The Effects of Manganese Overexposure On Brain Health.

Abstract

Manganese (Mn) is the twelfth most abundant element on the earth and an essential metal to human health. Mn is present at low concentrations in a variety of dietary sources, which provides adequate Mn content to sustain support various physiological processes in the human body. However, with the rise of Mn utility in a variety of industries, there is an increased risk of overexposure to this transition metal, which can have neurotoxic consequences. This risk includes occupational exposure of Mn to workers as well as overall increased Mn pollution affecting the general public. Here, we review exposure due to air pollution and inhalation in industrial settings; we also delve into the toxic effects of manganese on the brain such as oxidative stress, inflammatory response and transporter dysregulation. Additionally, we summarize current understandings underlying the mechanisms of Mn toxicity.

1. Introduction

Manganese (Mn) is a trace essential metal that is necessary for maintaining human health and function. Its presence can be found in a wide variety of foods such as legumes, rice, nuts, and whole grains as well as in sea foods, seeds, chocolates, teas, etc (Peres et al., 2016). It is also present in products such as fireworks, fertilizers, paints and cosmetics. The most common form of general manganese intake is via food consumption. Consuming small amounts of Mn daily is important to maintaining a variety of physiological functions in the body such as defense against oxidative stress via Mn superoxide dismutase (MnSOD) in mitochondria, proper development, digestion, immune response and others (Aschner and Aschner, 2005). In the event that not enough Mn is consumed, one may become Mn deficient and present with symptoms such as skeletal deficiency, decreased levels of fertility, impaired glucose tolerance, and impaired growth (Aschner et al., 2005). For a more extensive review on the essentiality of Mn, please see a previously published paper (Aschner and Aschner, 2005).

Conversely, exposure to high levels of Mn most commonly occurs through inhalation from industrial sources, and can result in elevated levels of the metal throughout the tissues of the body (Williams et al., 2012). Indeed, excessive Mn accumulation in the central nervous system (CNS) can cause a phenomenon known as manganism, which resembles idiopathic Parkinson’s Disease (IPD) in its clinical features and results in adverse neurological effects both in laboratory animals and humans (Dobson et al., 2004). Overexposure and inhalation of Mn can also bring about symptoms such as pneumonia, decreased libido, and sperm damage (Hobbesland et al., 1997; Williams et al., 2012; Yamada et al., 1986). This exposure can be serious and detrimental and while infants and children are thought to be particularly at risk, Mn exposure is not limited to any specific age bracket or gender. The accessibility of Mn in the environment via gasoline, fertilizers, water, food, and other substances renders it a viable source of contamination through inhalation and consumption for the general population (Oulhote et al., 2014). This review focuses on the neurological implications of overexposure to Mn, particularly via inhalation, and summarizes routes of exposure, toxic effects, and mechanisms of toxicity.

1.1. Methodology

We were guided in our literature search to build on previous reviews that touched on Mn in relation to specific neurological diseases (Martins et al., 2019), essentiality and toxicity (Erikson and Aschner, 2019), mechanism of toxicity (Chen et al., 2018; Chen et al., 2016), homeostasis (Chen et al., 2015b), or the like. Here, we aimed to provide a broad summary of Mn routes of exposure while giving specific attention to air Mn, give details on the neurological implications of Mn overexposure and the mechanisms of damage, as well as signaling pathways affected. The PubMed database was primarily used to conduct the literature search supplemented by searches on Google or Google Scholar search engines as necessary. Keywords included: air manganese, manganese inhalation brain, manganese neurotoxicity, manganese and brain, occupational manganese exposure. The last major update to the Toxicological Profile on Manganese by the Agency for Toxic Substances and Disease Registry (ATSDR) was in 2012 where much is compiled and summarized on the topic of Mn. Therefore, where we could, we preferred to reference literature from this past decade (2009-2019) to address and summarize newer findings.

2. Routes of Exposure

2.1. Non-Occupational Exposure

According to the National Academy of Sciences, an adequate quantity of daily intake of Mn per adult should be approximately 2-6 mg/day (Freeland-Graves et al., 2016). An excess of dietary Mn seldom causes toxicity in humans since homeostatic mechanisms are present to quickly regulate mild concentration perturbations (Horning et al., 2015). In addition to diet, environmental contamination in air, soil, and water are also routes of exposure to Mn (Bowler et al., 2011; Lucchini et al., 2014; Viana et al., 2014)

Mn can be introduced into the environment as byproducts of industrial processes or goods (Bowler et al., 2011; Lucchini et al., 2014; Viana et al., 2014). Recognized since the 19^th century, Mn contaminated air and drinking water serve as the main routes of toxic exposure (Khan et al., 2012; Normandin et al., 2004; Wasserman et al., 2006).

