Introduction

Epigenetics can be explained as the study related to alteration in gene expression with unchanged DNA sequences. Presently, the field of epigenetics is rapidly expanding. Understanding its role in various diseases and therapeutic interventions is an emerging field of research worldwide [22, 62]. Alteration in expression of target genes depends on the epigenetic status of the chromatin structure, involving DNA promoter methylation or hydroxymethylation, histone modifications, promoter–enhancer interactions, and noncoding RNA–mediated regulation. De-regulation of epigenetic machinery leads to inappropriate onset of genes causing either activation or inhibition of downstream protein complexes [22, 62].

Present researches on epigenetics can be divided into two key areas: epigenetic-regulation of nuclear DNA and mitochondrial DNA [89]. Epigenetic-regulation of nuclear DNA involves DNA methylation, post-translational histone modifications and non-coding RNAs; whereas epigenetic-regulation of mitochondrial DNA seems to be alike in the aspect of DNA methylation and noncoding RNAs but lacks histone moieties. Previously it has been established that mitochondrial epigenetic-mechanisms in coordination with nuclear epigenetic regulation influence transcription activation or repression of crucial genes and protein synthesis [20].

Mitochondria possess their own genome, along with its transcription-translation machinery; communicates intricately with the nuclear genome for maintaining normal cellular homeostasis [20]. Human mtDNA consists of ∼16,569 base pairs (bp) double stranded circular DNA that encompasses 37 genes. The Heavy strand (or H-strand) consisting of 28 genes is guanine rich; whereas the Light strand (or L-strand) consisting of 9 genes is cytosine rich. Out of these, 13 genes encode for polypeptides that comprises the electron transport chain (ETC). These include 7 mitochondrial genes encoding for complex I subunit (MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6), one for complex III subunit (MT-CYB), three for complex IV subunits (MT-CO1, MT-CO2, MT-CO3) and two for complex V subunits (MT-ATP6, MT-ATP8), along with two ribosomal RNAs (MT-RNR1 and MT-RNR2) and 22 transfer RNAs [20, 26, 90]. Nuclear DNA possess single promoter for the transcription of a target gene, whereas total mtDNA has only three promoter regions (LSP, HSP1 and HSP2) responsible for transcribing all the genes from both L-strand and H-strand. Mitochondrial displacement loop (D-loop) is a unique region which contains all three promoters along with the replication start site (or RSS) controlling mitochondrial gene expression. Replication and transcription of mtDNA is regulated by multiple nuclear encoded proteins, like TFAM, POLRMT, TFB1M, TFB2M and mTERF etc. [90].

Based on previously established facts, mitochondrial epigenetics comprises of four areas: mtDNA methylation/hydroxymethylation, mitochondrial nucleoid modifications, mitochondrial RNA modifications and mtDNA-derived or nuclear DNA-derived non-coding RNA modulations during mtDNA-encoded gene transcription and protein synthesis [26, 90]. In this review, we highlighted the role of mtDNA methylation in various diseases including cancer and in exposures to environmental pollutants in order to provide an in-depth mechanistic insight.

Mitochondrial epigenetics

Despite decades of research work, mitochondrial epigenetics remained a controversial area of study. Major modifications in mitochondrial epigenome include DNA methylation or hydroxymethylation, demethylation and alteration in mitochondrial non-coding RNAs [16, 43, 68, 88]. However, as in nuclear genome, mitochondrial genome lacks histone packaging; therefore, post translational histone modifications are not a part of mitochondrial epigenetic regulation as in nuclear epigenetics. It is already established that, there exists a cross-talk between mitochondria and nucleus which plays an important role in maintaining overall cellular homeostasis. Key regulatory genes for biogenesis, fission-fusion, mitophagy and also mtDNA methyltransferase enzymes (responsible for mito-epigenetic modification) are encoded by nuclear genome. In response to specific cellular micro-environmental condition, synchronized anterograde (nuclear to mitochondrial) and retrograde (mitochondrial to nuclear) signalling pathways are found to be activated. Epigenetic changes in mitochondrial DNA may induce alterations in the nuclear epigenome leading to an array of downstream activities including differential gene expression, genetic instability and tumorigenesis [34, 61, 71]. Furthermore, in response to extracellular signals like environmental exposure towards toxic substances, mitochondria may play an essential role in modification of nuclear epigenetic regulation, which in turn affect mitochondrial functionality [68]. Thus, the reciprocity between nuclear and mitochondrial epigenome thought to be a crucial factor in understanding the aspects of mitochondrial functionality under normal and pathological conditions [51].

