Oxidative Stress and Its Role in Cd-Induced Epigenetic Modiﬁcations: Use of Antioxidants as a Possible Preventive Strategy

: Oxidative stress (OS) represents one of the main mechanisms of toxicity induced by environmental pollutants such as cadmium (Cd). OS is a natural physiological process where the presence of oxidants, such as reactive oxygen-derived species (ROS), outweighs the strategy of antioxidant defenses, culminating in the interruption of signaling and redox control. It has been suggested that Cd increases ROS mainly by inducing damage to the electron transport chain and by increasing the activity of nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) oxidase (NOX) and the concentration of free iron (Fe), as well as causing a decrease in antioxidant defense. On the other hand, OS has been related to changes in the biology of the epigenome, causing adverse health effects. Recent studies show that Cd generates alterations in deoxyribonucleic acid (DNA) methylation, histone modiﬁcations, and noncoding RNA (ncRNA) expression. However, the role of OS in Cd-induced epigenetic modiﬁcations is still poorly explored. Therefore, this review provides an update on the basic concepts of OS and its relationship with Cd-induced epigenetic changes. Furthermore, the use of antioxidant compounds is proposed to mitigate Cd-induced epigenetic alterations. effects induced by Cd are due to a decrease in DNA methyltransferase (DNMT) activity and an increase in the ten-eleven translocation family (TET). Meanwhile, the effects of prolonged exposures have been linked to elevated levels of DNMT. SAH: S-adenosylhomocysteine, S-adenosylmethionine, methionine methionine GSH: glutathione, factors are involved in the expression of noncoding RNA (ncRNA). Transcriptional ncRNAs are classiﬁed into small ncRNAs and long ncRNAs (LncRNA). Small ncRNAs can be divided into microRNAs (miRNAs), P-element induced wimpy (PIWI)-RNAs of interference (piRNAs), and small RNAs of interference (siRNAs). Cd can alter LncRNA expression and miRNA biosynthesis. Drosha: subsequently processed in the nucleus by RNase III, DGCR8: DiGeorge syndrome critical region 8. Created with biorender.com, accessed on 25 April 2022 (published with permission from biorender.com, accessed on 25 April 2022). oxygen which interferes with the activity of DNA methyltransferases (DNMT); they also oxidize guanine from CpGs, which means that DNMT cannot methylate the cytosine or that binding to the methyl binding protein (MBP) complex does not take place. Guanine oxidation also makes the methylated cytosine more susceptible to oxidation by ten-eleven translocation (TET). In addition, ROS interfere with DNMT activity by depleting S-adenosylmethionine (SAM) by oxidizing methionine adenosyltransferase (MAT) and methionine synthase (MS) or by using homocysteine to regenerate glutathione (GSH). These effects cause DNA hypomethylation; however, ROS can also cause DNA to be hypermethylated by inhibiting TETs. glutathione, dehydrogenase, NADH: nicotinamide adenine dinucleotide, CoA: coenzyme A, SAH: S-adenosylhomocysteine, LSD: lysine-speciﬁc demethylases, FADH 2 : ﬂavin adenine dinucleotide, O 2 : oxygen, CO 2 : carbon dioxide 2OG: 2-oxoglutarate, H 2 O 2 : hydrogen peroxide, CoA: coenzyme A, Fe: iron.


Introduction
Cadmium (Cd) is a heavy metal with no biological function, which is highly toxic to living beings [1,2]. The primary sources of exposure for humans are water, food, and air pollution [3][4][5][6]. In addition, cigarette smoke also represents an essential source of Cd [7,8]. Exposure to Cd has been a cause for concern as it is associated with numerous health effects such as renal dysfunction, obstructive airway diseases, emphysema, dysregulated blood pressure, bone disorders, immunosuppression, reproductive and pregnancy disorders, and developmental toxicity [9,10]. Cd exposure has also been associated with the development of different types of cancer [11,12]; hence, it was classified as a human carcinogen in group with the ability of this metal to damage the electron transport chain (ETC), alter the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), increase free iron (Fe), and decrease antioxidant defense [18,19].
Several studies show that OS induced by environmental pollutants has an essential role in programming disease phenotypes through epigenetic processes [20]. Epigenetic changes associated with Cd exposure are methylation of deoxyribonucleic acid (DNA), histone modification, and impaired expression of noncoding ribonucleic acid (ncRNA) [21][22][23]. These Cd-induced changes in the epigenome have been primarily related to kidney and liver damage and cancer [24][25][26]. However, the role of OS in Cd-induced epigenetic modifications is still underexplored. Therefore, this review aims to provide an update on the basics of OS and its relationship to Cd-induced epigenetic changes. In addition, we focus on recent studies demonstrating the use of antioxidant compounds to mitigate Cd-induced epigenetic alterations.

Reactive Oxygen Species (ROS)
Living aerobic systems use oxygen (O 2 ) for various metabolic processes, including efficient energy production. For example, to produce energy in the form of adenosine triphosphate (ATP) in mitochondria, O 2 is needed as the final electron acceptor in the ETC [27]. However, O 2 can be toxic at concentrations >21% to aerobic organisms [28]. O 2 in its most abundant form and most stable or fundamental state is a diradical diatomic molecule. It has two unpaired electrons, each located in an antibonding orbital π*. Therefore, both have the same quantum spin number [28,29]. Because of this characteristic, O 2 can only react with compounds that have equally unpaired electrons and have a quantum spin number opposite to that of this molecule [29].
To overcome spin restriction, O 2 accepts electrons one at a time to form water (H 2 O); this process is known as univalent O 2 reduction ( Figure 1). In univalent reduction, O 2 undergoes four successive reductions with one electron. The addition of the first electron produces superoxide radical (O 2 •− ); adding the second electron plus two protons yields hydrogen peroxide (H 2 O 2 ); adding a third electron yields the hydroxyl radical ( • OH) plus the hydroxyl anion (−OH); finally, adding the fourth electron plus two protons produces two H 2 O molecules. This addition of electrons leads to the formation of O 2 reduction intermediates, also called ROS [30][31][32]. On the other hand, the cytochrome c oxidase (COX) complex or complex IV of the mitochondrial respiratory chain acts as a defense by preventing the univalent reduction of O 2 . COX carries out the tetravalent reduction of O 2 . In tetravalent O 2 reduction, the unique structure of the oxygen-binding site on subunit I of mitochondrial COX simultaneously transfers four electrons to O 2 without the release of ROS [33].
The toxic effects of O 2 are mainly due to ROS production [34]. ROS is a collective term used to define species derived from O 2 , which are more reactive than O 2 . ROS contain O 2 that is incompletely reduced (e.g., H 2 O 2 , • OH) or with a different electronic distribution (e.g., singlet oxygen [ 1 O 2 ]) [28,32,35]. Due to their biological importance in signaling and generating damage to biomolecules (such as DNA, proteins, and lipids), ROS are widely studied.
ROS can be produced from both endogenous and exogenous sources. Endogenous sources of ROS, under aerobic conditions, are generated as a product of metabolic biochemical reactions. These include mitochondria, peroxisomes, microsomes, cellular activation by inflammation, and other enzymatic sources. In humans, mitochondrial ETC has been described as a significant cellular source of ROS production in most tissues [35,36]. Electron leakage in the ETC of complexes I, II, and III reduces O 2 to O 2 •− [37]. Under physiological conditions, it is estimated that 1% to 2% of the electrons entering the ETC result in the production of O 2 •− [36,38]. Other ROS such as H 2 O 2 , • OH, and hypochlorous acid (HOCl) are derived from O 2 •− [37,39]. In contrast, exogenous ROS sources include environmental agents such as heavy metal ions (Cd, lead, arsenic, Fe, and mercury), ultraviolet light, ionizing radiation, or xenobiotics ( Figure 2) [34,[40][41][42][43].