Environmental contamination caused by the development of the Mn industry raises the concern for potential Mn overexposure for the general public. Indeed, the highest incidence of Mn toxicity cases outside of direct occupational exposure often includes people living close to mining, manufacturing and welding industries; elevated atmospheric Mn concentrations were reported in the vicinity of factories involving Mn production (Bowler et al., 2016; Cortez-Lugo et al., 2015; Crossgrove and Zheng, 2004; Lucchini et al., 2014; Menezes-Filho et al., 2018; Solis-Vivanco et al., 2009). The United States (US) Environmental Protection Agency (EPA) data from 1965 to 1982 demonstrated that rural air Mn was approximately 6.25 times lower than urban air Mn (Colledge et al., 2015). This data also showed that air Mn in areas with alloy smelters or other operations using Mn-containing products is significantly higher than what has been reported in both rural and urban environments (Colledge et al., 2015). Furthermore, the average annual air Mn concentration in the vicinity of a Mn alloy production plant in Beauharnois, Canada decreased by almost 50% after it shut down, showcasing how much pollution it was originally contributing to the local environment (Boudissa et al., 2006).

As previously noted, the US EPA has noted that environmental levels of air Mn differ from rural to urban locations. Rural locations have had readings of 5-60 ng/m³ while urban areas have higher Mn level readings around 33-110 ng/m³ (EPA, 2003). The EPA also summarized data from Canadian studies which showed that homes in Canadian urban locations had Mn levels of 7-12 ng/m³ (EPA, 2003). Cumulatively though, the exposure range remains in the ng/m³ region, many orders of magnitude below than what is seen in occupational settings (Howe et al., 1998; WHO, 2011). The US ATSDR used to limit non-occupational air Mn levels to 300 ng/m³ (ATSDR, 2019). However, individual locales can set lower limits. For example, the state of Minnesota sets its health risk value for chronic Mn exposure to 200 ng/m³ (Palmer, 2005). The state of Michigan sets its health benchmark according to the EPA Reference Concentration of 50 ng/m³(DEQ, 2012). Across the Atlantic Ocean, the Air Quality Guidelines for Europe sets a guideline of 150 ng/m³ for air Mn (WHO, 2000).

Another source of air Mn exposure is through methylcyclopentadienyl manganese tricarbonyl (MMT), which has been used to improve the quality of gasoline and serve as an antiknock agent (Beaupre et al., 2004). Combustion of MMT in the gasoline powered automobile engine emits Mn byproducts directly into the atmosphere. There are three main Mn byproducts of MMT combustion: a manganese phosphate known as hureaulite (Mn₅(PO₄)[PO₃(OH)]₂•4H₂O), manganese tetraoxide (Mn₃O₄) and manganese sulfate (MnSO₄•H₂O). Humans can inhale 90% of these byproduct particles, which have an average diameter of 0.5 to 1.0μm (Dorman et al., 2006; Lynam et al., 1999; Ressler et al., 1999; Zayed et al., 2003).

The United States Clean Air Act prohibits lead and manganese based additives in gasoline. However, the Act also allows for waivers. A waiver (Federal Register citation: 60 FR 36414) was granted in 1995 to Ethyl Corporation for a MMT product called HiTEC 3000 despite public health concerns for increased Mn particulate emissions (Sopata, 1995). Since then, Ethyl Corporation has continued to sell MMT. It now operates as Afton Chemical Corporation in the United States and markets its MMT products, such as HiTEC 3062 and HiTEC 3600 as “the only metallic gasoline additive[s] fully tested, and approved by the US EPA under the US Clean Air Act” (Afton, 2016a, b).

Afton Chemical Corporation has published a summary of locations where its products are sold, along with accepted regulations for those regions, giving insight into the current usage levels for MMT around the world (Afton, 2016c). Briefly, they state that their HiTEC 3000 Series of mmt® fuel additive is allowed up to the treat level of 18 mg Mn/L in Argentinian, Australian, and Canadian fuels. China allows for the product, as well, but limits the amount to 8 mg Mn/L. The European Union used to permit the fuel additive without restriction until 2011; it was set to 2 mg Mn/L in 2014. Finally, the US permits the MMT fuel additive up to 8.3 mg Mn/L. From this, we gather that different countries around the world have permitted MMT to various degrees. We can also conclude that Afton Chemical Corporation has a global reach in terms of customers willing to use MMT in their fuel.

Studies in Montreal, Canada, where MTT had been used for 10 years, found average total air Mn in Montreal to be 27 ng Mn/m³ for an area with low concentration of cars and 50 ng Mn/m³ for an area with a high concentration of cars (Crump, 2000; Loranger and Zayed, 1997). These numbers are comparable to the typical urban air Mn levels (33 ng Mn/m³) (Pellizzari et al., 2001). Traffic has contributed to the elevated air Mn level in urban areas. Data from studies in Paris show that Mn concentration in airborne particles is higher in areas with intense traffic (Poulakis et al., 2015).