Mitochondrial DNA methylation and associated methyltransferases

Earlier, DNA methylation occurring in the mitochondrial epigenome has been a topic of debate since 1970s. In 1973, Nass MM had first reported the evidences of mtDNA methylation in in-vivo and in-vitro model systems [73, 74]. Gradually, technical advancements along with new methodologies have evolved for quantifying methylation on CpG- or non-CpG-specific sites that substantiated the existence of mtDNA methylation mark [90]. The conclusive evidence suggesting the presence of methylated CpG islands came from the study of Infantino and co-workers [44] who used a mass spectrometry-based system which overcame the technical limitations of former studies. On the contrary, few studies showed the absence of mtDNA methylation mark [27, 42, 59, 69]. Several factors such as circular nature of mitochondrial genome, different experimental model systems, disease occurrences, exposures to various external and internal stimuli, stressors etc. play a vital role in determining the presence or absence of mitochondrial cytosine methylation [27, 42, 59, 69].

DNA methylation occurs on cytosine residues that are followed by guanine residues near the promoter regions or transcriptional start sites (CpG islands or CpG clusters or CpG sequences) catalyzed by DNA methyltransferases (DNMTs) in presence of methyl donor, S-adenosylmethionine or SAM [86, 87]. Presence of mtDNA methyltransferase activity was first recorded by Nass MM [73]. Nuclear DNA methyltransferase 1 (DNMT1), the most abundant methyltransferase, is translocated to the mitochondria via signalling by a mitochondrial targeting sequence (MTS) located immediately upstream of the translational start site (TSS). MTS has been found to be conserved across mammals, encodes a peptide required for translocation of nuclear DNMT1 to mitochondrial matrix. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. However, MTSs are not present in the primary sequences of de novo methyltransferases DNMT3A and DNMT3B that are specifically found in mitochondria [87].

Mitochondrial DNA methyltransferase 1 or mtDNMT1 plays a significant role in the epigenetic mechanism of mtDNA methylation and in the regulation of transcription of mitochondrial genes. MtDNMT1 regulates the mitochondrial gene expression through mtDNMT1 binding to the mitochondrial D-loop control region and promoting transcription of target genes [27, 42, 59, 69, 87]. Interestingly, recent studies have highlighted the role of DNMT1 mutations and its effect on mitochondrial epigenome [36, 67]. Dominant mutations in Replication foci targeting sequence (RFTS), present in the N-terminal domain of DNMT1 has been previously reported, deletion of which has been shown to alter DNMT1-dependent DNA methylation in cancerous cells [96]. Currently, a study by Maresca et al., 2020 showed that DNMT1 mutations lead to mitochondrial hyper-function and increased oxidative stress, resulting in neurodegeneration [67]. Also, DNMT1 mutation was reported to cause epigenetic de-repression of the γ-globin gene in β-thalassemia elevating the fetal hemoglobin levels [36]. In addition to this, mutations in DNMT3A and DNMT3B have also been reported to cause significant alterations in mtDNA methylation thereby affecting mitochondrial functionality [24, 31, 48, 52, 78, 80].

Apart from mtDNA methylation, hydroxymethylation has also been detected in mitochondrial genome. MtDNA hydroxymethylation occurs through direct addition of 5-hydroxymethyl group to cytosine residues catalyzed by mtDNMT1. Also, mitochondrial DNMT3A and DNMT3B contribute to mtDNA hydroxymethylation. Current reports also suggested the existence of N6-Deoxyadenosine methylation in mammalian mtDNA [40]. METTL4, a mammalian methyltransferase mediated mtDNA N6-Deoxyadenosine (6mA) methylation promotes reduced mtDNA transcription along with a decreased mitochondrial biogenesis and mtDNA copy number [40].

