroxisomal electron transfer leads to free electrons rather than ATP, which are transferred to H₂O to form H₂O₂. In peroxisomes, H₂O₂ is produced by various oxygen-consuming oxidases, including D-amino acid oxidase, xanthine oxidase, d-aspartate oxidase, polyamine oxidase and acyl-CoA oxidase. In addition, peroxisomal oxidases and xanthine oxidases generate ROS and nitric oxide (36). The lysosomal electron transport chain generates HO^• via proton translocation to maintain an optimal pH for acidic hydrolases (9).

Thus, ROS are produced as a byproduct of cellular events wherein the NOX family of proteins mediate the reduction of O₂ to O₂ ^•- and phagocytes NOX accounts for an increased amount of O₂ ^•- and H₂O₂ production by respiratory burst (3). Upon stimulation, the membrane-bound catalytic core assembles with proteins from the cytosol (p47 phox, p67 phox and small G protein Rac), activating O₂ ^•- production. ^•NO produced by Nitric oxide synthase (NOS) can migrate through the cell membrane via diffusion and mediate several signaling pathways in a dose-dependent manner (37). However, inflammatory activation of iNOS by cytokines or lipopolysaccharides enhances cellular levels of ^•NO and results in inflammatory diseases and septic shock (38). Both oxidative and nitrosative stress can hinder the functioning of intracellular redox buffer systems, resulting in decreased antioxidant capacity of affected cells (39). Thus, a proper balance between ROS-RNS is essential in regulating immunological response. A schematic representation of types and sources of ROS are presented in Figures 1A, B , respectively.

Macrophages are a heterogeneous population of immune cells that play a vital role in tissue homeostasis in response to pathogen infection by phagocytosis and mediate tissue repair during injury. They rapidly recognize, engulf and destroy pathogens or apoptotic cells, which can be attributed to their plasticity and heterogeneity (70). Through polarization events, macrophages adopt either a pro-inflammatory phenotype classified as M1 macrophage or an anti-inflammatory M2 phenotype that mediates wound healing and inflammation resolution (71). Recently, it has been observed that excess molecular stimuli induce diverse and partially overlapping macrophage phenotypes that are distinct from M1 and M2. Macrophage activation by ROS, cytokines and commensal lipopolysaccharide (LPS) results in the activation of NF-κB and PI3K/AKT signaling pathways (72, 73). Thus, upregulated NF-κB increases pro-inflammatory chemokines and cytokine transcription, inducible NO synthase (iNOS) and HIF1 α. These signaling pathways, enzymes, and transcription factors are essential in maintaining macrophage activation and M1 polarization by driving metabolic reprogramming (17).

ROS regulate the intracellular signalosome within a constantly evolving cellular microenvironment, thus underlying its role in polarization and the specialized function of immune cell populations (74). M1 and M2 macrophages differ from resting macrophages in their phenotype and exhibit distinct metabolic profiles (75). M1 macrophage metabolism is characterized by aerobic glycolysis, changes in pentose phosphate pathway (PPP), FAS, and truncated TCA cycle, while M2 mostly depends on oxidative metabolism, fatty acid oxidation (FAO) and decreased glycolysis (76) ( Figure 2 ).

Glycolysis is a crucial metabolic event for M1 macrophages, and its inhibition affects typical functions of their inflammatory phenotype, including phagocytosis, ROS production, and pro-inflammatory cytokine secretion. Lipopolysaccharides and Toll-like receptor (TLRs) mediated differentiation of M1 macrophages are associated with a metabolic shift towards glycolysis, blocking it by glucose derivate like 2-deoxyglucose impairs both ROS and pro-inflammatory cytokine production. Numerous transcription factors are known to be involved in maintaining metabolic changes associated with M1 macrophages, and notable factors were discussed in detail. Reports demonstrated the involvement of HIF1α in activating inflammatory macrophage through the glycolysis mechanism (77). They act as modulators of the methylation status of hypoxia-responsive elements in the promoter regions. Enhanced expression of HIF1α reduces mitochondrial activity by suppressing electron transport chain enzymes, resulting in mitochondrial autophagy. HIF1α exaggerates glycolytic flux, thereby increasing the expression of glucose transporters (GLUT1 and GLUT3) and inflammatory mediators. More specifically, M1 macrophages rely on glycolysis and accumulation of succinate from the TCA cycle to stabilize HIF1α, which in turn activates the transcription of glycolytic genes sustaining glycolytic metabolism in M1 macrophages (36). Increased levels of HIF1α were evident along the differentiation of monocytes into tissue macrophages and play a prominent role in the uptake of bacteria by macrophages under hypoxic conditions and expression of tumour necrosis factor (TNFα) and nitric oxide (NO) through inducible NO synthetase (iNOS). In macrophages, overexpression of glucose receptors enhances glycolysis, which induces ROS production and pro-inflammatory mediators (78). In M1 macrophages, aerobic glycolysis induction depends entirely on redox-sensitive transcription factor HIF1α activated by the NF-κB pathway during inflammation. HIF1α interacts with pyruvate kinase isoenzyme M2 (PKM2), thereby mediating the transcription of glycolytic enzymes and inflammatory factors like IL-1β. ROS-like NO-mediated prolyl hydroxylases (PHDs) inhibition induces HIF1α (79). Further, NO reduces oxidative phosphorylation by nitrosylation, inhibiting proteins involved in the mitochondrial electron transport chain. Thus, ROS production positively regulates and maintains the shift towards aerobic glycolysis in M1 macrophages.