2.2. Occupational Exposure

Due to its unique properties, Mn is used in a variety of industries, including iron (Fe) and stainless steel production, formation of aluminum alloys, as well as purification of oxygen and chlorine. For example, ferromanganese and silicomanganese are widely used for rust and corrosion prevention on steel. Mn is likewise a component of batteries, fungicides (i.e. Mn ethylene-bis-dithiocarbamate, MANEB), and as previously mentioned, antiknock additive (i.e. MMT). Mn dioxide, in particular, is used as the cathode material in batteries. Mn compounds are also used in pigments and for the coloring of ceramics and glass (Aschner et al., 2006; Blanc, 2018). Due to these varied usages of Mn compounds, those working in industrial settings are particularly at risk of increased exposure to Mn.

The concentrations of Mn in occupational settings can vary across regulatory agencies. For the American Conference of Governmental Industrial Hygienists (ACGIH), the occupational guideline for respirable Mn is approximately 20 μg/m³ and 100 μg/m³ for inhalable Mn. The recommended exposure limit from the National Institute for Occupational Safety and Health (NIOSH) is 1000 μg/m³. The US Federal Occupational Safety and Health Administration (OSHA) puts their exposure ceiling at 5000 μg/m³ (Bailey et al., 2018). OSHA for the state of California suggests a lower permissible exposure limit (PEL) of 200 μg/m³ which is 25 times lower than the Federal OHSA limit (Cal/OSHA, 2000). We can observe from this that there are some disagreements among agencies and their reference numbers for acceptable occupational Mn exposure.

Occupational Mn exposure occurs in a variety of occupational settings such as mining, welding, and ferroalloy production (Smith et al., 2007). Occupations such as these are linked to increased risk for Mn neurotoxicity (Lee et al., 2016; Lee et al., 2018). Exposure risk to air Mn differs between various industrial activities. For example, when Mn serves as the main material being interacted with, workers are subjected to higher levels of air Mn exposure: these include the likes of welding activities; the mining, crushing, transfer and storage of manganese ore; ferroalloy production; and MnO₂ battery production (NIOSH, 2019; Williams et al., 2012). In contrast, workers in other industries, such as those working as iron foundry workers or steel workers, are also at risk to Mn exposure but to comparatively lower levels of air Mn levels (Kendzia et al., 2017; Lander et al., 1999; Williams et al., 2012). Collectively, factors such as the duration and quantity of exposure, whether one is working in a confined space, along with the route of exposure will determine the amount of risk to workers in a particular industry. Neurotoxicity due to inhalation exposure to airborne Mn has been reported in miners who worked in Mn dioxide mines (Rodier, 1955), workers in dry alkaline battery factories (Roels et al., 1992), smelters (Dydak et al., 2011), and steel manufacturing workers (Bowler et al., 2011; Racette et al., 2012). Occupational Mn-induced parkinsonism may occur after prolonged inhalation of Mn dusts (Racette et al., 2012).

Welding is a major activity in manufacturing and construction work in modern industrial sectors. In 2016, the number of workers whose duties involved welding was around 400,000 in the US (Abe et al.). Exposure to welding fumes include multiple Mn species, both soluble and insoluble (Dorman et al., 2006). Biological markers of exposure are critical for evaluations of health effects pertinent to metal exposure (Awata et al., 2017). In the case of Mn, studies have utilized a number of different biological markers of exposure, such as Mn levels in whole blood, urine, and hair in demonstrating neurological deficits from occupational and environmental exposures (Ge et al., 2018; Reiss et al., 2016). In ferroalloy workers, blood Mn was associated with air Mn levels in subjects exposed to low (0.42 mg/m³) and moderate (4.2 mg/m³) air Mn levels, but not in workers exposed to the highest Mn levels (292 mg/m³) (Smith et al., 2007). In bridge welders, blood Mn, but not plasma or urine Mn, was significantly associated with cumulative respiratory exposure index (Smith et al., 2007). In welders, a small percentage of whole blood Mn (6%) was from the plasma fraction, and the researchers found that there was no association between whole blood and plasma Mn levels (Smith et al., 2007). Another study showed that the airborne Mn levels were significantly associated with Mn/Fe ratio (MIR) of erythrocytes (eMIR) and plasma (pMIR) (Cowan et al., 2009)

A study carried out by Racette, et al. (2016) followed Mn-exposed welders for up to 9.9 years after baseline evaluation; they demonstrated that parkinsonian symptoms such as impairment of speech and facial expression, upper limb bradykinesia, limb rigidity increased in Mn-exposed welders for each mg Mn/m³ of air inhaled per year. In line with this, Lee et al. (2018) also demonstrated that welders with low-level exposure to Mn also showed Mn accumulation in the brain and motor function deficit; in chronic exposure cases, microstructural changes were also noted.