OS
The first OS concept was formulated by Sies in 1985 [44]. He defined OS as "a disturbance in the prooxidant-antioxidant balance in favor of the former", and countless authors have accepted it; however, Jones [45] proposed to redefine the term and argued that a more helpful definition is that OS is "an interruption of redox signaling and control". Finally, an updated definition was reformulated by Sies and Jones in 2007 [46] as "an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage"; at the moment, this definition the most accepted.

Oxidant Damage Caused by OS
Just as ROS has been observed to have physiological and signaling functions, the dysregulated production of ROS can damage biological molecules. The persistence of OS leads to oxidative damage, which is considered damage to chemical structures (oxidation modifications) of nucleic acids, proteins, lipids, and carbohydrates [47]. ROS that damage nucleic acids affect the nitrogenous bases of purine (adenine, guanine) or pyrimidine (cytosine, thymine) and/or the sugar 2-deoxyribose. Guanine is the DNA base most susceptible to oxidation. DNA damaged by oxidation can generate mutations, can decrease protein synthesis, and may be linked to cancer, the development of aging, or degenerative diseases. The effects depend on the damage's extent, location, and repair [48].
On the other hand, the most common oxidant damage to proteins is the oxidation of thiol groups (-SH) of proteins (amino acids with sulfur, cysteine, and methionine are the most susceptible) and the formation of protein carbonyls (aldehydes and ketones). The amino-acid residues most vulnerable to oxidation are lysine, arginine, proline, and threonine. Protein oxidation leads to inhibition of their function through inhibition of enzyme activity, conformational changes, crosslinking, or aggregation ( Figure 2) [32,58]. Therefore, the establishment of cellular antioxidant responses must be fast and efficient to neutralize the possible oxidizing effects of ROS.

Antioxidants
Antioxidants are molecules vital in reducing ROS's oxidizing processes and harmful effects [59]. They include any substance that delays, prevent, or eliminates oxidative damage from a target molecule [28]. Antioxidants can act directly by neutralizing ROS or indirectly by regulating endogenous antioxidant defenses and inhibiting ROS production [60].

Classification and Description of Antioxidants
Antioxidants can be classified depending on the level of antioxidant action, i.e., the role they play against OS, and they are classified as the first line, second line, third line, and fourth line of defense [61]. They can also be classified by their molecular weight as high or low, by their nature as enzymatic and nonenzymatic, and by their origin as endogenous and exogenous [61,62]. In this article, we focus on the last two classifications.

Endogenous Antioxidants
The endogenous antioxidant system comprises enzymatic antioxidants and nonenzymatic antioxidants [63].

Nonenzymatic Antioxidants
Nonenzymatic antioxidants work by interrupting free-radical chain reactions to minimize the damage caused. Examples of nonenzymatic antioxidants are glutathione (GSH), bilirubin (BR), uric acid, and melatonin [72][73][74]. Some of these antioxidants are water-soluble, found mainly in the cytosol or cytoplasmic matrix. Others are lipid-soluble and are present in cell membranes [75].

Exogenous Antioxidants
Exogenous antioxidants or dietary antioxidants (DAs) can be obtained mainly through the diet by consuming fruits, vegetables, byproducts, and/or food supplements. Some examples are vitamins such as A, C, and E, some phenols, and flavonoids [76]. DAs together with endogenous antioxidants can counteract OS and maintain redox homeostasis [62,77]. Previously, in vitro studies suggested that the primary mechanism of DA was through free-radical scavenging, but this is not possible in vivo because DAs, except vitamin E, are not in sufficient concentration and their reactions are not as fast as ROS reactions. It is now known that the main mechanisms of DAs are the maintenance of nucleophilic tone through parahormesis, understood as the generation of signals for the induction of antioxidant enzymes (e.g., induction of nuclear factor E2-related factor 2 (Nrf2)) [78]. In addition, supplementation of essential metals such as zinc (Zn) and magnesium (Mg) has been shown to have beneficial pro-antioxidant effects [79]. Zn has been proposed to act as a proantioxidant agent or cofactor through three mechanisms: (1) protection of the free sulfhydryl group in proteins, (2) outperforming redox-active metals, and (3) specific induction of the antioxidant system response [80]. Mg participates in energy metabolism since intracellular adenosine triphosphate (ATP) is in the form of ATP-Mg [81], and its deficiency has been associated with an increase in ROS and a decrease in antioxidant enzymes [81].

Role of OS in Cd Toxicity
OS plays an essential role in Cd-induced toxicity, in a wide variety of cell culture systems [82][83][84], in intact animals [85][86][87], or in humans through all routes of exposure [88,89]. However, it has been seen that Cd generates OS via indirect mechanisms since it is not an active redox metal and cannot carry out the Fenton reaction like other metals [90]. The mechanisms via which Cd increases ROS production include (1) damage to ETC, (2) increased NOX activity, (3) increased concentration of free Fe, and (4) decreased antioxidant defense ( Figure 3) [18,19].
Excessive production of ROS or reactive nitrogen species (RNS) in the mitochondria is a consequence of Cd-induced ETC damage. Cd inhibits mitochondrial complexes causing electron leakage and the collapse of mitochondrial membrane potential (∆Ψm), which increases ROS and RNS [19]. The primary proposed mechanism via which Cd could be inhibiting the mitochondrial complexes of the ETC is by binding to cysteine residues, Fe-S groups, and the SH groups forming the complexes [91]. In addition, it has been observed that Cd generates unstable semiubiquinones and the accumulation of these promotes the production of O 2 •− [92]. On the other hand, the increase in NOX expression due to Cd exposure has been reported in different studies [93]. NOX is a family of enzymes that use NADPH as a reducing agent to generate O 2 •− [94]. NOX helps regulate metabolism and homeostasis; however, an excess in the production of O 2 •− can be harmful to different cellular components. Cd has been reported to increase NOX1 expression in rats treated with 1 mg/kg Cd chloride (CdCl 2 ) [93]. In addition, Cd also increases ROS production by raising the concentration of active redox metals such as Fe. Cd replaces Fe in several cytoplasmic and membrane proteins, such as ferritin and apoferritin, causing the concentration of the free Fe ion to increase. Being an active redox metal, Fe participates in the Fenton reaction, generating an increase in ROS production [18,95].
fore, OS [18,95]. Cd also alters the activity of SOD, CAT, and GPx. The alterations of these enzymes by Cd are given by the fact that this metal can change the enzymatic topography fundamental for the catalytic function of the enzymes [97], replace the metals that are part of the active centers of enzymes [18,98], or deplete the cofactors necessary for functioning; for example, selenium depletion due to the formation of the Cd-Se-Cys complex decreases GPx activity [18,[99][100][101]. In addition, Cd indirectly decreases the concentration of antioxidant enzymes by altering the expression of Nrf2 [102][103][104]. Cd is a non-redox metal that causes OS via different indirect mechanisms. The mechanisms via which Cd increases reactive oxygen species (ROS) production include damage to the electron transport chain (ETC). Cd binds to the thiol groups (-SH or -S-) of the complexes, generating a decrease in the mitochondrial membrane potential (ΔΨm) and mitochondrial uncoupling that leads to lower production of triphosphate (ATP). Moreover, Cd increases the activity of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), which increases the production of superoxide radical (O2 •− ). Likewise, Cd generates OS by increasing the concentration of redox metals such as iron (Fe 2+ ). Fe 2+ can participate in the Fenton reaction and increase radical hydroxyl ( • OH) production. Lastly, Cd also causes a decrease in the activity of antioxidant enzymes such as superoxide dismutase (SOD), manganese superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase (GPx). Ultimately  Cd is a non-redox metal that causes OS via different indirect mechanisms. The mechanisms via which Cd increases reactive oxygen species (ROS) production include damage to the electron transport chain (ETC). Cd binds to the thiol groups (-SH or -S-) of the complexes, generating a decrease in the mitochondrial membrane potential (∆Ψm) and mitochondrial uncoupling that leads to lower production of triphosphate (ATP). Moreover, Cd increases the activity of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), which increases the production of superoxide radical (O 2 •− ). Likewise, Cd generates OS by increasing the concentration of redox metals such as iron (Fe 2+ ). Fe 2+ can participate in the Fenton reaction and increase radical hydroxyl ( • OH) production. Lastly, Cd also causes a decrease in the activity of antioxidant enzymes such as superoxide dismutase (SOD), manganese superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase (GPx Another important mechanism via which Cd generates OS is the decrease in antioxidant defenses, which both direct and indirect interactions achieve. GSH is one of the antioxidants that is decreased, and this decrease occurs due to the affinity of Cd for the thiol group of GSH [96]. GSH deficiency causes alterations in the redox state and, therefore, OS [18,95]. Cd also alters the activity of SOD, CAT, and GPx. The alterations of these enzymes by Cd are given by the fact that this metal can change the enzymatic topography fundamental for the catalytic function of the enzymes [97], replace the metals that are part of the active centers of enzymes [18,98], or deplete the cofactors necessary for func-tioning; for example, selenium depletion due to the formation of the Cd-Se-Cys complex decreases GPx activity [18,[99][100][101]. In addition, Cd indirectly decreases the concentration of antioxidant enzymes by altering the expression of Nrf2 [102][103][104]. In the end, the OS generated by exposure to Cd causes damage to biomolecules such as lipids (formation of MDA and 4 HNE) [85,87,105] and DNA (8-hydroxydeoxyguanosine (8-OHdG) adduct formation) [106,107], causing cell death by apoptosis or necrosis [108,109]. Therefore, OS has been described as one of the causes of several conditions generated by Cd, such as nephrotoxicity, hepatoxicity, and cancer [110]. Furthermore, it is thought to play a role in the epigenetic changes reported by Cd exposure.