Technologies involved in the detection of mtDNA methylation

MtDNA methylation is associated with various physiological aspects such as cardiovascular diseases, neurodegenerative diseases, cancer and many more. Various technical approaches are involved for the assessment of methylation of mtDNA. The most widely used technique for the study of mtDNA methylation is bisulphite sequencing. Prior to this, bisulphite conversion of mtDNA has been done that converts the unmethylated cytosine into uracil, whereas the methylated cytosines remain unchanged. The bisulphite converted DNA undergoes PCR amplification prior to bisulphite sequencing procedure. The methylation level is assessed using the percentage of methylated cytosine over the total number of methylated and non-methylated cytosine residues. MtDNA methylation analysis has been commonly done through bisulphite PCR pyrosequencing [46]. Global DNA m5C content can be assessed using ELISA assay by capture and detection antibodies [21, 37]. Such technique allows the detection of methylated mtDNA fluorimetrically by measuring the intensity of fluorescence. MtDNA methylation can also be evaluated using targeted next-generation bisulphite sequencing (TNGBS) [87, 98]. MassARRAY platform also can be used to detect the methylation of mtDNA which is more efficient as compared to methylation specific PCR and bisulphite sequencing. hMeDIP Assay can be performed using hMeDIP kit which provide three spikes or internal immunoprecipitation controls such as, an unmethylated DNA, a methylated DNA, and a hydroxymethylated DNA [6]. Post bisulphite conversion, the DNA fragments can be hybridized to the Infinium Human Methylation 450 BeadChip array [95]. In addition to this, there are targeted bisulphite deep sequencing and shotgun bisulphite deep sequencing methods to assess the methylation pattern of mtDNA [75]. Another option for evaluation mtDNAmethylation pattern is to use immunoprecipitation reaction using specific antibodies for 5mC followed by microarray hybridization technique [90]. Sequence specific restriction enzymes may be helpful in detecting the methylation sites residing in mtDNA as these enzymes are inhibited by 5mC, they can indicate the methylation pattern more effectively [90]. In combination with high-throughput sequencing, methylation specific restriction enzyme digestion has also been reported for the detection of specific methylation sites [38]. An alternative approach to hybridization and sequencing is to use mass spectrometry; however, it requires gene specific amplification. Also, nanopore sequencing is one of the most effective methods which allow direct sequencing of 5mC without the need for the bisulphite conversion of mtDNA [89, 90]. New approaches such as ELISA along with restriction digestion procedure results in better yield of mitochondrial methylation pattern [90]. Recent advancements include Bisulphite-PCR–single stranded DNA conformation polymorphism (SSCP) and methylation sensitive-high resolution melting (MSHRM) that uses a CFX96 Real-Time PCR detection system [70, 92]. Recently identified easiest methods for mtDNA methylation analysis is Illumina Infinium Bead Chip array and Methylation Epic Bead Chip [79]. Interestingly, it has been found that the closed circular topology of mtDNA inhibits bisulphite conversion resulting in false-positive detection of mtDNA methylation [75]. At times, incomplete bisulphite conversion produces false-positive measurement which becomes a major drawback [75]. The adequacy of mtDNA methylation can be affected by the purity of the bisulphite converted pyrosequencing templates, presence of incompletely converted DNA and the topology of the mtDNA. To avoid these discrepancies and false-positive results it is suggested to use selective primers and the evaluation of conversion efficiency must be calculated accurately for identification and analysis of mtDNAmethylation [75].

Regulation of mtDNA methylation in response to environmental pollutants

Humans are being continuously exposed to environmental toxins in daily life which is a growing concern affecting cellular and genomic integrity. Nuclear and mitochondrial DNA damage is the most widely studied impact of environmental or occupational exposures to xenobiotics [12, 23, 32, 65, 66]. Mitochondria play a crucial role in the cellular response to environmental pollutants or stressors, including both intrinsic and extrinsic pathways. Earlier, few reports have revealed how mitochondrial epigenome gets frequently altered primarily through DNA methylation in response to various environmental toxins [2, 53, 72, 81, 83]. Differential tissue specific mitochondrial DNA methylation has been previously reported in placenta, brain, liver and blood cells as a result of environmental exposures involving heavy metal exposures, air particulate matter, various chemical compounds [2, 53, 72, 81, 83]. In the following sections, a detailed account of association of altered mtDNA methylation and its downstream cellular effects have been discussed.

MtDNA methylation in response to air particulate matter exposure

Epigenetic mechanism tune gene expression in response to various environmental and occupational pollutants such as different air particulate matters [3, 10, 13, 19, 84]. Such exposuresare associated with several human diseases including neurodegeneration, diabetes, obesity, cardiovascular diseases and different types of cancers. Environmental exposures to air particulate matter (PM) or smoke has been previously reported to induce ROS generation that is shown to cause oxidative stress and promote mitochondrial damage. Evidences support alteration in methylation pattern of mitochondrial DNA affects mitochondrial biogenesis [15, 17, 47, 94].