Mn may deposit in the body in high doses, over 1 mg Mn/m³ (usually through chronic inhalation), and subsequently cause neurological toxicity by overwhelming Mn homeostasis mechanisms in the CNS (Andersen et al., 2010; Crossgrove and Zheng, 2004; Hochberg et al., 1996; Roels et al., 1999). The accumulation of Mn occurs mainly in the hippocampus, cerebral cortex, striatum, and basal ganglia (Barbeau, 1984; Calne et al., 1994; Iregren, 1999) leading to the appearance of neurasthenic syndrome and later, disorders of the extrapyramidal system like IPD and other parkinsonian diseases (Laohaudomchok et al., 2011). Thus, according to these reports, air inhalation with high concentrations of Mn is a public health problem and must be an important wellspring for additional studies.

3. Manganese Toxicity

The route of exposure of Mn plays a crucial role in determining the extent of physiological damage initiated within the body. This damage is related to the degree of exposure, duration, rate of influx/efflux, concentration, symptoms exhibited and organs affected (Williams et al., 2012; Zhang et al., 2017). Factors that determine susceptibility to Mn toxicity include; age, sex, ethnicity, genetics and co-morbid medical conditions. All these, combined with the fast absorption and long half-life of the element in tissues, raises concerns on its health impact on vital organs (O’Neal and Zheng, 2015). Mn is not easily absorbed from the skin, though slight excretion has been seen to occur via sweat in pregnant women (WHO, 2011; Williams et al., 2012). It is essential in preventing the phenotypic features of reactive oxygen species (ROS)-induced aging and in promoting wound healing in the skin via the MnSOD complex (Treiber et al., 2012). High Mn concentrations can disrupt the regular anti-oxidative activity of the MnSOD complex within the mitochondria (Koh et al., 2014). MnSOD expression has also been shown to increase in inflammatory skin diseases like psoriasis though no direct association with the disease condition and Mn overexposure was found (Li and Zhou, 2011). Nonetheless, low dose diet supplementation of artificial Mn has been shown to result in mild skin irritation and lesions ve (Crossgrove and Zheng, 2004; Williams et al., 2012).

The liver is responsible for redistribution and excretion of Mn; regular homeostasis is needed to avoid accumulation. Such accumulation can result from transport gene mutations which further impede the rate of Mn excretion, increases ROS production and leads to hepatocyte injury (Huang et al., 2011; Tillman, 2019). A previous epidemiological study in North Carolina, USA found a significant correlation between chronic liver disease mortality and increased air Mn concentrations (Spangler, 2012). Hepatic and renal failures characterised by necrosis and tubular proteinosis, accompanied by other debilitating side effects, have also been documented in other mammals (specifically dogs) exposed to high Mn intake (Borchers et al., 2014).

Furthermore, plasma levels of Mn in pre-dialysis patients were found to increase with progression in treatment. This progression had a negative association with glomerular filtration rate and lowered micronutrient retention (Sánchez-González et al., 2015). Subsequent malnutrition and anemia have been demonstrated in patients with Chronic Kidney Disease (Kim et al., 2017). In rhesus monkeys, inhalation of high doses of Mn sulphate induces inflammatory changes in small airways, bronchiolitis and proliferation of bronchus-associated lymphoid tissue. When compared with the occupational threshold limit, there was no significant pathology (Dorman et al., 2005). Similar pulmonary effects of smoking and inhaled Mn dust exist, which presents as decreased lung function. Mn impaired pulmonary function is predominant amongst males than females (Wang et al., 2015). Welding rods and automotive emissions contain a substantial percent of Mn that has a dose-dependent adverse effect on respiratory and neurological health (Bowler et al., 2007; Hassani et al., 2012; Mulyana et al., 2016). Individuals exposed to welding fumes have been seen to manifest with parkinsonian syndrome including cognitive impairment, gait abnormalities, hallucinations, rigidity, dystonia and bradykinesia. These symptoms are caused by Mn accumulation in specific brain regions such as substantia nigra, striatum and globus pallidus resulting in a condition called “manganism” (Guilarte and Gonzales, 2015; Kwakye et al., 2015). A slight distinguishable factor of manganism from IDP and other parkinsonian diseases is its unresponsiveness to dopamine precursor treatment therapy. The neurodegenerative effect that manifests afterwards lead to various neurobehavioral declines; therefore, an in-depth understanding of the mechanisms in which Mn induces brain damage is imperative (Fordahl et al., 2012; Peres et al., 2016).

3.1. Neurotoxic effects of Mn

Several factors interplay to form the cascade of events involved in Mn neurotoxicity; including oxidative stress, neuroinflammation, transporter dysregulation and metal imbalance (Harischandra et al., 2019).