Epigenetic Effects of Cd Exposure
Growing scientific evidence shows that environmental exposure to heavy metals generates significant epigenetic alterations [111]. In particular, it has been reported that exposure to Cd induces alterations in DNA methylation, post-translational histone modifications, and ncRNA expression.

DNA Methylation
DNA methylation is the best-studied epigenetic alteration and can be defined as a covalent modification that occurs almost exclusively in the cytosine of the cytosineguanosine (CpG) dinucleotide sequences [112]. The DNA methylation process is carried out by the DNA methyltransferase (DNMT) family, including DNMT3A, DNMT3B, and DNMT1. DNMT3A and DNMT3B methylate sites that were not previously methylated; that is, they are responsible for de novo methylation [113]. On the other hand, DNMT1 is responsible for maintaining the methylation marks during the mitotic division process; therefore, it is said to be a maintenance methyltransferase [113].
DNA methylation occurs when DNMT transfers a methyl group of the S-adenosylmethionine (SAM) cofactor to cytosine forming 5-methylcytosine (5-mC). In turn, SAM can regenerate again from methionine. When SAM donates the methyl group to cytosine, it is transformed into S-adenosylhomocysteine (SAH), which is hydrolyzed to homocysteine. Homocysteine can generate methionine by the action of methionine synthase (MS). Finally, methionine regenerates SAM via the action of methionine adenosyltransferase (MAT) [114][115][116].
CpG methylation has been mainly associated with gene silencing. Methylation prevents transcription factors from accessing their binding sites directly by methylating transcription factor binding sequences or indirectly by recruiting methyl-CpG binding domain (MBD) proteins. MBD proteins block the binding between transcription factors and DNA or recruit chromatin modifiers that alter chromatin structure in a more transcriptionally repressive environment [117].
In the context of environmental pollution, it has been shown that several metals can have a profound impact on DNA methylation with subsequent effects on gene expression [111]. Several research groups have been dedicated to explaining the association between Cd exposure and DNA methylation, and this metal has been found to induce both hypomethylation and hypermethylation [119,120].
DNA hypomethylation by Cd is mainly carried out by decreasing DNMT activity and increasing TETs. A study conducted on nonsmoking women from the Argentinian Andes showed that environmental and dietary exposure to Cd is associated with a decrease in methylation of the long intercalated element-1 (LINE-1) and decreased expression of DNMT3B [121]. LINE-1 is a repetitive DNA retrotransposon that makes up about 17% of the human genome; thus, methylation or demethylation of LINE-1 is considered a marker of global DNA methylation [122].
TET expression also plays an essential role in Cd-induced DNA hypomethylation. It was previously reported that human embryonic kidney cells (HEK293) exposed to CdCl 2 in the short term increased messenger RNA (mRNA) and TET protein levels, altering the methylation of complete genomic DNA [123].
On the other hand, DNA hypermethylation by Cd has also been widely reported. In a study conducted in pairs of mothers and children, alterations in the methylation of 61 genes in fetal blood were observed, associated with the Cd of maternal blood at the time of delivery [124]. In addition, Cd exposure was associated with a decrease in gestational age through an increase in DNA methylation at a specific CpG site, cg21010642 [125]. The CpG site was noted in a gene involved in early embryonic development. Therefore, irregular methylation patterns at this site may contribute to preterm birth by mediating irregular biological mechanisms [125].
Change-induced hypermethylation has been widely linked to increased expression of DNMT proteins. In human bronchial epithelial cells (16HBE), Cd elevated the expression of DNMT1 and DNMT3A, causing DNA hypermethylation, inactivating tumor suppressors and genes associated with DNA repair [126]. Likewise, in lymphocytes treated with Cd, hypermethylation of the promoter p16 associated with the increase in DNMT1 and DNMT3B expression levels was observed [127]. Overexpression of DNMT3B is also demonstrated after chronic Cd-induced toxicity in the human prostate [25]. Expression levels of DNMT3B and DNMT3L are crucial in DNA methylation in placentas exposed to Cd [128].
Discrepancies between whether Cd increases or decreases DNA methylation are associated with the duration of Cd exposure. Evidence shows that acute exposures to Cd induce DNA hypomethylation, whereas chronic exposures cause DNA hypermethylation [21,129]. A study conducted on TRL1215 cells derived from rat liver reported that short exposure to Cd (1 week) decreases the activity of DNMT, causing decreased DNA methylation, while prolonged exposure to Cd (10 weeks) produces DNA hypermethylation due to increased DNMT activity [21]. Similar effects were also reported in rat livers [130]. It has also been reported that 24 h to 1 week exposure of Cd in chronic myelogenous leukemia (K562) cells led to DNA hypomethylation through noncompetitive inhibition of DNMT activity by Cd [131]. In contrast, exposure of 8 to 10 weeks to Cd caused global DNA hypermethylation and increased DNMT activity in human embryonic lung fibroblast (HLF) cells [120]. In addition, studies carried out in Lumbricus terrestrial earthworms proved that hypermethylation by Cd has long-lasting effects since it can be extended up to a period of 7 months at a low environmentally relevant concentration of Cd (10 mg/kg) [132].
Alterations in DNA methylation due to Cd exposure are associated with different diseases such as cancer, Alzheimer's, diabetes, multiple sclerosis, hepatic and renal alterations, and the reproduction and development of organisms [133]. In particular, DNA hypermethylation induced by prolonged exposure to Cd has been associated with malignant transformation in rat liver-derived TRL1215 cells, human prostate epithelial cells, human lung fibroblasts, and human bronchial epithelial cells (BEAS-2B) [21,25,120,134]. Cd has also been shown to silence the expression of enzymes that repair DNA in a dose-dependent manner through hypermethylation [25,120,126].
On the other hand, Cd-induced DNA hypomethylation has been proposed as a cause of alterations in the reproduction and development of organisms. Exposure to Cd for 24 h caused hypomethylation of the HSD11B2 gene in cultured human choriocarcinoma cells, causing increased expression of its protein hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2), which regulates the steroid hormone cortisol [135]. Likewise, DNA hypomethylation has been observed in babies' umbilical cord blood with managerial exposure to Cd, which can persist until 9 years of age [136]. Alterations in methylation from exposure to Cd are also associated with alterations in size at birth [137,138], and specific differences have been reported depending on sex [139].
Multiple sclerosis has also been described as a consequence of DNA methylation as an epigenetic change influenced by environmental factors, such as heavy metal contamination. A study conducted with patients with multiple sclerosis reported the correlation of a high concentration of Cd with increased expression of the atypical chemokine receptor 3 (ACKR3) gene by hypomethylation and decreased expression of the apolipoprotein E (APOE) gene by hypermethylation. This indicates that epigenetic changes induced by Cd may be an essential factor in increasing and decreasing the expression of genes involved in the appearance and/or progression of inflammatory processes in multiple sclerosis [140].
Lastly, alterations in DNA methylation have also been associated with liver diseases. It was found that there is a positive relationship between the degree of DNA methylation and the concentration of Cd, and there were many differential sites of methylation promotion in DNA from rat liver tissue [24].
In summary, the alterations in DNA methylation induced by Cd are related to the time of exposure to this metal. The short-term effects induced by Cd are due to a decrease in DNMT activity and an increase in TETs. Meanwhile, the effects of prolonged exposures have been linked to increased levels of DNMT. In addition, Cd-induced DNA hypomethylation and hypermethylation are related to various diseases; thus, understanding how Cd generates these alterations in DNA methylation helps to understand the toxic mechanisms of Cd exposure ( Figure 4).
Oxygen 2022, 2, FOR PEER REVIEW 10 tion from exposure to Cd are also associated with alterations in size at birth [137,138], and specific differences have been reported depending on sex [139]. Multiple sclerosis has also been described as a consequence of DNA methylation as an epigenetic change influenced by environmental factors, such as heavy metal contamination. A study conducted with patients with multiple sclerosis reported the correlation of a high concentration of Cd with increased expression of the atypical chemokine receptor 3 (ACKR3) gene by hypomethylation and decreased expression of the apolipoprotein E (APOE) gene by hypermethylation. This indicates that epigenetic changes induced by Cd may be an essential factor in increasing and decreasing the expression of genes involved in the appearance and/or progression of inflammatory processes in multiple sclerosis [140].
Lastly, alterations in DNA methylation have also been associated with liver diseases. It was found that there is a positive relationship between the degree of DNA methylation and the concentration of Cd, and there were many differential sites of methylation promotion in DNA from rat liver tissue [24].
In summary, the alterations in DNA methylation induced by Cd are related to the time of exposure to this metal. The short-term effects induced by Cd are due to a decrease in DNMT activity and an increase in TETs. Meanwhile, the effects of prolonged exposures have been linked to increased levels of DNMT. In addition, Cd-induced DNA hypomethylation and hypermethylation are related to various diseases; thus, understanding how Cd generates these alterations in DNA methylation helps to understand the toxic mechanisms of Cd exposure ( Figure 4).