Interestingly, some studies (Table 1) on PM exposure have previously reported a higher level of mtDNA methylationalong with increase in mtDNA copy number [15, 47, 94]. Earlier, a study was performed on steel workers exposed to metal-rich particulate matter demonstrated higher mtDNA methylation(in MT-TF and MT-RNR1 genes) detected through bisulphite pyrosequencing and higher mtDNA copy number [16]. Consistent with the earlier results, exposures to PM (2.5) induced higher oxidative stress and mtDNA methylation that affects mitochondrial biogenesis with increased risk for cardiovascular diseases [15]. However, mother-newborn pairs exposed to PM (2.5) during the gestational period revealed hypermethylation of placental mtDNA tested through bisulphite pyrosequencing which resulted in lower mtDNA content [46]. An in-vivo and in-vitro study performed by Breton et al., 2019 reported that exposure to PM (2.5) caused the hypermethylation of mitochondrial D loop regions that affect mitochondrial respiratory function and mtDNA in SH-SY5Y cells and in liver tissues from C57B1/6 males [10].

Table 1 Mitochondrial DNA methylation in response to airborne particulate matter exposure
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MtDNA methylation in response to chemical exposures

Exposure to different chemical compounds has impactful effects on mtDNA methylation and thus in turn can influence various disease progressions (Table 2). Earlier, an in vivo study by Byun et al., 2015 [13] experimentally demonstrated the exposures to PBDEs (Polybrominated diphenyl ethers) and mtDNA methylation in rats PBDE induced ROS generation and hypomethylation of mitochondrial Mt-CO2 (Cytochrome c oxidase gene) led to mitochondrial dysfunction in the frontal brain of rats [13]. Maternal smoking during pregnancy has been reported to affect placental trophoblast tissues, placental activity and foetal development by altering the methylation pattern of mtDNA identified by bisulphite pyrosequencing [3]. Doxorubicin, a well-known chemotherapeutic drug, was found to be associated with decreased SAM levels and consequently the hypomethylation of PGC1α and TFAM genes [29]. Next generation bisulphite sequencing (TNGBS) and ELISA of 5mc DNA content has helped the identification of methylation alteration of mtDNA by the psycho stimulant drug cocaine [25]. Cocaine has been reported to alter mtDNA methylation pattern, lowering the protein levels of DNMT3A and DNMT3B affecting mitochondrial biogenesis [25]. Also, tobacco smoking and air pollution during pregnancy has been reported to cause epigenetic modifications of mtDNA. Hypermethylation of mtDNA in two regions of displacement loop control region (D-loop and LDLR2) has been reported to lower the mtDNA content in placental tissues [94]. Exposures to PAH (Polycyclic aromatic hydrocarbons), also caused significant alterations in the expression of epigenetic modifiers and thus is responsible for the altered level of methylation of mtDNA. Methyl Flash Global DNA methylation (5-mC) ELISA is used for the determination of this epigenetic modification of mtDNA [7].

Table 2 Mitochondrial DNA methylationin response to environmental exposure of several chemical compounds
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However, the exact mechanisms responsible for changes in mitochondrial methylation due to different exposures need to be investigated and further studies are needed to substantiate the role of DNA methylation as potential mechanism for different adverse health effects and toxicities.

MtDNA methylation in response to heavy metal exposure

Environmental or occupational heavy metal exposures are associated with diverse mitochondrial dysfunctions. Extensive research has been performed in the field of heavy metal exposure and nuclear DNA epigenetics. However, information regarding how heavy metal contaminants affect mitochondrial epigenome through DNA methylation remains very limited (Table 3). Previous research work demonstrated the role of mtDNA hypomethylation through Mass ARRAY Platform in response to chromium in chrome-plate workers. The methylation levels of MT-TF and MT-RNR1 genes were negatively associated with blood Cr ion concentrations; however, no change in mitochondrial copy number was observed [58]. A recent study by a group reported a significant association between arsenic contamination and hypomethylation (of D-loop region and ND6) in exposed individuals, as detected through Methylation specific PCR. Hypomethylation of D loop and ND6 led to the transcriptional activation of mitochondrial genes along with higher mitochondrial copy number in arsenic exposed individuals [2, 81]. Consistent with the above results, significant hypomethylation of both TFAM and PGC1α in arsenic exposed individuals along with the higher level of gene expression and increased mtDNA copy number was also observed [83]. Transcriptional activation of these crucial genes are important mediating factors for mitochondrial biogenesis and are associated with tumour cell proliferation and development of arsenical skin cancer [81, 83]. Another study have reported Cd2+ and Cr6+ induced methylation in the mitochondrial DNA in an invertebrate, Exopalaemon carinicauda. Heavy metal stress in E. Carinicauda has resulted in been reduced gene expression through mtDNA methylation [63].

