Genetics and Epigenetics of Manganese Toxicity

Purpose of Review At elevated levels, the essential element manganese (Mn) is neurotoxic and increasing evidence indicates that environmental Mn exposure early in life negatively affects neurodevelopment. In this review, we describe how underlying genetics may confer susceptibility to elevated Mn concentrations and how the epigenetic effects of Mn may explain the association between Mn exposure early in life and its toxic effects later in life. Recent Findings Common polymorphisms in the Mn transporter genes SLC30A10 and SLC39A8 seem to have a large impact on intracellular Mn levels and, in turn, neurotoxicity. Genetic variation in iron regulatory genes may to lesser extent also influence Mn levels and toxicity. Recent studies on Mn and epigenetic mechanisms indicate that Mn-related changes in DNA methylation occur early in life. One human and two animal studies found persistent changes from in utero exposure to Mn but whether these changes have functional effects remains unknown. Summary Genetics seems to play a major role in susceptibility to Mn toxicity and should therefore be considered in risk assessment. Mn appears to interfere with epigenetic processes, potentially leading to persistent changes in developmental programming, which warrants further study.


Introduction
Manganese (Mn) is an essential element for living organisms, including humans. It is a required cofactor for many enzymes that have critical functions in diverse processes such as forming cartilage and bone, excreting waste via the urea cycle, maintaining mitochondria, antioxidant defenses, producing glucose, brain development, and wound healing [1]. Humans mainly get Mn from dietary intake and Mn deficiency is very rare. However, excess Mn causes severe deleterious health effects in humans. These effects are observed especially in the central nervous system, since Mn accumulates in the brain [2,3]. Mn exposure was first associated with adverse health outcomes in adults, including Mn-induced Parkinsonism and other neurodegenerative conditions, due to occupational exposures from mining, battery production, welding, and ferromanganese alloy plants [2,4,5]. Environmental Mn exposure has become a public health concern in recent years due to emerging evidence that children may be exposed to harmful levels of Mn from multiple sources, including drinking water, soil and dust, and possibly their diet [1]. Epidemiological studies have shown that elevated Mn exposure is associated with reductions in full scale IQ, along with adverse behavior and fine motor function in children and adolescents [6][7][8][9]; however, others have found no adverse association [10,11]. Mechanisms linking Mn exposure to neurodevelopmental outcomes include oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, apoptosis, neuroinflammation, and interference with neurotransmitter metabolism [12]. Recent studies have reported Mn-related alterations in the epigenetic regulation of gene expression, indicating that Mn can target the programming of cells and tissues. Epigenetic alterations may be long-term and of importance for neurodevelopment and vulnerability to brain disorders [13,14].
Examining susceptibility factors can provide insights on the mechanisms of toxicity. One susceptibility factor for Mn toxicity is sex. Several studies have shown different associations between Mn exposure and neurological effects between girls and boys, suggesting that there could be sex-related differences in Mn sensitivity [15][16][17]. Another susceptibility factor is low iron (Fe) stores. Mn and Fe compete for the same protein, divalent metal transporter 1 (DMT1) [18] and blood Mn and Fe levels are therefore often inversely correlated [19]. Recent data suggest that underlying genetics is also a susceptibility factor.
In this review, we provide an overview of the genetic factors of Mn metabolism and toxicity. Further, we review epigenetic effects of early life and adult exposure to Mn and hypothesize those effects are persistent when occurring early in life.