3.1.1. Oxidative stress

A primary end-product of nearly all metabolic pathways within the biological system is the group of redox messengers called reactive oxygen species (ROS). The ROS family is primarily ^•O⁻₂, H₂O₂ and ^•OH, which can damage mitochondria molecules (Martinez-Finley et al., 2013). These are highly reactive molecules that elevate the level of cellular oxidative stress, which can be generated by pathologic processes which disrupt normal cellular processes (Sarkar et al., 2018). A common oxidative scavenger that mediates the ROS family of molecules is the reduced glutathione (GSH). GSH has been demonstrated to be depleted via disrupted synthesis in the brain after exposure to Mn (Yang et al., 2018). This increases the oxidized glutathione disulfide level (GSSG) and decreases the GSH/GSSG ratio thereby inhibiting the rapid detoxification of excessive hydroxyl radical (Rose et al., 2012). Also, inhibition of the antioxidant activity by superoxide dismutase (SOD), coupled with increase GSSG and accumulation of ferrous iron (Fe²⁺) have been experimentally demonstrated (Fernsebner et al., 2014). This causes neuronal injury by excessive ROS production and promotes neurodegeneration (Aoyama and Nakaki, 2013).

Some previous reports had contrary opinions on the involvement of ROS in Mn-induced neurotoxicity (Taylor et al., 2006). These reports may be due to the suppressive ability of Mn on oxidative stress via scavenging capacity of MnSOD complex, which further renders relief to oxidative stress within the mitochondria.

The oxidative state of Mn explored in these studies is mostly in the Mn⁺² form. Mn exists in a range of oxidation states from −3 to +7. Although Mn⁺² and Mn⁺³ are the common forms of Mn in biological tissues, humans are exposed to other oxidative states of Mn, which have more cytotoxic impacts due to their unstable structures. The Mn⁺² and Mn⁺³ states of Mn in biological systems are sometimes found interchangeably in the MnSOD complex (Gunter et al., 2004; Smith et al., 2017). Irrespective of the oxidative state, the solubility of the Mn containing compound determines its level of reactivity. Exposure to environmental Mn in the form of MMT have been demonstrated to exhibit characteristics similar to Mn⁺² after degradation of the compound, and high ROS production which have apoptotic effects on dopaminergic neurons (Carmona et al., 2014). The Mn⁺² group of molecules is generally more soluble and stable amongst all others. Some of the Mn found in the mitochondria of organs as heart, liver and brain are in Mn⁺² complexes. This could then be further oxidized into Mn⁺³ and Mn⁺⁴ that are most involved in ROS production (Butterfield et al., 2013; Majestic et al., 2007). Therefore, subsequent accumulation of Mn⁺² being oxidized into other states and direct accumulation of higher oxidative states of Mn could lead to rapid ROS generation. Furthermore, this divalent metal promotes dopaminergic neurotoxicity by continually facilitating the release of H₂O₂ (a ROS metabolite in the mitochondria) by microglial cells (Liu et al., 2013).

High Mn levels promote excessive production of ROS above physiological limits, thereby inducing oxidative stress (Kaur et al., 2017; Li and Yang, 2018; Owumi and Dim, 2019). Oxidative stress is detected by a reduction in several anti-oxidative enzymes, including glutathione, superoxide dismutase and catalase (Salim, 2017). The extreme effect of ROS initiates neuronal cell death indicated by activation of specific signalling pathways such as caspases, protein kinase C and mitogen-activated protein kinases (Cordova et al., 2013; Harischandra et al., 2019). Brain susceptibility to Mn-induced oxidative stress is increased by the disruptive influence of Mn to antioxidant activity, especially in dopaminergic-rich neuronal brain areas and synaptic clefts (Harischandra et al., 2019).

3.1.2. Transporter dysregulation

To avoid neurotoxicity, proper balance of Mn is required. Mn homeostasis in the brain is a tightly regulated mechanism that controls its uptake, storage and secretion. One of the factors that may cause excess Mn is transporter dysregulation (Harischandra et al., 2019). Meanwhile, a Mn-specific transporter has yet to be identified (Harischandra et al., 2019; Peres et al., 2016). Divalent metal transporter-1 (DMT1) is one of the first active transporters to be identified by which Mn is transported to the portal system via transferrin-(Tf) cycle endosomes (Erikson and Aschner, 2006; Fitsanakis et al., 2007). The neuronal transport of Mn depends mainly on DMT1 without the Tf pathway (Huang et al., 2004). Accumulation and neurotoxicity of Mn in the brain may be due to the higher expression of DMT1 in the basal ganglia, putamen, cortex and the olfactory epithelium (Salazar et al., 2008; Thompson et al., 2007); overexpression of DMT1 heightened cytoplasmic accumulation of Mn²⁺ and results in cell viability reduction (Tai et al., 2016).