Histone Modification
Histones are proteins that provide structural support to DNA within units known as nucleosomes [141]. Each nucleosome comprises two identical subunits containing four histones: H2A, H2B, H3, and H4 [142]. In addition, there is another histone, H1, which is not part of the nucleosome itself, but its function is to stabilize the internucleosomal DNA [143]. Post-translational modifications in these histones decide DNA's interaction with the different components. For example, modifications that disrupt the histone-DNA interaction cause relaxed chromatin, known as euchromatin, where DNA is accessible for transcriptional machinery binding and subsequent gene activation. In contrast, modifications that strengthen histone-DNA interactions create a tightly packed chromatin structure called heterochromatin, where the transcriptional machinery cannot access DNA, causing gene silencing [144].
There are different post-translational modifications in histones, which occur mainly in the tail domains of these proteins. The best-known histone modifications are methylation, phosphorylation, acetylation, and ubiquitylation; however, O-GlcNAcylation, citrullination, nitration, ribosylation, crotonylation, sumoylation, and isomerization are also found [145][146][147][148]. Each modification is added to or removed from histone amino-acid residues by a specific set of enzymes. Dysregulation of histone-modifying enzymes will alter post-histone modification patterns and cause various diseases, including cancer. Consequently, histone-modifying enzymes have become promising biomarkers for disease diagnosis and prognosis [149].
Cd-induced histone modifications have not been very studied compared to other epigenetic modifications. However, few studies have shown that Cd exposure affects histone acetylation, methylation, and phosphorylation primarily by altering the expression of enzymes that modify them.
Histone acetylation involves the addition of a negatively charged acetyl group to lysine residues in the tails of histone proteins [150]. When a histone is acetylated, the histone-DNA interaction is weakened; hence, it has been associated with transcriptional activation [151]. There are two enzymes responsible for regulating this process, histone acetyl transferases (HAT), which are responsible for adding acetyl groups, and histone deacetylases (HDAC), which eliminate acetyl groups [152]. A current study reported that Cd exposure increases HDAC2 expression and impairs the learning and memory of rats exposed to 5 ppm of Cd in drinking during weaning and up to three months of age. These effects were most significant when Cd exposure was combined with lead [153]. Likewise, it was observed that maternal exposure to Cd in mice impairs embryonic development and increases the expression of HDAC1 [154].
Lastly, another modification in histones observed by exposure to Cd includes phosphorylation. Histone phosphorylation confers a negative charge on the histone, resulting in more open chromatin conformation. This modification has been associated with gene expression and is involved in DNA damage repair and chromatin remodeling [163]. Cd has been shown to decrease H3 phosphorylation by inhibiting the human vaccinia-related kinase (VRK1/2) in an in vitro model [164].
In summary, Cd causes histones' methylation, phosphorylation, and acetylation (Figure 5). Because histones are the most common chromatin proteins, any change in their abundance, structure, or post-translational modifications (PTMs) will severely impact the overall structure of chromatin, influencing gene expression, as well as genome stability and replication.
Oxygen 2022, 2, FOR PEER REVIEW 12 with gene expression and is involved in DNA damage repair and chromatin remodeling [163]. Cd has been shown to decrease H3 phosphorylation by inhibiting the human vaccinia-related kinase (VRK1/2) in an in vitro model [164]. In summary, Cd causes histones' methylation, phosphorylation, and acetylation ( Figure 5). Because histones are the most common chromatin proteins, any change in their abundance, structure, or post-translational modifications (PTMs) will severely impact the overall structure of chromatin, influencing gene expression, as well as genome stability and replication.