NADPH generated by PPP regulates the inflammatory response of M1 macrophages. They mediate ROS and NO production through NOX and iNOS, respectively, to kill invading pathogens and sustain the functionality of TRX and GSH antioxidant systems. Results presented by Nguyen and co-workers demonstrate that deletion of the TRX1 system impairs NLRP3 inflammasome formation and binding of NF- κB to target DNA in monocytes and macrophages (10). It leads to defective production of pro-inflammatory cytokines and ROS accumulation. Additionally, mitochondrial ROS-induced DNA damage aid in a significant drop in NAD+ levels in M1 macrophages. Suppression of PPP in macrophages attenuates LPS-induced inflammatory and oxidative stress response. Compared to glycolysis, intermediates of the TCA cycle, namely succinate and citrate, support biosynthesis in M1 macrophages (11). In LPS-stimulated macrophages, two breaks in the TCA cycle result in the accumulation of succinate and citrate, stabilizing HIF-1α and the subsequent increase in IL-1β transcription. Furthermore, succinate dehydrogenase-mediated oxidation of succinate and increased mitochondrial membrane potential drive ROS production (72). Another TCA cycle metabolite, citrate, gets transported into the cytosol and utilized for fatty acid synthesis to support membrane biogenesis and synthesis of pro-inflammatory lipid mediators, namely prostaglandins. In LPS-activated macrophages, ROS-dependent oxidation of unsaturated phospholipids results in glutamine utilization to feed the TCA cycle and lead to cytoplasmic accumulation of oxaloacetate. These metabolites stabilize HIF-1α, enhancing IL-1β secretion in atherosclerosis (13).

In M1 macrophages, ROS-mediated activation of NRF2 is essential for PPP maintenance and NADPH production, which is necessary for FAS, TRX, and GSH systems. IL-1β is produced as an inactive precursor in response to pathogens, and thus, further processing of it into a biologically active form requires the formation of multiprotein complexes termed inflammasomes. Mitochondrial ROS and its oxidation products are known to play a prominent role in inflammasome activation, whereas the role of NOX is highly dispensable (14).

Unlike M1 macrophages, M2 macrophages hold an intact TCA cycle and enhanced mitochondrial OXPHOS. CD36 internalizes circulating lipoproteins and fatty acids, mediating fatty acid uptake and fueling OXPHOS. The increased cellular concentration of IL-4 and IL-13 drives M1 macrophages towards anti-inflammatory and healing phenotypes described as M2 macrophages. Tyrosine phosphorylation and signal transducer and activator of transcription 6 (STAT6) activation mediate polarization of macrophages into the M2 phase. IL-4 and IL-13 suppress pro-inflammatory cytokine production by upregulating transforming growth factor beta (TGF-β) activity (70). Adenosine 5′-monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) are found to play a vital role in the transition of macrophage polarization states through IL-13 and IL-4. AMPK inhibits NF-κB and stimulates OXPHOS and FAO, reducing HIF1α levels and inflammation and terminating aerobic glycolysis (80). AMPK negatively regulates LPS-induced inflammatory response in macrophages by inhibiting NF-κB activity and activating the PI3K/Akt signaling pathway. Enhanced IL-10 expression promotes TAM activation by PPARα/β, resulting in a polarization of M2 macrophages (12). The importance of FAO in M2 polarization was highlighted in several studies wherein blocking FAO with inhibitors against mitochondrial carnitine palmitoyl-transferase 1 inhibited the activation of M2 macrophages. Results from various studies confirm the PPARγ-mediated activation of M2 signature genes either through oleic acid and IL-4 stimulation or by promoting glutamine oxidation fueling OXPHOS (81). Modification in arginine metabolism emphasizes the transition from M1 to M2 polarization. Increased activity of iNOS mediates arginine metabolism to produce NO, which maintains the switch towards aerobic glycolysis in M1 macrophages. In the case of M2 macrophages, arginine gets metabolized into ornithine and urea due to increased transcription of arginase-1. Both urea and ornithine are essential in M2 macrophage proliferation and survival. Additionally, glutamine metabolism is particularly interesting since glutamine oxidation depletes extracellular glucose levels in an inflammatory environment, maintaining TCA activity and activating the glutamine–UDP-N-acetylglucosamine (GlcNAc) pathway to reinforce M2 polarization (82).

