BH4 is a pteridine that acts as a mandatory cofactor for the activity of phenylalanine, tyrosine, and tryptophan hydroxylases, for alkylglycerol monooxygenase, and all isoforms of nitric-oxide synthases (NOS) [73]. Therefore, BH4 is essential for the biosynthesis of the neurotransmitters, dopamine and serotonin, for the catabolism of phenylalanine and ether lipids, and for the formation of nitric oxide.

Three biosynthetic pathways are responsible for tuning the intracellular concentrations of BH4: the de novo, the salvage, and the recycling pathways. The de novo pathway synthesizes BH4 from guanosine triphosphate through the sequential action of guanosine triphosphate cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase (PTPS), and sepiapterin reductase (SPR) [73]. GTPCH is the rate-limiting enzyme of the de novo pathway and is transcriptionally regulated by inflammatory mediators, including interferon-γ (IFN-γ), lipopolysaccharide (LPS), interleukin-1β (IL-1β), and hydrogen peroxide [74]. Thus, under inflammatory conditions, the expression of GCH1, the gene that encodes for GTPCH, is up-regulated several times; however, since the other enzymes in the pathway are not inducible by inflammation, a metabolic pseudo-blockage is generated, resulting in the production of neopterin [75]. Indeed, neopterin has been used as a sensitive marker of immune-system activation for several decades [76]. The salvage pathway synthesizes BH4 by utilizing intermediates from the de novo pathway to form sepiapterin, which is later converted to BH4 by the action of the enzymes SPR and dihydrofolate reductase (DHFR) [74]. The recycling pathway is a mechanism that maintains adequate intracellular concentrations of BH4 without the need for energy expenditure in tissues with a high demand for this pteridine, i.e., in the liver for the proper metabolism of phenylalanine. After BH4 is used as an essential cofactor, the molecule is oxidized to quinonoid dihydrobiopterin (qBH2) and recycled back to BH4 by the action of dihydropteridin reductase (DHPR) [73].

BH4 is traditionally known due to its activity as an enzyme cofactor [73]. However, our group and others have shown that the BH4 pathway is essential for maintaining the activity of mitochondria and the antioxidant system, and for inducing an anti-inflammatory scenario [77,78,79]. This has positioned BH4 metabolism as a potential new target to prevent or attenuate the cytotoxicity linked to chronic inflammatory diseases. In this context, our lab has shown that a single intracerebroventricular administration of neopterin (a dose that will slightly increase the levels of the compound in the cerebrospinal fluid) to naïve mice provoked the increase of the antioxidant response by augmenting glutathione levels and the activity of GPx, which are downstream components of the NRF2/ARE-pathway activation, in the brain [80]. In addition, the treatment also prevented the brain’s massive increase of pro-inflammatory cytokines after an intraperitoneal LPS challenge, suggesting that neopterin also maintains the balance between NRF2 and the master regulator of inflammation, nuclear factor-κB (NF-kB) [81].

To try to dissect the mechanisms involved in the antioxidant effect of neopterin, our group also exposed nerve cells obtained from mammals, humans, and rodents to neopterin. We observed that the pre-conditioning with neopterin prevented the activation of the inflammasome, which is a macromolecular protein complex that mediates the synthesis of IL1-β through the activation of pro-inflammatory caspase [82], and also the production of reactive oxygen species (ROS) induced by LPS and IFN-γ [81]. The treatment with neopterin to naïve cells provoked the rapid nuclear translocation of NRF2, the production of HO-1, and increased mitochondrial activity [76,81]. The latter was evidenced by increased activity of complexes I and IV and by increased basal respiration. The enhanced mitochondrial activity was accompanied by reduced lactate formation, indicating that neopterin increased mitochondrial oxidative metabolism and reduced anaerobic glycolysis. Furthermore, our group also observed that neopterin exposure provoked the formation of very low concentrations of superoxide radicals, which can be responsible for an electrophilic attack and consequent activation of the NRF2/ARE pathway [81].

The antioxidant, anti-inflammatory, and mitochondrial-activator properties shown by neopterin might also be responsible for the mnemonic effects of the molecule. Our group demonstrated that neopterin enhances aversive memory acquisition by reducing the threshold to generate hippocampal long-term potentiation, which is an essential mechanism for memory formation [83].

It is widely described that all the above-mentioned cytoprotective mechanisms are induced by the regular practice of moderate-intensity physical exercise. These effects have been described in the blood, muscle, liver, and brain of animals and also in the blood and urine of humans [58,64]. Recently, we have shown that moderate running exercise increases urinary neopterin levels under basal conditions and prevents exacerbated immune-system activation under an inflammatory scenario [84].