Table 3 Mitochondrial DNA methylation in response to environmental exposures of heavy metal pollutants
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Mitochondrial epigenetics and its relation with different health disorders

Nuclear epigenetics and its relation with various diseases have been studied thoroughly; however, the association between mitochondrial epigenetics and different diseases has not been given much importance. Here in, we have tried to shed light over this area to look into various means of association between altered mtDNA methylation and different human diseases (Table 4). Mitochondrial epigenetic alterations have been linked to various disease types including Type 2 Diabetes mellitus, neurological disorders (Alzheimer’s disease, Parkinson disease), Down Syndrome, NASH (Nonalcoholic Steatohepatitis), cardiovascular diseases (CVD), cancer etc. [5, 8, 45, 55, 77]. Increased oxidative stress has been shown to damage to mitochondrial genome and epigenome leading to mitochondrial dysfunction as observed in neurodegenerative disorders [8, 47, 97].

Table 4 Mitochondrial DNA methylation in response to various diseases
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Mitochondrial DNA methylation and its association in disease outcomes have evolved as a new area of research in the last few years. Higher mtDNA methylation was observed in ND6 and D loop region of mitochondria during the early stage of diabetes through Methylation specific-PCR. Altered mtDNA methylation was found to be responsible for the development of insulin resistance in individuals with diabetes [99, 100]. The abnormalities in methyl group donors such as S-adenosylmethionine (SAM) and mtDNA methylation status probably led tomitochondrial dysfunction as widely documented through mass spectrometry in patients with Down syndrome [44]. Higher mtDNA methylation at ND6 region due to high level of DNMT1 mRNA in the liver of NASH (Nonalcoholic steatohepatitis) patients has been reported by Pirola et al., 2013 [77] along with increased oxidative stress. Hypermethylation of ND6 gene in the mitochondrial genome caused its transcriptional inactivation that promoted mitochondrial dysfunction. Higher mitochondrial methylation in platelets has been reported to promote the dysfunction of mitochondria and in turn has been shown tobe responsible for platelet dysfunction and cardiovascular risks [5]. These mitochondrial epigenetic de-regulations are associated with the prognosis of the various diseases. A greater understanding of how DNA methylation in these regions of mitochondria affects mtDNA genes, evolution that needs to be investigated and pharmacologic manipulations is needed which could provide better insights to the patho-physiology of the diseases.