Manganese Transporters
Rare variants of genes involved in Mn homeostasis can result in increased intracellular Mn levels. During the last 10 years, identification of inherited Mn transportopathies has highlighted a network of solute carrier transporters that are required for Mn homeostasis in humans. Solute carrier family 30 member 10 (SLC30A10) and solute carrier family 39 member 14 (SLC39A14) act in conjunction to excrete Mn into the bile and intestine (Fig. 1). SLC30A10 is a Mn efflux transporter, which transports Mn from the cytosol to the cell exterior and protects against Mn toxicity [20 ••]. SLC39A14 is a divalent metal efflux transporter, which transports zinc, Mn, Fe, and cadmium [21••]. Rare homozygous loss-offunction mutations in either gene result in Mn accumulation, even in the absence of external Mn exposure, in the basal ganglia, particularly the globus pallidus, causing Mn neurotoxicity and progressive dystonia-Parkinsonism [ High-penetrance mutations associated with disease confer a high absolute risk irrespective of environmental factors and are generally rare, occurring at frequencies lower than 1% in the population. More common genetic variants in SLC30A10 (non-coding rs2275707, rs1776029, rs12064812; Table 1) and SLC39A8 (rs13107325, A391T) influence Mn concentrations in healthy individuals from various populations and age groups (genome-wide association study in [25•, 26, 27]), and despite being common alleles, they have a substantial effect on Mn concentrations.  By contrast, alleles of the major arsenic methylating gene arsenite methyltransferase (AS3MT) only explain 7% of the variation in the urinary fraction of dimethylated arsenic, a metabolite involved in arsenic excretion [29]. Mendelian randomization analysis also showed that the same polymorphisms in SLC30A10 and SLC39A8 were associated with neurodevelopmental outcomes, particularly test scores for ADHD-related behavioral problems [28•] and contributed to differences in Mn sensitivity, particularly in girls [30]. The rs2275707 and rs12064812 variants of SLC30A10 are classified as functional as they are expression quantitative trait loci (eQTL, GTEx database) that show lower and higher expression, respectively, in parts of the basal ganglia where Mn accumulates. Furthermore, rs2275707 was associated with significant differences in expression in blood where the variant allele correlated with lower gene expression [26]. Further support for the effect of SLC39A8 rs13107325 (which encodes an Ala-391-Thr in the SLC39A8 protein) on Mn regulation comes from a recent animal and human study [44]. CRISPR/Cas9-mediated knock-in was used to generate a mouse model carrying the SLC39A8 amino acid substitution (Ala-391-Thr) variant and mice carrying this variant had lower blood Mn levels than mice carrying the Ala variant. These mice lines exhibited tissue-specific abnormalities in Mn homeostasis, with decreases in liver and kidney Mn levels and increased biliary Mn levels, providing in vivo evidence of altered transporter function. SLC39A8 391-Thr was also associated with reduced triantennary plasma N-glycan species in a populationbased human cohort. The SLC39A8 rs13107325 variant is one of the most pleiotropic polymorphisms known so far and has been repeatedly associated with neurological and metabolic disorders [45,46]. Interestingly, SLC39A8 polymorphisms, including rs13107325, and polymorphisms in linkage disequilibrium with rs13107325, are associated with different magnetic resonance imaging phenotypes of the brain [47].
So far, no studies have reported associations between common polymorphisms in the Mn transporter gene SLC39A14 and Mn levels or Mn toxicity.

Iron Transporters
Earlier studies investigated the role of genes involved in Fe metabolism in Mn susceptibility because of the well-established inverse correlation between Fe stores and Mn absorption [19]. Indeed, Fe and Mn likely share some transporters and regulatory proteins. Type 1 hereditary hemochromatosis is caused by being a homozygous carrier of missense mutations (His-63-Asp or Cys-282-Tyr) in the homeostatic iron regulator (HFE) gene; these mutations lead to increased Fe uptake from the gastrointestinal tract. Mice carrying the His-67-Asp Hfe mutation, which is homologous to the His-63-Asp mutation in humans, had lower Mn levels in the blood, liver, and brain after Mn inhalation, and lower toxicity of inhaled Mn [33]. Similar results were found when analyzing Mn levels in Hfe −/− and Hfe +/+ mice, revealing that Hfe −/− mice had lower Mn and higher Fe concentrations [35]. A pilot study of 141 human individuals living near a ferro-Mn refinery in the USA only detected a significant association between hair Mn levels and estimated ambient air Mn levels when polymorphisms in both HFE and the Fe storage gene transferrin (TF) were included in linear models, but not with either gene alone [48] (Table 1). Furthermore, among 332 pregnant Mexican women exposed to Mn from the environment, heterozygous carriers of either of the HFE polymorphisms (Cys-282-Tyr or His-63-Asp) had 12% lower blood Mn levels than women with no HFE variants [35].

Other Genes
In the juvenile form of Parkinsonism, mutations are found in ATPase cation transporting 13A2I (ATP13A2), which encodes a P5-type ATPase pump recently shown to transport polyamines [49]. Polymorphisms in ATP13A2 significantly modified the effects of Mn exposure on motor coordination in elderly people in Italy [38] ( Table 1).
The possible consequences of Mn exposure early in life have been explored in the context of birth outcomes and underlying genetics. In a Chinese nested case-control study, higher maternal (collected in gestational weeks 4-22) serum Mn concentrations were associated with preterm birth (before week 37), and this association was modified by the genotype of genes encoding antioxidant proteins including superoxide dismutases (SOD2 and SOD3) and catalase (CAT ) [39].
Further, Rahbar et al. [40] evaluated 266 age-and sexmatched pairs of Jamaican children with autism spectrum disorder and normally developing controls (2-8 years) to determine whether copy number variation of the xenobiotic metabolizing gene glutathione S-transferase theta 1 (GSTT1) modifies the association between blood Mn concentrations and autism spectrum disorder. They found a significant interaction between GSTT1 copy number and blood Mn concentrations: compared to controls, autism spectrum disorder cases with GSTT1 homozygous for deletion of the gene on both chromosomes had 4.35 times higher odds of blood Mn concentrations above 12 μg/l vs. below 8.3 μg/l. However, the confidence interval was very wide.
Trdin et al. [43] did not find any association between apolipoprotein E polymorphism ε4 and Mn concentrations in mothers and newborns.