ncRNA
ncRNAs are RNA molecules that are not translated into proteins, and it has recently been described that they can participate in epigenetic regulation by adjusting gene expression without altering the DNA sequence [165]. Transcriptional ncRNAs are classified into small ncRNAs and long ncRNAs (LncRNAs). Small ncRNAs can be divided into

ncRNA
ncRNAs are RNA molecules that are not translated into proteins, and it has recently been described that they can participate in epigenetic regulation by adjusting gene expression without altering the DNA sequence [165]. Transcriptional ncRNAs are classified into small ncRNAs and long ncRNAs (LncRNAs). Small ncRNAs can be divided into microRNAs (miRNAs), P-element induced wimpy (PIWI)-RNAs of interference (piRNAs), and small RNAs of interference (siRNAs) [165].
LncRNAs have a length of more than 200 nucleotides and are transcribed by RNA polymerase II (POLII). Depending on their location and their specific interactions with DNA, RNA, and proteins, LncRNAs can modulate chromatin function, obstruct transcriptional machinery, maintain nuclear speckle structure, and regulate nuclear body assembly and function. Without a membrane, they act as molecular sponges for miRNAs, alter the stability and translation of cytoplasmic mRNAs, and interfere with signaling pathways [166,167]. These functions affect gene expression in various biological and pathophysiological contexts, such as neuronal disorders, immune responses, and cancer. Therefore, the expression of LncRNAs can be used as potential biomarkers and justify attacking them clinically [167].
miRNAs are the smallest ncRNAs,~21 to 23 nucleotides in length, that regulate posttranscriptional gene expression through their complementary binding to untranslated regions 3 or 5 (UTR 3 or 5 ) of mRNAs, causing their degradation or translation blocking [168]. miRNAs can also regulate transcription by directly binding to gene promoter sequences and inducing chromatin remodeling [169]. The miRNAs are transcribed by the action of POLII and subsequently processed in the nucleus by RNase III (Drosha), within a nuclear protein complex called Microprocessor, and DiGeorge syndrome critical region 8 (DGCR8) to form structures of hairpin known as pre-miRNAs [170]. The pre-miRNAs are then exported to the cytoplasm, cleaving them by Dicer1 to generate a mature miRNA. More than 17,000 distinct mature miRNAs have been identified in more than 140 species [171]. Alteration in miRNA expression can be associated with various diseases such as cardiovascular and genetic disorders and cancer [172]. Therefore, it has been proposed that they can be used as biomarkers of exposure and disease due to the availability of circulating extracellular miRNAs found in body fluids, such as amniotic fluid, saliva, serum, and plasma [173].
On the other hand, piRNAs have a length of 26 to 32 nucleotides, and their name comes from the fact that they interact with PIWI proteins of the Argonaut family, which have endonuclease properties [174,175]. piRNAs act on somatic cells and are crucial for guiding epigenetic regulation and inducing the silencing of transposons [175,176]. They are synthesized from repetitive intergenic regions, and their processing is carried out independently of Dicer/Drosha. Abnormal piRNA expression is associated with several types of cancer, such as gastric, breast, renal, colorectal, and lung cancer [175].
siRNAs have a length of 20 to 25 nucleotides and, like miRNAs, rely on Dicer enzymes to separate them from their precursors [177]. siRNAs bind to the nucleotide sequence in their target mRNA and interfere with the expression of the respective gene, primarily by degrading mRNA. In addition, they also participate in the organization of the structure of chromatin in a genome and defend the cell from foreign nucleic acids [178]. siRNAs resemble miRNAs; when miRNAs are presented with a substrate with perfect complementarity, they can act as siRNA and guide multiple rounds of mRNA degradation [179].
In the context of environmental pollution, Cd has been shown to affect the expression of ncRNAs. Recent evidence shows that heavy-metal toxicity is related to aberrant alterations of endogenous miRNAs [26]. For example, exposure to Cd telluride (CdTe) induced new miRNAs in mouse embryonic fibroblast cells (NIH/3T3). These results were associated with the ability of CdTe to reprogram gene expression even after the initial signal was removed. In addition, alterations in miRNA biogenesis suggest that these molecules participate in the cytotoxicity of CdTe quantum dots by inducing apoptosis-like cell death [180]. In another study, 4 h exposure to Cd in murine ovarian granulosa cells resulted in the altered expression of five miRNAs involved in alternative splicing of kit ligand (kitl) pre-mRNA [181]. Furthermore, it has been seen that the accumulation of Cd can generate preeclampsia. miRNA-26a and miRNA-155 were affected in preeclamptic placentas and trophoblasts treated with Cd [182].
Alterations in miRNAs may also be associated with Cd-induced kidney damage [26,183]. A study conducted on the renal cortex of Sprague-Dawley rats subcutaneously exposed to CdCl 2 (0.6 mg/kg 5 days a week for 12 weeks) showed increased miRNA-34a-5p and miRNA-224-5p and decreased miRNA-455-3p [184]. Increased miRNA-132-3p was also found in primary human proximal tubular epithelial cells treated with CdCl 2 [185]. These findings demonstrate that Cd significantly alters the expression profile of miRNA in the kid-ney and raises the possibility that these alterations play an essential role in the physiology of Cd-induced kidney injury.
Cancer is another pathology associated with alterations in miRNAs induced by Cd. Cd causes malignant transformation of RWPE-1 prostate epithelial cells along with increased levels of Kirsten rat viral sarcoma oncogene homolog (KRAS) and alteration in miRNAs [186]. It was observed that about 12 miRNAs decreased (miRNA-373 was the lowest) in the transformed RWPE-1 cells [186]. In addition, Cd exposure affects the expression of p21 (Cip1/WAF-1) in a hepatoma cell line (HepG2) by increasing miRNA-372 [187]. p21 is a protein downstream of p53, a protein with an essential role in regulating apoptosis and preventing cancer development. Therefore, Cd-induced changes in p21 also alter the functions of p53 [188]. Similarly, exposure to Cd through smoking in bronchial epithelial cells modified multiple miRNAs via both up-and downregulations [189]. Lastly, it was found that exposure to Cd is also related to pancreatic ductal adenocarcinoma (PDAC). An in vitro study reported that exposure to CdCl 2 was overexpressed to miRNA-221 and miRNA-155, while miRNA-126 was downregulated [23].
Other ncRNAs that have been affected by Cd exposure are LncRNAs. A study conducted on workers exposed to Cd found a significant positive correlation between the level of Cd in the blood and LncRNA-ENST00000414355 and serine/threonine kinase (ATM) expression and a significant negative correlation between the level of Cd in the blood and the ∆Ψm [190]. Another study reported that a maternally expressed tumor suppressor LncRNA 3 (MEG3) is significantly downregulated in BEAS-2B cells transformed with Cd [191]. Cd exposure was also reported to alter LncRNAs expression during T-cell activation [192].
In summary, it can be said that Cd alters the expression of ncRNAs, particularly miRNAs and LncRNAs, which is related to kidney and liver damage and cancer progression ( Figure 6).