Neutrophils are the first responders against invading pathogens through innate and humoral immunity. Activated neutrophils rely on glycolysis as their primary source of energy under both physiological and inflammatory environments; however, they can regulate their metabolism to carry out its effector functions, namely phagocytosis, oxidative burst, degranulation, extracellular trap formation and chemotaxis (83). Neutrophil extracellular trap (NETs) formation by neutrophils relies on glycolysis and PPP as a source of NADPH, resulting in free oxygen radical production. Superoxide anion radical, thus produced, further induces the formation of ROS and HOCl used by neutrophils in oxidative bursts following phagocytosis of invading pathogens. ROS promote several steps of NETosis, including releasing neutrophil elastase from granules by increasing membrane permeability and degradation of H1 linker and core histones resulting in chromatin decondensation (84). The morphological changes associated with NETosis were promoted by ROS, which in turn inactivated caspases to block apoptosis and trigger autophagy. Secondary oxidants, namely HOCl, mediate PMA-induced NETosis, and it entirely depends on NOX activity ( Figure 3 ). The absence of extracellular Cl^-, a substrate for Myeloperoxidase (MPO) in vitro results in decreased NET production (85). On the contrary, calcium ionophores induced NETosis are NOX independent and rely on mtROS. NOX-independent Netosis depends on calcium which in turn activates peptidyl arginase deiminases resulting in cellular hypercitrullination (86).

Neutrophil functions are highly influenced by cellular redox status, which includes both ROS/RNS production and cellular antioxidant systems (87). Enhanced ROS production is reported to comprise phagocytosis, resulting in dysregulated oxidative burst events and NET production. ROS levels determine the sensing of pathogens by neutrophils and their subsequent activation of NLRP3 inflammasome and cytokine synthesis (88). Additionally, chronically upregulated ROS and cytokine production lowers neutrophil migration by internalizing CXCR2, a membrane chemokine receptor. Neutrophil functions, namely oxidative burst and NET formation, were sustained by the glutathione system (GSH). Its basal activity was reported to be lower in neutrophils compared to other myeloid cells. Prolonged neutrophil activity and excessive production of MPO due to chronic nitrooxidative stress and inflammation lead to the depletion in GSH levels (89). Thus, depleted GSH levels in neutrophils affect their chemotaxis, transmigration and cytoskeletal reorganization, resulting in early apoptosis and impaired degranulation. Redox factors involved in macrophage and neutrophil function are presented in Table 2 .

Multiple transcriptional and epigenetic modification factors are known to be involved in macrophage differentiation and its activation. Epigenetic changes within macrophages allow them to switch between cellular programs affecting phenotype plasticity. DNA demethylation is known to be involved in the process of monocyte-to-macrophage differentiation. DNA demethylation affects specific genes that regulate actin cytoskeleton and phagocytosis (19). ROS-mediated oxidation of amino acid residues in histone H3 results in chromatin relaxation and accumulation of transcription factors. Cysteine residues at histone H3 sense redox changes and mediate further opening of chromatin structures (95). In LPS-stimulated macrophages, lipid peroxidation products are reported to form lysine adducts with H2, H3 and H4, including H3K23 and H3K27. These modifications at histone acetylation and methylation sites are known to be associated with the epigenetic patterning of cardiovascular diseases (96). LPS stimulation and TLR-4-dependent activation of inflammatory genes primarily depend on H3 and H4 acetylation. ROS-mediated post-translational modification of both class I and II histone deacetylases impair its enzymatic function, resulting in open chromatin structure.