Epigenetic Effects of Manganese
Epigenetic changes are heritable changes in gene expression and regulation that are not coded by the DNA sequence, but by various modifications of the DNA. For example, DNA methylation, the specific methylation of cytosine residues directly upstream of a guanine, is essential for embryogenesis and for the maintenance of cell lineage-specific gene expression throughout life [50]. Transcript levels may also be regulated by non-coding RNAs, such as microRNAs, which regulate gene expression post-transcriptionally by hybridizing to messenger RNAs, leading to translational repression or degradation of the target RNA [51]. Another regulatory mechanism of gene expression entails histone modifications, which affect chromatin structure; an open chromatin structure facilitates active transcription, while a closed structure limits transcription. However, the histone-based "epigenetic code" has recently been challenged [52].
The pre-and postnatal environments are important determinants of disease susceptibility later in life [53] and this influence is thought to be mediated mainly through alterations in DNA methylation, which subsequently alter the epigenetic programming of the child and can lead to longterm negative health outcomes. Epigenetic modification by external factors was clearly demonstrated for smoking [54]. Increasing evidence also shows that early-life metal exposure may modulate the epigenetic landscape (e.g., as shown for methylmercury in [55] and for arsenic in [56]).
The symptoms of hypermanganesemia syndromes are partially reversible, i.e., the Mn load and disease progression can be ameliorated in carriers of loss-of-function mutations in SLC30A10 and SLC39A14 with chelation therapy together with Fe supplementation [57,58]. However, we do not know the long-term effects of external Mn exposure and whether Mn changes cellular programming, such as via epigenetic modifications, also remains unclear. Additional research will also be needed to test the influence of Mn on DNA methylation and whether epigenetic factors change the individual's predisposition to Mn toxicity. However, increasing evidence suggests that Mn targets the epigenetic machinery, by a yet-unknown mechanism. Below, we summarize several studies in humans and animals that explore the effect of Mn on epigenetic factors.
Only one study has, to our knowledge, evaluated Mn exposure in relation to histone modifications. In a crosssectional study of steel workers, estimated air metal concentrations were correlated with histone modification in blood leukocytes [59] (Table 2). However, no association was found between Mn concentration in air (mean 11.26 μg/m 3 SD ± 30.41) and histone modifications.
Nwanaji-Enwerem et al. [60] examined the relationship of urine Mn levels in elderly men (Normative Aging Study) over a 24-h period with three DNA methylation-based measures of biological aging: DNAmAge, GrimAge, and PhenoAge. Urine Mn (mean 1.4 ng/ml ± 0.4 SD) was linked to PhenoAge. A 1 ng/ml increase in urine Mn was associated with a 9.93-year increase in DNA-methylation based biological age. Because Mn is normally excreted via bile, not urine, this finding may be explained by a partial shift to excretion of Mn in urine related to kidney disease, which in turn accelerates biological aging. The study did adjust for kidney function but there may be residual confounding and further studies are warranted to clarify this finding.
In the same cohort of elderly men, estimated dietary Mn intake (categorized in quartiles from ≤ 2.68 to ≥ 5.48 mg/day) from food/beverages and supplements were correlated with circulating biomarkers of inflammation and DNA methylation of genes involved in the production of biomarkers of inflammation [61]. No strong evidence was found for increasing Mn intake and altered DNA methylation of the genes, but trends (non-significant after adjustment for multiple comparisons) were found for methylation of non-promoter CpG sites in genes encoding NF-κβ member activators.
Bozack et al. [62••] analyzed whether Mn in maternal erythrocytes (median 15.80 ng/g IQR 13.10, 19.70) during the first trimester was associated with differentially methylated positions (DMPs) and regions (DMRs) in cord blood and tested if associations persisted in blood collected in midchildhood (6-10 years old) in a cohort of 361 children. Mn was associated with increased methylation of cg02042823 in the gene RNA binding fox-1 homolog 1 (RBFOX1, also called A2BP1) in cord blood, and this association was still significant, but attenuated in blood collected at mid-childhood. Two and nine Mn-associated DMPs were identified in male and female infants, respectively, with two and six persisting in mid-childhood. The DMPs identified in males and females did not overlap. This finding supports that prenatal exposure to Mn may result in changes in DNA methylation that persist into childhood and that the changes may be sex-specific. In cord blood, Mn exposure was associated with a DMR annotated with tenascin XB (TNXB) in the human leukocyte antigen region, but this did not persist into childhood. In maternal blood of 97 non-smoking pregnant women, maternal Mn (geometric mean 12.67 µg/l) concentrations were non-significantly associated with hypermethylation at four DNA methylation sites, one of which was near the gene ATrich interaction domain 2 (ARID2) [63]. Genes encompassing Mn-associated methylated sites were enriched for cellular nitrogen metabolism, cell cycle process, nucleic acid metabolism, and negative regulation of response to DNA damage stimulus.