Relationship of Epigenetic Modifications with Cd-Induced OS
Exposure to different environmental factors, such as heavy metals, can change the biology of the epigenome and lead to adverse health effects. As discussed in the last section, Cd exposure causes epigenetic changes, including DNA methylation, histone modifications, and ncRNA expression. OS may play an essential role in the epigenetic changes induced by Cd [26,193,194].

DNA Methylation
OS is an indirect mechanism via which Cd overexposure influences DNA methylation. Exposure to Cd has been associated with both hypomethylation and hypermethylation of DNA (Figure 7). In hypomethylation, there are different mechanisms via which OS could be participating [195,196]. One of the mechanisms is the depletion of SAM levels. It has been seen that OS can inhibit the activity of MAT and MS by oxidizing them, which causes a decrease in SAM [114][115][116]. Because SAM is the substrate for DNMT, its decrease causes less DNA to be methylated. Moreover, the trans-sulfuration pathway to regenerate GSH under OS conditions causes SAM depletion because this molecule is an intermediate of this pathway [197,198]. ; they also oxidize guanine from CpGs, which means that DNMT cannot methylate the cytosine or that binding to the methyl binding protein (MBP) complex does not take place. Guanine oxidation also makes the methylated cytosine more susceptible to oxidation by ten-eleven translocation (TET). In addition, ROS interfere with DNMT activity by depleting S-adenosylmethionine (SAM) by oxidizing methionine adenosyltransferase (MAT) and methionine synthase (MS) or by using homocysteine to regenerate glu- by Cd is involved in DNA methylation. Reactive oxygen species (ROS) cause DNA damage, which interferes with the activity of DNA methyltransferases (DNMT); they also oxidize guanine from CpGs, which means that DNMT cannot methylate the cytosine or that binding to the methyl binding protein (MBP) complex does not take place. Guanine oxidation also makes the methylated cytosine more susceptible to oxidation by ten-eleven translocation (TET). In addition, ROS interfere with DNMT activity by depleting S-adenosylmethionine (SAM) by oxidizing methionine adenosyltransferase (MAT) and methionine synthase (MS) or by using homocysteine to regenerate glutathione (GSH). These effects cause DNA hypomethylation; however, ROS can also cause DNA to be hypermethylated by inhibiting TETs. SAH: S-adenosylhomocysteine, GSH: glutathione, GSSG: glutathione disulfide; CpG: cytosine-guanosine dinucleotide sequences. Created with biorender.com, accessed on 25 April 2022 (published with permission from biorender.com, accessed on 25 April 2022).
Another mechanism of hypomethylation by OS is the generation of DNA lesions due to increased free radicals, mainly the • OH radical. Excessive • OH production is known to cause DNA damage, including base modifications, deletions, strand breaks, and chromosomal rearrangements [199,200]. Because DNA is damaged in these lesions, it cannot be used as a substrate for DNMT, resulting in global hypomethylation [201].
DNA hypomethylation may also be related to the oxidation of DNA bases in CpGs. Guanine is the preferred nucleotide for ROS to be oxidized, forming mainly (but not exclusively) 8-OHdG [202][203][204]. Guanine oxidation prevents methylation of the neighboring cytokine at CpG due to inhibition of the binding of DNMT enzymes to DNA [205,206]. In addition, the oxidation of guanine prevents the binding of the MBP complex to DNA because the formation of 8-OHdG causes the N7 position of guanine to be converted into a hydrogen bond donor instead of a hydrogen bond acceptor [193,207]. Furthermore, if the CpG cytokine where guanine is oxidized is methylated, then it becomes more susceptible to TET action [208].
A study conducted in Nile tilapias (Oreochromis niloticus) showed that exposure to Cd induces OS, including reduced antioxidant activities and increased MDA and 8-OHdG content, which was related to DNA hypomethylation. A decrease in DNMT and increased expression of TET1 and TET2 were also observed [209]. It should be noted that this study carried out in tilapia was chronic (45 and 90 days); thus, it contradicts the statement that acute Cd exposures are related to hypomethylation, while chronic exposures are related to hypermethylation [21,129]. However, the hypomethylation in the tilapia model could be explained because the OS was evident, and this is more related to DNA hypomethylation than to hypermethylation due to the mechanisms explained previously in this section.
In other words, OS has been shown to appear on acute exposures to Cd, and acute exposures are related to DNA hypomethylation, whereas prolonged exposures to Cd are more related to DNA hypermethylation; however, ROS production in chronic exposures becomes absent [210]. This could be because chronic exposure induces adaptation mechanisms to compensate for the ROS induced by Cd and OS, such as the induction of MT, the increase in cellular GSH, and the activation of the antioxidant transcription factor Nrf2 and other antioxidant components [210]. For example, it has been reported that DNA hypermethylation in prostate cells exposed to Cd is associated with increased DNMT activity and decreased OS and redox-sensitive signal transduction pathways, such as the c-Jun N-terminal kinase (JNK) [25,211].
Although most of the evidence shows that the OS in chronic exposures to Cd is low, there are studies where the excessive production of ROS has been reported. In a study carried out on zebrafish liver, it was found that heavy-metal contamination, mainly Cd, in the Le'an river caused OS and a significant increase in global methylation [212]. Furthermore, overproduction of O 2 •− has been shown to increase DNA methylation in some types of cancer, such as melanoma and possibly endometriosis [213]. In Cd-transformed human bronchial epithelial cells, promoter hypermethylation resulted in decreased expression of DNA repair genes [126]. In addition, it has been seen that the OS could inhibit the activity of the TETs. It is known that these proteins are dependent on Fe(II), which, during the TET catalytic cycle, is oxidized to Fe(III) and Fe(IV) [214], while it is regenerated by the action of ascorbate [215]. Under OS conditions, ascorbate levels are reduced; hence, Fe(II) is not regenerated, and TETs are inhibited, causing hypermethylation [216]. In a study performed on TRL1215 rat liver cells exposed to 2.5 µM Cd for 10 weeks and then cultured in a Cd-free medium for an additional 4 weeks, harmful modulation of TET1 was found. In addition, the expression of tissue inhibitors of metalloproteinases 2 and 3 (TIMP2 and TIMP3), which are positively regulated by TET1, was decreased by Cd. All of the above caused a decrease in the expression of apolipoprotein E (apoE), possibly due to DNA hypermethylation [217].
In summary, it can be said that Cd-induced OS plays a crucial role in DNA methylation and demethylation, particularly in acute exposures. However, the relationship of OS to DNA methylation in chronic Cd exposures needs further investigation.