Mitochondrial DNA methylation and cancer

Nuclear epigenetics and its association with diseases especially cancer has been studied widely and given much importance till date; however, the field of mitochondrial epigenetics and its relation with cancer has not been studied and reported thoroughly. Herein, we have made an attempt to elaborate the relationship between mtDNA methylation and cancer progressions (Table 5) elucidated in recent studies [28, 33, 45, 91, 93]. Previously, studies have reported that colorectal cancer tissues with hypomethylated mitochondrial D-loop might be associated with higher expression of mtDNA along with higher mtDNA copy number [28, 33]. MtDNA methylation study was done using Methylation specific PCR and mitochondrial copy number was estimated using quantitative PCR (qPCR). A study by Feng et al., 2012 [28] showed increased expression of ND2 (a subunit of NADH, encoded by mtDNA) is associated with hypomethylation of mtDNA D-loop region, a crucial factor for mtDNA replication and transcription. Hypomethylation of D loop region is associated with the binding of TFAM on mtDNAand thus the expression of ND2 is upregulated leading to oxidative phosphorylation which is required for the enhanced growth of cancer tissues [28]. Consistent with the earlier results, similar finding was observed in colorectal cancer cases and Caco-2 cell line (colorectal cancer cell line) in a study by Gao et al., 2015 [33]. In this study, the demethylation of the D-loop region has facilitated the binding of the TFAM thus increasing the mtDNA expression i.e., the upregulation of ND2. Increased ND2 expression is responsible for higher oxidative phosphorylation thus enhancing the growth of the colorectal cancer cells [33]. Further validation was done by a DNA hypomethylating agent, 5-aza-2’-deoxycytidine (5-AZA). It was performed to examine the effect of DNA methylation on the mtDNA copy number and the biological behaviours of cells by Sequenom MassARRAY platform using various colorectal cell lines [93]. 5-AZA treatment induced hypomethylation of mitochondrial D loop region at specific sites on CpG islands were found to be associated with higher mtDNA copy number that in turn is probably a triggering factor for increased cell proliferation in colorectal cancer cell lines [93]. Similarly, in breast cancer, it has been revealed that DNA methylation in D loop region is maternally inherited and eight aberrant methylation sites are associated with cancer progression detected through whole genome DNA methylation array and bisulphite sequencing[39]. According to a study by Lee et al., 2015 [54], cancer stem-like cells were found to be hypermethylated at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) that accounted for low mtDNA copy number. Also, a study by Sun et al., 2018 [91], revealed a negative correlation between mtDNA methylation and mtDNA copy number during the progression of tumorigenesis in osteosarcoma and glioblastoma. Mitochondria, being a prognostic marker in carcinogenesis, further studies on the relationship between mtDNAepigenetic regulation and cancer should be clearly elucidated to better understand the clinical significance, function and mode of actions of mitochondrial epigenomein response to disease progression.

Table 5 Mitochondrial DNA methylation and cancer:
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Mitoepigenetics, especially mtDNA methylation directly regulate gene expression, which was previously studied in different cancers [28, 33, 39, 54, 91, 93]. MtDNA possess only 13 protein coding genes and all of them encode the subunits of respiratory complexes associated with ETC. As a result, any small change in these gene expressions affects the cellular respiration. For malignant transformation and cancer progression, alterations in glucose metabolism pathway are one of the major phenomena that have been reported earlier [14, 30]. Up-regulation in the glycolytic pathway under hypoxic conditions is an important measure for tumour cell survival. Interestingly, even in the presence of sufficient oxygen, tumour cells preferentially use glycolysis rather than oxidative phosphorylation for energy production known as Warburg effect, leading to a shift in metabolism from aerobic respiration toward glycolysis [11, 30, 35]. This occurs via the induction of HIF-1 (hypoxia-inducible factor-1) which was shown to suppress mitochondrial respiratory activity, as a consequence, to suppress Krebs cycle and mitochondrial respiration [50, 76]. Also, mitoepigenetics may play essential role in mitochondria mediated biological processes. Alteration of mitoepigenetics alters the mtDNA encoded proteins which play essential roles in ETC/OXPHOS including glucose, lipid and amino acid metabolism [50, 76]. MtDNA of cancer stem-like cells has shown hypermethylation and low mtDNA copy number which provokes them to use glycolysis for cell proliferation [55]. It is reported that methylation of mtDNA may differentially suppress mtDNA encoded genes that are necessary for oxidative phosphorylation [85]. Oxidative stress such as ROS can trigger cellular proliferation and carcinogenesis by inducing mutation in mtDNA which further reduce the efficiency of ETC and OXPHOS [18, 56, 60]. Taken together, changes in mitoepigenetics affect downstream gene expression, which in turn may cause altered cellular respiration associated with malignant transformation under certain circumstances.