Since we have characterized the cytoprotective effects of the BH4 metabolic pathway on the brain of experimental systems and cultured cells, it is feasible that part of the effects induced by exercise might be mediated by the activation of BH4 metabolism. The relationship between BH4 metabolism and NRF2 activation remains unclear, but in vitro studies using Gch1-deficient macrophages indicated the existence of a NRF2/GCH1/BH4 axis, which has the function of protecting against oxidative stress, with GCH1 being one effector switch [77,96]. Furthermore, our group demonstrated that neopterin can activate the expression, content, and activity of NRF2 in vitro [81], and increase the content of the downstream proteins of the pathway [81]. Independently, it has been also shown in an in vitro study with macrophages that NRF2 requires BH4 for its activation [96]. The correlation between neopterin and the beneficial effects of physical exercise has led sport and exercise medicine to use it as an indicator of immune-system activation. Its use as a biomarker is also growing when compared with other traditional inflammatory markers [97,98].

Regarding plasma levels of BH4 increase, it has been shown that the levels can increase rapidly and can be sustained for up to 2 h after the practice of strong physical exercise in young and middle-aged individuals, pointing to a temporal response of this metabolic pathway with exercise intensity [85,86].

During physical exercise, there is an inherent consumption of energy, generation of ROS, and consequent activation of the immune system [99]. Increased plasma levels of neopterin have been demonstrated after running [87], ergometer [88,89], and even after ultra-endurance competition [90]. This increase has also been reported in urine after running [91], rugby [92], bodybuilding competition [93], triathlon [94] and ultra-marathon [95]. The rapid and transient increase in BH4 and neopterin after exhausting exercises can be also interpreted as the result of an oxidative burst followed by the activation of monocytes and macrophages, reflecting the immune activation stimulated in this context [88]. The presented scenario indicates that BH4 metabolism can behave as a biomarker of inflammation induced by high-intensity physical exercise, but also as a cytoprotective and neurological mediator of the beneficial effect generated by physical exercise on the antioxidant system, including the activation of NRF2 [3].

The term epigenetics was conceived by Conrad Waddington in 1940 to describe the possible causal processes acting on genes that regulate phenotype [100]. Over the years, the definition and concept of epigenetics have gradually evolved to mean the existence of a process that alters gene activity without changing the nucleotide sequences [101]. Epigenetic profiles are controlled by several biochemical processes, including DNA methylation, histone modification, and non-coding-RNA-modulated expression. These mechanisms mainly control gene expression at the transcriptional level through chromatin compaction and/or relaxation, thereby blocking/allowing the accessibility of transcription factors to the promoter region [102]. Epigenetic processes can also prevent protein translation by inactivating or degrading messenger RNA (mRNA) through the action of interfering microRNAs (miRNA) [103,104].

DNA methylation is the most characterized epigenetic alteration and consists of the covalent addition of a methyl group catalyzed by DNA methyltransferases (DNMTs). DNMTs transfer a methyl group from S-adenosylmethionine (SAM) to the 5′ carbon of a cytosine that usually precedes guanine (CpG dinucleotide), forming 5-methyl cytosine (5-meC). DNA methylation of CpG regions, called CpG islands, is usually associated with the inhibition of gene expression [105]. DNA methylation can be also modified by a family of 2-oxoglutarate- and Fe (II)-dependent dioxygenase enzymes named TET translocation proteins (TET-eleven-translocation). These proteins, TET1–3, can oxidize 5-meC into 5-hydroxymethylcytosine (5-hmC) and 5-carboxycytosine (5-caC). The decarboxylation of 5caC will provoke the demethylation of the DNA [106]. Studies have suggested a direct action of CpG-island hypermethylation in the regulation of NRF2 transcriptional activity [107]. The downregulation of NRF2 by DNA methylation has been described in a cellular model of Alzheimer’s disease [108], diabetic cardiomyopathy [109], and especially in different types of cancers [107]. Furthermore, DNA methylation has been associated with the protective effect of physical exercise [110]. Although exercise-induced redox disturbances can act as downstream modulators of the epigenetic machinery, data demonstrating a direct exercise-induced epigenetic modulation of NRF2 gene expression are scarce [111]. The increased activation of NRF2 has been attributed to the hypermethylation of KEAP1, favoring NRF2translocation to the cell nucleus [112,113]. In agreement, it has been shown that running exercise can reverse NRF2 promoter hypermethylation in a pre-clinical osteoporosis model, thereby attenuating the suppression of antioxidant enzymes [114]. In addition, it is well established that physical exercise increases ROS production, and recent studies indicate that ROS can activate TET DNA demethylases and cause hypomethylation of the NFE2L2 promoter, resulting in NRF2 activation [115,116].

Histone modification is another key mechanism in the regulation of gene expression. An octamer of histone proteins makes up the main repeating element of chromatin, the nucleosome. Histones have N-terminal tails that are prone to a variety of post-translational changes, with histones H3 and H4 being the most studied concerning gene-expression regulation [102,117,118]. These modifications are controlled by four groups of enzymes: histone acetyltransferases (HATs), histone methyltransferases (HMTs), histone deacetylases (HDACs), and histone demethylases [119,120]. In this scenario, it has been demonstrated that increased histone acetylation occurred in the hippocampus of rats that were subjected to physical exercise. This epigenetic modification was associated with improved neurocognition and aversive-memory performance [121,122].