Histone Modification
The chemical modification of histones is another epigenetic mechanism that can be altered by OS induced by environmental factors such as Cd [218,219]. It has been shown that OS and nitrosative stress (excessive increase in RNS) modify histones, affecting their folding and stability and their ability to modify themselves post-translationally ( Figure 8) [219].   In particular, it has been observed that Cd alters histone acetylation, and ROS production could be a cause [153,220,221]. Previously, maternal exposure to Cd was reported to affect embryo development prior to implantation by inducing DNA damage and increasing HDAC1 levels and ROS production [154]. The increase in HDAC could be attributed to the production of H 2 O 2 . It is known that H 2 O 2 generates an increase in lactate dehydrogenase (LDH), an enzyme that catalyzes the reduction of lactate to pyruvate, producing NAD + [222]. NAD + is used by HDACs, which stimulates deacetylation [222]. Notably, other studies have reported that ROS can also inhibit HDACs, which is achieved via different mechanisms, including carbonylation, phosphorylation, nitrosylation, or glutathionylation, due to ROS increase [223] and reactive aldehydes [224][225][226]. The inactivation of HDAC2 results in its ubiquitination and proteasome degradation, which increases histone acetylation. Cd-induced stress has also been associated with H4K5 acetylation and increased DNA damage in bean seedlings (V. faba) [227].
Cd also affects histone methylation, and OS could be involved. OS is related to increased histone methylation by inhibiting JmjC by oxidizing Fe(II) to Fe(III), which makes Fe(II) unavailable for use by JmjC [216]. Another way to inhibit this type of protein is when • NO binds directly to the catalytic Fe [228,229]. However, the production of ROS also generates a decrease in histone methylation. One of the reasons is that ROS decreases SAM levels. As explained above, under OS conditions, enzymes involved in SAM synthesis are inhibited and, thus, histone methyltransferases (HMTs) are blocked [197,230].
OS could also be involved in histone phosphorylation caused by Cd. ROS production generally generates breaks in double-stranded DNA, causing histone phosphorylation to trigger DNA repair [194,231]. In addition, it is known that H 2 O 2 increases histone phosphorylation via an ATR serine/threonine kinase (ATR)-dependent pathway [232]. However, it has also been found that OS can oxidize the catalytic metal ion within protein phosphatases, causing their inhibition [233].
Lastly, OS is related to other histone modifications; however, these have not yet been much explored with Cd exposure. One of them is the nitration of histones. ONOO − is known to nitrate histones, leading to increased β-sheet structures and increased thermostability. Furthermore, it is known that the in vivo nitration-denitration activities of histones could be involved in the control of numerous vital cellular events to maintain apoptosis [234,235]. Cd has been shown to increase ONOO − production by generating protein nitration [236].
On the other hand, another modification associated with OS is the modification of histones by the action of oxidized lipids. It has been reported that 4-oxo-2-nonenal (4-ONE) forms adducts with histones causing inhibition of nucleosome assembly [237]. In addition, 4-HNE has been shown to alter the binding of histones to DNA [238].
In summary, OS could have an essential role in Cd-induced histone modifications; however, there is a great need for more studies in this area. In addition, like DNA methylation, the few studies that have seen alterations in histone modifications due to OS are only in studies of acute exposure to Cd (24 and 48 h) [154,212].

ncRNA
ncRNAs are a newly identified group of epigenetic regulators sensitive to ROS and function according to cellular redox status (Figure 9) [165]. For example, the anormal expression of ncRNAs, mainly miRNAs, is partly attributed to the dysregulation of their transcription factors generated by increased ROS production. Some of these ROS-sensitive transcription factors are c-Myc, p53, and nuclear kappa light chain enhancer of activated B cells (NF-κB) [239,240]. A study conducted on Daphnia pulex found that exposure to Cd plus hypoxia increases ROS production by triggering increased extracellular signal-regulated kinases (ERK), protein kinase B (Akt), and hypoxia-inducible factor 1α (HIF1α), which in turn promotes miRNA-210 expression [241].
In miRNAs, biosynthesis may be affected by ROS exposure. The formation of pre-miRNA is carried out by the Drosha-DGCR8 complex, which depends on Fe(III) for its action. The pre-miRNAs then leave the core and mature by Dicer processing. ROS has been shown to promote the processing power of DGRC8 [239,242]. However, ROS inhibits Dicer activity to delay the production of mature miRNAs, thereby coordinating cellular behavior [239]. Oxidative modifications can also influence pri-miRNA protrusions and loops [243].
miRNA expression may also be affected by increased ROS. ROS has been reported to affect the methylation status of specific promoter regions of miRNA genes [244]. Previously, the expression of miRNA-199a and miRNA-125b was shown to be decreased in the presence of ROS, mainly by upregulation of DNMT1 [245]. In addition, inhibition of miRNA-122 expression in tilapia livers by increased ROS production induced by Cd exposure has been demonstrated. The decrease in miRNA-122 was related to increased metallothionein levels [246]. In miRNAs, biosynthesis may be affected by ROS exposure. The formation of pre-miRNA is carried out by the Drosha-DGCR8 complex, which depends on Fe(III) for its action. The pre-miRNAs then leave the core and mature by Dicer processing. ROS has been shown to promote the processing power of DGRC8 [239,242]. However, ROS inhibits Dicer activity to delay the production of mature miRNAs, thereby coordinating cel- On the other hand, it has been observed that most miRNAs that respond to ROS influence the Nrf2 system [247]. A study conducted on the human hepatocellular carcinoma (HepG2) cell line showed that Cd induces the expression of transcription factor MTF1, which activates the expression of MT1DP, a pseudogene in the MT family. Subsequently, MT1DP raises the levels of miRNA-365, which causes a decrease in Nrf2 levels, generating OS [248].
In general, it could be said that OS plays an essential role in the maturation of ncRNAs, and, in turn, some of the ncRNAs are also involved in the cellular response to OS. In addition, most of the studies in which alterations in ncRNA expression have been seen as a consequence of OS were carried out with acute exposures to Cd (6 and 24 h) [154,227].