In a birth cohort, Mn concentrations measured in infant toenails were correlated with genome-wide DNA methylation in 61 placental samples [64]. The Mn levels ranged from 0.131 to 5.666 µg/g toenail where the second, or    3 Kresovich et al. [61] used average DNA methylation of CpGs in each gene 4 Kresovich et al. [61] estimated total dietary intakes of Mn and other macro-and micronutrients using a self-administered, semi-quantitative food frequency questionnaire; responses were processed through a nutrient database to estimate usual daily nutrient intakes 5 Ery, erythrocyte 6 Appleton et al. [65 • ] computed mean M-values across the interrogated CpG regions. The M-value is calculated as the log2 ratio of the intensities of methylated probe vs. unmethylated probe referent, tertile ranged from 0.394 to 0771 µg/g. Five significantly differentially methylated loci (annotated genes LINC00908 (LOC284276), FTO, EMX2OS, ATAD2B, and EN1) reside in neurodevelopmental, fetal growth, and cancer-related genes. cg22284422, located within LOC284276, was associated with birth weight; for every 10% increase in methylation, lower birth weights were observed. The observations suggest a link between prenatal micronutrient levels, placental epigenetic status, and birth weight. A cohort of healthy-term singleton pregnancies [65•] studied prenatal Mn exposure and DNA methylation in placentas, focusing on methylation of nuclear receptor subfamily 3 group C member 1 (NR3C1), encoding the glucocorticoid receptor essential for the body's stress response. Mn concentrations (median 0.56 μg/g) were measured in infant toenails, which reflect long-term external exposure at a fairly reproducible level [66]. Compared to the lowest exposure tertile, the highest tertile of Mn in toenails was associated with a small (0.80%) but significant increase in placental NR3C1 methylation. Whether this small effect has functional consequences is unknown but there is some evidence that higher NR3C1 methylation is the epigenetic nexus between early life stress and later life psychiatric disorders [67].
Animal studies have also been performed. Pregnant mice were treated with 800 ppm MnCl 2 in their diet from gestational day 10 through day 21 [68]. Following 800-ppm Mn exposure, a CpG promoter microarray study found hypermethylation of the promoter regions of 24 genes in the hippocampal dentate gyrus of male offspring. After 800-ppm Mn exposure through the adult stage, hypermethylation and transcript downregulation was confirmed in Pvalb, Mid1, Atp1a3, and Nr2f1. These results suggest that Mn exposure alters epigenetic gene regulation and programming of cellular populations related to neurogenesis. Still, how the Mn dose translates to human exposure is unclear.
In a later animal study, pregnant mice were given drinking water with high concentration of Mn (MnCl 2 of 10 mg/l in the water, to compare with the US EPA health advisory value for Mn in drinking water of 0.3 mg/l) from gestational days 1-10 and young male offspring were tested for behavioral deficits [69]. In utero exposure to Mn resulted in multiple behavioral abnormalities that persisted into adulthood. Brain samples from three Mn-treated and three control animals were evaluated for changes in the frontal cortex of CpG island methylation in promoter regions and associated changes in gene expression. In Mn-exposed animals compared to water-treated controls, the chromodomain helicase DNA binding protein 7 (Chd7) gene, essential for neural crest cell migration and patterning, was found to be hypomethylated and showed higher gene expression. However, this study should be interpreted with caution, as the Mn level was very high, and the study group was small.

Conclusions
Underlying genetics clearly plays a critical role in Mn metabolism and toxicity. The genotypes of the Mn transporter genes SLC39A8 and SLC30A10 have repeatedly been shown to influence Mn homeostasis and susceptibility to Mn neurotoxicity, and the association between the common variants of these genes and intracellular Mn concentrations is one of the strongest gene-environment interactions reported so far. Genes involved in Fe uptake and metabolism may modify Mn levels as well, although to a lesser extent.
The epigenetic effect of Mn is a new and growing research field. Thus far, one human study reported Mnrelated changes in DNA methylation from birth to childhood. This finding suggests that prenatal exposure to Mn may result in changes in DNA methylation that persist into childhood. Still, no gene has consistently, and across studies, been found to be altered in relation to Mn exposure. DNA methylation is the predominant epigenetic factor evaluated so far and further studies on histone modification and noncoding RNA in relation to Mn are warranted.
Funding Open access funding provided by Lund University.

Conflict of Interest
The authors declare no competing interests.

Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.
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