ZIP-8 and ZIP-14 are Zrt-/Irt-related protein (ZIP) family metal transporters, encoded by gene solute carrier family 39 member 8 and 14 (SLC39A8 and SLC39A14a) respectively, are major transporter for Zinc (Jeong and Eide, 2013). However, SLC39A8 has been found to have higher affinity for transporting Mn than zinc in mammalian cells (He et al., 2006). Additionally, SCL39A8 knockdown mice displayed decreased tissue Mn level (Lin et al., 2017). Patients with Leigh-like mitochondrial diseases with deficient SLC39A8 were observed to have clinical pathologies which included basal ganglia dysfunction and progressive neurodegeneration of the brainstem (Lake et al., 2016). Also, autosomal recessive intellectual disability with cerebral atrophy syndrome has been reported to be associated with mutation in SLC39A8 (Boycott et al., 2015). Likewise, another study also reported that genetic mutation of the wild-type hSLC39A8 may lead to loss of function of its intrinsic metal transport activity; this can interfere with Mn uptake and cause mitochondrial dysfunction and oxidative stress (Choi et al., 2018).

Similarly, genetic overload syndrome features increased accumulation of Mn found in the basal ganglia and thalamus in CNS associated with a mutation in solute carrier family 30 member 10 (SLC30A10) (Taylor et al., 2019) that serves as a Mn transporter within the brain (Chen et al., 2015a). Mutation in this transporter also includes neurologic, hematologic and hepatic disturbance (Quadri et al., 2012).

3.1.3. Metal imbalance

Mn neurotoxicity as a result of excessive accumulation of Mn in the brain has been implicated in the dysregulation of essential metal homeostasis. Mn accumulates in brain regions that are normally rich in iron, most likely due to the fact that the two metals share common transport mechanisms (Tf and DMT1) (Dos Santos et al., 2011). These transporters also regulate influx of other metals, such as copper (Cu), zinc (Zn), calcium (Ca), cadmiun (Cd), lead (Pb), cobalt (Co), and nickel (Ni) (Chen et al., 2018; Chen et al., 2015b). Given the structural and chemical similarities between Mn and Fe, homeostasis of both metals is interdependent (Peres et al., 2016; Ye et al., 2017) resulting in an inverse relationship between Mn and Fe (Garcia et al., 2006). That is, since Mn and Fe share and compete for the same transporters, alterations in the regulation (influx / efflux) of either of the metals will result in the dyshomeostatis of the other (Chen et al., 2018). Thus, excessive accumulation of Mn may lead to Fe deficiency and vice versa.

Additionally, dyshomeostasis of Fe and Mn has an effect on oxidative stress. There are several intracellular antioxidative agents including glutathione, superoxide dismutase, flavonoids, vitamin E, catalase, glutathione peroxidase (GPX) and ascorbic acid (Abdal Dayem et al., 2017). The SOD2 subtype of superoxide dismutase forms the MnSOD complex in the mitochondria. This complex aids the conversion of superoxide ion into the stable state of hydrogen peroxide (Abdal Dayem et al., 2017; Thomas et al., 2009). However, another mechanism by which the MnSOD complex may aggravate the ROS level is through the competitive nature of Fe to replace Mn in its active binding sites (Aguirre and Culotta, 2012). Fe fosters the generation of ROS via the Fenton and Haber Weiss reaction. This overall two-step reaction process involves the initial combination of ferrous iron and hydrogen peroxide to produce a final product of ferric iron, hydroxyl radical and hydroxyl anion. The intermediate superoxide produced is reduced to ferric iron (Das et al., 2015). Under oxidative stress conditions of the mitochondria, one of the very reactive ROS metabolites, the hydroxyl radical formed in the Fenton reaction, can promote a sustained state of oxidative stress leading to neurodegeneration (Martinez-Finley et al., 2013; Thomas et al., 2009). Oxidative damage to neuronal cells and destruction of essential molecules have been implied to occur at a higher rate as a result of this inclusion of Fe via this process (Collin, 2019). Research by Venkataramani and colleagues (2018) demonstrated how the dysregulation of iron homeostatic proteins; amyloid precursor protein and heavy chain ferritin led to the uncoordinated accumulation of ferrous iron and increased oxidative state. This dysregulation was induced by exposure of human blastoma cell line to manganese-(II)-chloride tetrahydrate and manganese-(II)-acetate. Results from this experiment further elucidates the shared binding sites of Mn and Fe and subsequent displacement or accumulation of either in the condition of increased exposure (Venkataramani et al., 2018). Therefore, both monitoring of Fe stores and regulatory molecules should be done alongside to curtail Mn exposure aftermath in oxidative stress conditions (Imam et al., 2017). In relation to other metals, Mn accumulation in the brain results in the increased influx of metals such as chromium, zinc and calcium. An animal model study revealed that accumulation of Mn in the brain of Mn-exposed pups resulted in an increase in brain chromium and zinc concurrent with a decrease in brain Fe. Also, there was enhanced protein expression of DMT1 and transferrin receptor (TfR) overall in the brain, especially in the cerebellum, cortex, hippocampus, midbrain, and striatum (Garcia et al., 2006).