The HDACs family is composed of sirtuins (Sirts), which due to their NAD+-dependence on the deacetylase activity, can regulate redox reactions by modulating transcription factors that control the expression of antioxidant enzymes [123]. NRF2 has been suggested to be a downstream regulator of Sirt1 in a cardiac-ischemia model [124]. On the other hand, Sirt2 has been associated with the deacetylation of NRF2 and consequent reduction of its total cellular and nuclear levels, leading to a decrease in its transcriptional activity [125]. NRF2 levels can also be modulated by Sirt2 through Akt phosphorylation, leading to the regulation of glutathione concentrations, suggesting a role in the NRF2/ARE system [126]. Although studies demonstrating the association between physical exercise, Sirts and NRF2 are scarce, the available evidence that exercise modulates sirtuins [127,128,129] and that they can act in the regulation of NRF2 [124,125,126] reveal an area to be studied and a possible mechanism generated by the practice of physical exercise.

Recently, non-coding RNAs (ncRNAs), especially long non-coding RNAs (lncRNAs), have been implicated as important epigenetic modulators due to the ability to neutralize miRNAs by their sponge activity. LncRNAs are also capable of directing DNA methylation and histone modifications, thereby modulating gene expression [130]. NcRNAs can act as competitive endogenous RNAs to absorb and suppress the activity of bound miRNAs, effectively derepressing other targets of these miRNAs [131]. The regulation of gene expression by lncRNAs at the epigenetic, transcriptional and post-transcriptional levels have been widely studied, and there are strong indications that the expression of certain lncRNAs can modulate the effects of physical exercise (Table 3).

Folate and BH4 are chemically defined as pterins due to the presence of the heterocycle ring pteridine. Different from BH4, folate is an essential vitamin that needs to be included in the diet in order to modulate metabolism as a micronutrient. Dietary folate requires the activity of DHFR to be converted first into dihydrofolate and then into tetrahydrofolate (THF), a universal one-carbon unit acceptor. DHFR is also an active enzyme in the BH4 salvage pathway, where it catalyzes the reduction of BH2 into BH4 [74]. THF accepts one-carbon units derived from the amino acids, serine and glycine, and the resulting methylated-THF exists in several interchangeable forms with varying chemical structures. These include formyl-THF, methyl-THF, and methylene-THF, which, respectively, donate their one-carbon units to purine synthesis, the methionine recycling pathway (via homocysteine methylation), and thymidylate synthesis [142].

Several studies have associated folate metabolism with increased DNA methylation in blood cells [143], liver [144], kidney [145], and gut [146], as well as, with increased concentrations of SAM in erythrocytes [147], a key metabolite involved in DNA methylation. On the other hand, it has been shown that the reduction of DHFR activity diminishes the cellular THF pool, altering the folate-dependent enzyme activity, and therefore, epigenetic profiles [148]. Folate deficits have been extensively associated with an increased risk of cardiovascular diseases, multiple cancers, and neural-tube defects due to deficient DNA methylation [149,150,151,152]. In addition, BH4 non-hereditable or genetic deficiencies have also been related to conditions where folate metabolism was reported to be compromised, including brain-maturation defects [153] and cardiovascular diseases [154]. Moreover, DHFR inhibition is essential to the action of antifolate medications used to treat cancer and some inflammatory diseases, and it is well described that methotrexate reduces BH4 levels [155], denoting the intricate association between these two metabolic pathways in regulating DNA methylation.

The practice of physical exercise is known to modulate DNA methylation, favoring the hypermethylation of some DNA regions and the hypomethylation of others. The outcome is the permissive gene expression of genes beneficial for cell health, i.e., anti-inflammatory and antioxidant genes. In this scenario, it has been shown that acute resistance exercise used to stimulate hypertrophy can induce different epigenetic modifications in human skeletal muscle, including the hypermethylation of GPAM and SREBF2 genes [156]. GPAM and SREBF2 encode for enzymes involved in the biosynthesis of lipids, a metabolism that has been proposed to be dependent on appropriate intracellular levels of BH4 [157].

The practice of light-intensity physical activity by a general cohort of a healthy middle-aged population generated hypermethylation of the gene speck-like protein containing a caspase recruitment domain (ASC) in peripheral blood mononuclear cells, resulting in a decrease in expression [158]. ASC encodes an adapter protein that is necessary for inflammasome formation and the consequent activation of pro-caspase 1 and IL1-β synthesis [159]. ASC hypermethylation was correlated with a decrease in the pro-inflammatory cytokines IL-6, IL-8, IL-15, and TNF-a, resulting in a decrease in systemic inflammation in middle-aged individuals [158]. Furthermore, hypermethylation of NFκB in peripheral blood cells has also been demonstrated after low-intensity walking exercise by elderly individuals, also demonstrating a decrease in exercise-induced systemic inflammation mediated by epigenetic mechanism [160]. This scenario suggests a possible relationship between BH4 metabolism and the effects induced by exercise on DNA methylation, favoring the antioxidant response (Figure 1); however, studies in this area have not been performed to date.

4. Conclusions