Additionally, lipid peroxidation products mediate the carbonylation of HDACs, resulting in ubiquitination and proteasomal degradation of HDAC function, increased acetylation of histones in macrophages and release of pro-inflammatory cytokines. HDACs are predominant in regulating immunological pathways, more precisely in M1 activation. The difference in the expression pattern of almost all HDAC classes was observed in cells stimulated with LPS. Studies concerning macrophage stimulation with LPS result in an initial decrease in HDAC 4,5 and 7 expression, thereby leading to cyclooxygenase-2 gene activation (97). On the contrary, HDAC6 aids in the expression of pro-inflammatory genes in macrophages stimulated with LPS and thus, its inhibition limits macrophage activation. Concerning the link between DNA methylation and LPS stimulation, SOCS1, a negative regulator of cytokine signals, has been found. DNMT1-mediated hypermethylation of SOCS1 results in the loss of its activity, thereby enhancing the expression of LPS-induced Pro-inflammatory cytokines, namely TNF-α and IL-6. DNMT1 has also been reported to improve the demethylation and trimethylation events of H3K9 in regulator proteins like Notch1 and KIF4 and mediate their polarization towards M1 macrophages. Furthermore, DNA methyltransferase (DNMTs) is known to be involved in M2 differentiation and phenotypic regulation. Individuals with atherosclerosis and apolipoprotein E knockout mice fed an atherogenic diet displayed enhanced DNMT levels in macrophages (98). Since the activity of PPAR-γ is reduced, the macrophage transition from M1 to M2 is affected, and thus, the progression of atherosclerosis is marked by increased pro-inflammatory cytokine production. Oxidative stress is linked to increased histone acetyltransferase (HATs) activity of p300/CBP along with NFĸB DNA binding, promoting pro-inflammatory gene expression. In endothelial cells and hyperglycemic adipocytes, enhanced expression of HAT GCN5 and H3 acetylation is associated with increased ROS production, as confirmed in diabetic models. p300/CBP mediated acetylation of H3K9 at NOX2 promoter encourages ROS generation underlying complexity of epigenetic modifications in ROS balance and response (16).

Upon activation, neutrophils generate a range of ROS and O₂ ^•- generated were reported to damage proteins and are limited to the compartment site it has been generated as its rate of dismutation was enhanced by SOD to H₂O₂. Neutrophil heme protein myeloperoxidase utilizes H₂O₂ to oxidize the halides chloride (Cl^-), iodide (I^-), and bromide (Br^-), or pseudohalide anion thiocyanate (SCN^-), into hypochlorous acid (HOCl) (99). Being a potent oxidant, HOCl poses a high reactivity towards biological macromolecules targeting free and protein-associated cysteine, methionine residues and low molecular weight thiols, and its ability to diffuse from the site of generation is very short. Thus, HOCl reacts with amines and forms chloramines, which are less reactive than HOCl and diffuse further from the generation site. The reaction of HOCl and chloramines with cytosine produces 5-chlorocytosine (5-clC), directly incorporated into DNA as a chlorinated nucleotide (100). Studies carried out reported gene silencing, and no significant changes were observed in global methylation levels due to the incorporation of 5-clC. Chloramines are also reported to interact with histone amine groups, thereby preventing methylation or acetylation events (101). However, the lack of in vivo experimental evidence makes it unclear how 5-clC and chloramine levels are regulated under physiological conditions. Neutrophil oxidants can react with cellular targets, including small molecules and redox-sensitive components of epigenetic pathways. Intracellular availability of methionine and ascorbate was depleted by neutrophil oxidants and reported to impact methylation by disrupting S-adenosylmethionine (SAM) levels (102). Mass spectrometry analysis to investigate the oxidant effects of methylation reported impaired cytosine methylation on newly replicated DNA in the Jurkat T-lymphoma cell line upon sub-lethal level exposure to glycine chloramines (103). The study reported DNMT1 inhibition and depletion of SAM levels at doses, which had minimal effect on cell proliferation. Though H₂O₂ treatment inhibited DNMT1, it did not reduce SAM or global methylation levels (104). Further experimentation is required to determine whether the methylation and demethylation effects observed are heritable to subsequent generations.