Mn neurotoxicity also results in calcium dyshomeostasis by increasing the mitochondrial calcium level. Reports revealed that elevated calcium levels results in ROS production, which allows for the opening of the mitochondrial permeability transition pore (MPT) (Ávila et al., 2014; Zoratti and Szabò, 1995). This causes the loss of inner membrane potential with resultant mitochondrial swelling, impairment of oxidative phosphorylation and inhibition of ATP synthesis (Ávila et al., 2014) leading to further generation of ROS associated with Mn neurotoxicity.

4. Mechanisms of Toxicity in the Brain: Regulation and Dysregulation

The mechanisms by which Mn induces toxic effects and contributes to neurodegenerative diseases are complex, could involve interaction of multiple mechanisms and ultimately remain unclear (Harischandra et al., 2019; Roth, 2009). Several studies have demonstrated that toxic and sub-toxic levels of Mn may affect important cell signaling pathways that regulates cell survival, differentiation, and apoptosis, and that are critically implicated in many neurodegenerative diseases, such as Parksinson’s, Alzheimer’s and Huntington’s disease (Bryan and Bowman, 2017; Ling et al., 2018; Roth, 2014). Mn-induced inflammatory processes have also been implicated in several neurodegenerative disorders. Neuroinflammation has been reported to be an important pathophysiological process involved in neurodegeneration (Yang et al., 2011) and other pathological conditions in the brain.

Activation of glial cells, such as astrocytes and microglia, by the release of inflammatory mediators, including pro-inflammatory cytokines by these glial cells, plays a vital role in Mn neurotoxicity (Cordova et al., 2013; Perl and Olanow, 2007; Sarkar et al., 2018). A 2007 study has shown that Mn treatment of astrocytes resulted in inflammation and swelling of astrocytes; the researchers concluded that this result could be due to the oxidative stress induced by Mn and that the swelling could be related to brain edema that is normally seen in patients with chronic hepatic encephalopathy (Rao et al., 2007). Exposure of Mn to astrocytes induced the release of pro-inflammatory cytokines and aggravated the inflammatory response induced by aggregated α-synuclein (Harischandra et al., 2014; Sarkar et al., 2018). Genes encoding pro-inflammatory chemokines and cytokines were altered in the astrocytes treated with Mn, implicating involvement of an inflammatory response in Mn neurotoxicity (Sengupta et al., 2007). Pro-inflammatory cytokines such as tumor necrotic factor (TNF-α) and interleukin 1-beta (IL-1β), primarily produced by glial cells in response to activation of environmental toxicants, increased in Mn-neuroinflammatory-induced injury (Guilarte, 2010). Interleukin-6 (IL-6) cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) is also present in Mn-induced neuroinflammation (Bahar et al., 2017). Decreased expression in the production of these inflammatory cytokines and nitric oxide protects astrocytes from Mn-induced damage (Streifel et al., 2011). Increase in the expression levels of nuclear factor kappa B (NF-κB), heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA, which are involved in inflammation, has also been reported to be implicated in the mechanism of Mn-induced neurotoxicity (Bahar et al., 2017). Also, activation of mitogen activated protein kinase (MAPK) signalling pathway in microglia plays a major role in Mn-induced inflammatory response through the regulation of cytokines and chemokines that amplify the activation of astrocytes (Kirkley et al., 2017; Tjalkens et al., 2017).

In addition, production of pro-inflammatory cytokine TNF-α and reactive oxygen species was attenuated by the deletion of Leucine-rich repeat kinase 2 (LRRK2) (Kim et al., 2019). LRRK2 is highly expressed in macrophages and microglia and has been reported to contribute to inflammation and neurotoxicity in IPD which has symptoms similar to manganism (Di Maio et al., 2018; Russo et al., 2014).

NF-κB signaling pathway plays a major role in inflammation. Although inflammatory responses are essential for the maintenance and defense of tissues, uncontrolled or chronic inflammation can be detrimental to tissue homeostasis, especially in sensitive tissues like the nervous system. Normally, NF-κB is present in the cytosol and bound to its inhibitory protein called IκBα. Activation of this inhibitory protein leads to phosphorylation and ultimately degradation of IκBα. This allows for the phosphorylation of NF-κB and its translocation from the cytosol to the nucleus. Once bound to the nucleus, NF-κB can activate transcription of target genes such as TNF-α, IL-1β, and IL-6 (Lu et al., 2018). Previous studies have highlighted the critical role of Mn in neuroinflammatory diseases via NF-κB pathway (Aschner et al., 2009; Kirkley et al., 2017; Sarkar et al., 2018). Recently, it was demonstrated that Mn activates NF-κB signaling pathways in the hippocampus and striatum of adult rats, which in turn regulate genes involved in inflammation (Nkpaa et al., 2019).