Therapeutic interventions targeting mtDNA methylation

Epigenetic regulation has a reversible mode of action and can be regulated through direct epigenetic modifications. Several on-going drug therapies under research might provide a novel way to regulate mitochondrial epigenetic regulation particularly focussing on mitochondrial DNA methylation mark. Though several therapeutic drugs are validated that Potential ‘epitherapeutic’ drugs that are currently available include 5-Azacytosine, Decitabine, Guadecitabine, Belinostat, Panobinostat, Vorinostat, and Romidepsin have shown promise in clinical and preclinical trials [49]. Azacitidine and Decitabine have been approved by FDA (Food and Drug Administration) and European Medicines Agency (EMA), are known to function as potent DNA demethylating agents [1]. Certain dietary components play a significant role and have a promising field of research in modulating epigenetic phenomenon in lessening the disease burden. Specific dietary factors and supplements can activate or inhibit DNA methyltransferase activity directly or indirectly to influence mitochondrial DNA methylation [9, 41].Previous studies have suggested the role of folate and Vitamin B12 supplementation that can regulate mitochondrial DNA methylation [64]. Few DNA methyltransferase inhibitors (DNMT inhibitors) include dietary polyphenols, (-)-epigallocatechin-3-gallate (EGCG, from green tea), genistein (from soybean) and isothiocyanates (from plant foods), may be used for cancer prevention and therapy [57]. Implementation of these dietary factors in clinical trials against mitochondrial epigenetic de-regulations might prove to be helpful for therapeutic requirements. An important study done by Lee et al., 2015 [54] have demonstrated that cancerous cells with hypermethylated human mitochondrial POLGA region at exon 2 has been demethylated by applying DNA demethylating agent, 5-azacytidine. Various therapeutic drugs that are in medical practice have been reported to trigger mitochondrial toxicity leading to a wide range of clinical symptoms; hence development of non-toxic mitochondrial epitherapeutic drugs must be a primary concern. Further in-depth high-throughput studies are warranted in this field along with several preclinical and clinical trials and accurate validation is required to obtain maximum health benefits.

Discussion

DNA methylation is a vital epigenetic mark affecting our physiological and biochemical process. Methylation-demethylation phenomenon influence gene expression of crucial signalling pathways required for normal functioning of cells. This review primarily aims to bring a holistic picture of the crosstalk between environmental factors, lifestyle disorders and adverse health outcomes including cancer with altered mtDNA methylation. MtDNA methylation is largely influenced by various environmental toxicants such as airborne particulate matter, exposures to heavy metals or chemical compounds or diseases [87]. These factors perturb the normal cellular homeostasis by genetic and epigenetic de-regulation of mitochondrial genome affecting the transcriptional-translational machinery and protein synthesis [4]. Mitochondrial dysfunction indirectly elevates ROS generation leading to an increased oxidative stress and in-turn damage to the mitochondrial genome [17]. However, in-depth mechanistic insight about how these exposures alter the methylation pattern of mitochondrial DNA needs to be explored more in future studies. A global (country-wise) representation of methylation changes in mitochondrial genome in human studies has been shown (Fig. 1).

Fig. 1
figure 1

A global (Country-wise) representation of methylation changes in mitochondrial DNA in human studies

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Environmental or occupational exposure to different chemical compounds or drugs, heavy metal pollutants possesses potential risk factors for alterations in mitochondrial epigenome through differential mtDNA methylation pattern that affect the expression of significant mitochondrial genes [12, 53, 66]. Extensive studies are needed to comprehend the exact mechanisms responsible for methylation of mitochondrial genes to understand the role of ‘methylation mark’ as a potential risk factor. Previous studies have reported the association between mtDNA methylation and altered mtDNA copy number in response to various diseases, environmental exposures and cancer. Both hyper and hypo methylation of specific regions in mtDNA was found to be associated with higher mtDNA copy number [47, 70]. Those findings clearly indicate towards a complex mechanism involving not only the mitochondrial epigenetic modifications but also involvement of other confounding factors like nucleus-mitochondrial retrograde and anterograde signalling, accumulation of ROS, rate of mitochondrial biogenesis, fission-fusion, mitophagy status and individual genetic make-up, environmental exposure, cancer microenvironment (in case of malignant transformations) etc. in mtDNA copy number regulation. However, the exact intracellular molecular signalling pathway and identification of regulatory factors still remain obscure as majority of the studies regarding this area were associative in nature. Evident from previous studies, that differential mtDNA methylation pattern can be used as a prognostic marker for cancer detection [28, 33, 93]. MtDNA methylation also play crucial roles in several other diseases like cardiovascular diseases, neurodegenerative diseases etc. which revealed that how disease progression is often associated with the altered mtDNA methylation pattern leading to an alteration in mitochondrial structural and functionality [5, 44, 77]. As epigenetic changes are known to be reversible, accurate implication of epitherapeutic drugs might reverse the alterations associated with neurodegenerative diseases, cancers etc.

Conclusions

MtDNA methylation plays an important role in disease development and progression. Further in-depth studies are needed to comprehend the alterations of mtDNA methylation pattern in response to various environmental stressors influencing various disease outcomes. Detailed research in the evolving field of mtDNA methylation may pave its way towards the development of potential epitherapeutic approaches in the immediate future.