Use of Antioxidants to Mitigate Cd-Induced Epigenetic Alterations
There is no specific or 100% effective treatment for chronic Cd intoxication. However, metal chelators have been widely used [1,252]. Chelators are chemical agents that work by binding tightly to metals in the bloodstream, leading to sequestration of the metal for later excretion [253]. Chelators used for Cd poisoning include ethylenediaminetetraacetic acid (EDTA), penicillamine (DPA), dimercaprol (anti-British Lewisite (BAL)), 2,3-dimercaptopropanesulfonic acid (DMPS), and dimercaptosuccinic acid (DMSA) [1]. However, the use of chelators in Cd intoxication faces various problems, among which the increase in the body load of Cd in the kidney and the competition of chelators with albumin, macroglobulin, and MT stand out.
Another strategy used to treat Cd toxicity is to enhance the binding of this metal to MT. The latter can be achieved by Zn supplementation [254]. Zn is an essential metal that has been shown to attenuate Cd-induced OS because Zn functions as a cofactor for the antioxidant enzyme copper/zinc superoxide dismutase (Cu/Zn SOD) [255]. Furthermore, Zn increases the expression of MT [256]. MTs are low-molecular-weight proteins with a high affinity for Cd and other heavy metals due to the -SH on their cysteine residues, which aid detoxification [257]. However, given the critical role that OS plays in Cd toxicity, the use of compounds with antioxidant properties has become one of the most promising strategies [258]. In addition, the use of natural compounds represents advantages compared to chelating compounds, including high safety and few or no side-effects.
Among the compounds or plant extracts with antioxidant properties used to attenuate the toxic effects induced by Cd are green and black tea (Camellia sinensis), blueberries (Aronia melanocarpa), garlic (Allium sativum), onion (Allium cepa), holy basil (Ocimum sanctum), cape gooseberry (Physalis peruviana), ginger (Zingiber officinale), quercetin, lupeol, S-adenosylmethionine, lipoic acid, glutathione, selenium, N-acetylcysteine (NAC), methionine, cysteine, alpha-tocopherol, ascorbic acid, and resveratrol [258]. Many of these compounds have direct antioxidant effects by trapping ROS, while others exert their effects indirectly by inducing Nrf2 expression [258]. In addition, it should be noted that some of these compounds, such as epigallocatechin-3-gallate, have functional groups within their structure capable of chelating metals such as Cd [259]. Table 1 shows the antioxidants that have already been tested against epigenetic alterations induced by Cd.
Resveratrol CD-1 mice and JEG-3 cells CdCl 2  Selenium is one of the antioxidants with an antagonistic effect against Cd-induced carcinogenesis [260]. Selenium is an essential trace element in humans involved in numerous processes, including immune function and antioxidant defense. Selenium supplementation is protective against an extensive range of harmful factors, including heavy metals [265]. It has been shown that selenium can alleviate the proliferative effect of Cd on human breast cancer cells (MCF-7). One of selenium's mechanisms against carcinogenesis was the epigenetic regulation of 10 genes that Cd had modified. Within these genes, selenium downregulated APBA2 and KIAA0895 while upregulating DHX35, CPEB3, SVIL, MYLK, ZFYVE28, ABLIM2, GRB10, and PCDH9 [260]. Analyses of biological functions suggested that these epigenetically regulated genes are involved in multiple cancer-related pathways, such as focal adhesion and the PI3K/Akt pathway [260]. The main mechanisms via which selenium modified these genes were the regulation of DNA methylation and the expression of miRNA and LncRNA.
Quercetin has also been used against Cd toxicity. Quercetin is a polyphenolic flavonoid abundant in cabbages, onions, berries, apples, red grapes, broccoli, cherries, tea, and red wine [266]. Previously, 50 mg/kg quercetin treatment in male rats prevented hepatic steatosis and CdCl 2 -induced fibrosis by downregulating miRNA-21 transcription via an increase in ROS production [261]. miRNA-21 has a crucial role in various biological functions and diseases, including development, cancer, cardiovascular disease, and inflammation [267]. In addition, it has been shown to stimulate fibrosis in different organs by promoting fibroblast activation and subsequent deposition of extracellular matrix protein (e.g., collagen and fibronectin) [268], whereas quercetin can decrease ROS production and increase levels of Nrf2 and antioxidant enzymes [261]. Therefore, it could be said that quercetin protects against epigenetic alterations in the liver induced by Cd, thanks to its potent antioxidant potential.
On the other hand, N-acetyl-l-cysteine (NAC) is a soluble component of garlic, to which various properties, including antioxidant effects, have been attributed [269]. In addition, NAC has been shown to protect against Cd-induced carcinogenesis. Cd causes malignant transformation of rat liver cells (TRL1215) simultaneously with a negative regulation of ApoE through DNA hypermethylation inhibiting TET activity [217]. However, it has been observed that treatment with NAC restores TET levels, which have been associated with NAC properties to decrease the production of ROS generated by Cd [217].
Resveratrol is another antioxidant that has been used to attenuate epigenetic changes induced by Cd exposure. Resveratrol is a phytoalexin inedible material, including grape skin, peanuts, and red wine [270]. NF-κB, pi3K/Akt pathway, mTOR signaling, MAPK signaling, cyclooxygenases, phosphodiesterases, estrogen receptors, microRNAs, and various protein kinases are targets of this antioxidant [271][272][273]. These targets are involved in several biochemical pathways, including inflammation, cellular metabolism, cell-cycle regulation, cell signaling, and post-translational modification [274]. In vivo and in vitro studies indicate that resveratrol regulates epigenetic mechanisms, including enzymes such as DNMT, HDAC, and LSD1 [275]. Previously, a study conducted in rats exposed to 4.5 mg/kg CdCl 2 intraperitoneally and JEG-3 cells with 20 µM CdCl 2 showed that resveratrol could decrease Cd-induced hypermethylation. This effect was associated with decreased PI3K/Akt pathway [262].
Another antioxidant used against Cd-induced damage is flavone isoorientin (ISO), an extract of traditional Chinese medicine with proven antioxidant and anti-inflammatory properties. ISO is extracted from plants such as valerian and many fruits, foods, and herbs [276]. It has been proven that ISO can also protect from DNA damage. A study in rat proximal tubular cells (NRK2E and rPT) showed that 80 µM ISO administration decreased ROS production, H2AX histone phosphorylation, and oxidative DNA damage induced by exposure to 2.5 µM Cd. In addition, ISO relieved cell-cycle arrest caused by this metal. [263]. This indicates that ISO protects from Cd-induced OS and DNA damage.
Cyanidin-3-O-glucoside (C3G), an anthocyanin present in fruits, vegetables, and grains, has also been considered a potential antagonist of Cd toxicity [277]. In particular, C3G has been shown to protect against Cd-induced male reproductive dysfunc-tion. A study in CdCl 2 -fed male pubertal mice showed that treatment with 500 mg/kg C3G effectively protected spermatogenesis by normalizing histone modification, restoring histone to protamine exchanges, and enhancing the antioxidant system alleviating Cd-induced apoptosis [264].
Methionine has also been studied to mitigate the epigenetic effect of Cd [131]. Previously, Cd was shown to increase ROS production and DNA damage, stimulate cell proliferation, and induce global DNA hypomethylation in the K562 chronic myelogenous leukemia cell line. However, treatment with 1.0 mM of methionine significantly suppressed cell proliferation and raised the level of overall DNA methylation [131]. Methionine is one of the sulfur amino acids found in proteins and to which antioxidant properties have been attributed [278]. Methionine is thought to raise methylation levels by increasing the availability of SAM, the methyl donor for methylation, which has been seen to decrease with Cd treatment [279,280].
In summary, the data discussed in this section suggest that compounds with antioxidant properties are a promising treatment to decrease epigenetic alterations and the consequences of Cd exposure. The primary mechanism associated with this protection is the ability of these compounds to reduce the OS. However, it should also be taken into account that, in some instances, antioxidants not only do not help to counteract the toxicity of Cd, but can enhance it. A study in human HaCaT cells found a cumulative toxic effect with the coadministration of NAC and Cd [281]. In addition, it was observed that NAC does not act as a chelator, nor does it reduce the toxic effects induced by Cd in a model of Caenorhabditis elegans [282]. Something similar happened with the joint exposure of CdCl 2 and vitamin C in a zebrafish cell line (Z3 cells). In this investigation, the authors found that Cd is responsible for inhibiting the enzyme δ-aminolevulinate dehydratase in the rat lung and vitamin C increases the inhibitory effect [283].

Final Comments and Future Perspectives
Cd is one of the most toxic heavy metals associated with epigenetic alterations, including DNA methylation, histone modification, and ncRNA expression. OS is one of the mechanisms involved in these epigenetic alterations induced by exposure to Cd. The OS generated by Cd modifies DNA methylation by causing DNA damage, increasing the oxidation of nitrogenous bases, decreasing the concentration of SAM, and interfering with Fe homeostasis. It has also been related to histone modification since increased ROS production causes acetylation, methylation, phosphorylation, nitration, ribosylation, ubiquitination, sumoylation, or glycosylation of these proteins. Likewise, the OS induced by Cd alters the expression of ncRNAs by modulating their transcription factors and the enzymes necessary for their production and maturation. In addition, studies have shown that acute exposures to Cd are related to the induction of OS and the possible association with epigenetic alterations (DNA hypomethylation, histone modification, and alteration in ncRNA), while very few studies have related chronic exposures to OS. The alterations induced by Cd result in health problems such as alterations in development and reproduction, as well as kidney and liver diseases, multiple sclerosis, and cancer. Therefore, epigenetic marks associated with Cd exposure could be used to predict adverse health outcomes. In addition, taking into account the critical role of OS in the alterations induced by Cd and the evidence shown to date, the use of antioxidants should be considered a promising therapy to prevent these epigenetic alterations.