Mn alone can increase pro-inflammatory cytokine production very slightly and at high concentrations. However, studies have shown that Mn working in tandem with lipopolysaccharides (LPS) could induce expression of cytokines such as IL-6 and TNF-α in microglial cells via a NF-κB-dependent mechanism (Chen et al., 2006; Filipov et al., 2005). At present, it is not well established whether the potentiation of inflammatory cytokine production by Mn occurs at the level of NF-kB or further upstream in the intracellular signaling cascade.

Phosphatidylinositol 3 kinase (PI3K) is an intracellular phosphatidylinositol kinase that is interrelated to oxidative stress and regulation of cell differentiation, growth and apoptosis (Vanhaesebroeck et al., 2016). Serine-threonine protein kinase (Akt) is an important downstream mediator of PI3K-initiated cell survival signaling. It exerts anti-apoptotic activity by preventing the release of cytochrome C from mitochondria and inactivating forkhead box transcription factors (FOX) (Brazil et al., 2004; Song et al., 2005). Additionally, Akt can activate or inhibit its downstream target proteins such as FoxO3a, BAD, caspase-9, NF-κB, etc (Exil et al., 2014; Jia et al., 2014). Recently, Cheng et al. (2018) reported that chronic Mn exposure activates PI3K/Akt signaling pathway in hippocampus of rats. They observed a decrease of mRNA levels of Akt-1 and FoxO3a, accompanied by increased phosphorylation of Akt to p-Akt. This indicated that chronic Mn exposure could activate PI3K/Akt signaling through p-Akt, which inhibits the transcription function of apoptosis genes. Likewise, mRNA levels of Bcl-2 and caspase-3 decreased after Mn exposure while Bax levels had increased, indicating that apoptosis of rats’ hippocampal cells could affect the cognitive function of these animals (Cheng et al., 2018). Similarly, in vivo Mn treatment resulted in Akt phosphorylation and activation in striatum, 48 h after the last Mn administration, suggesting that changes in Akt signaling were related with impaired physiological neurodevelopment (Cordova et al., 2012). Mn activation of AKT and mTOR signaling was shown to be downstream of PI3K activation in cell-based studies (Bryan et al., 2018). Moreover, corroborating these findings, an in vitro study using microglia reported increases of phosphorylation level of Akt in the cell after treatment with Mn (Bae et al., 2006). Interestingly, PI3K signaling itself also has been reported to alter cellular Mn homeostasis and transport (Bryan et al., 2018). Also, PI3K has been shown to modulate Mn homeostasis by activating Akt and ATM/p53 signaling. Mn permits ATM to phosphorylate p53, a tumor suppressor gene related with DNA repair and neurodegeneration (Bryan et al., 2018; Chan et al., 2000; Tidball et al., 2015). Of interest, p53 and Akt/mTOR pathways are correlated with neurodegenerative diseases such as Parkinson’s Disease, including IPD, and Huntington’s Disease (Williams et al., 2010). Several studies have described possible molecular mechanisms and signaling pathways by which Mn induces neurotoxicity and neurodegeneration (Bryan et al., 2018; Cheng et al., 2018; Ling et al., 2018; Ma et al., 2015). Using the model organism, C. elegans, it was demonstrated that Mn exposure can increase expression of Akt in worms (Avila et al., 2012). It was reported that strains with loss of Akt (akt-1 and akt-2) had higher resistance to Mn exposure compared to wild-type strain. These results suggest, at least in part, that Akt could be related with Mn toxicity and indicate that Akt may be a potential target for future studies in Mn neurotoxicity (Peres et al., 2018).

Additionally, several studies have shown that Mn exposure may affect the αSyn expression, aggregation and cytotoxicity. In this regard, Cai et al. (2010) demonstrated that Mn-induced overexpression of αSyn in PC12 cells. This led to an upregulation of αSyn levels and αSyn aggregation, which is associated with cytotoxicity (Cai et al., 2010). Moreover, it was reported in a C. elegans model that exposure by Mn caused neurodegeneration by way of αSyn overexpression and misfolding (Vijayan et al., 2019). In another study, it was demonstrated that Mn exposure could accelerate cell-to-cell transmission of αSyn and increase release of misfolded αSyn that could increase pro-inflammatory and neurodegenerative response; this ultimately resulted in dopaminergic neurotoxicity (Harischandra et al., 2019). These studies suggest an important role of αSyn in Mn toxicity in the CNS.

Taken together, we see a whole host of signaling pathways affected by overexposure to Mn; putting the different pieces of the puzzle together invites further investigation.

5. Conclusion

Mn is an essential metal necessary for maintaining proper human health. This requirement is taken care of with a normal diet. However, increased exposure to Mn can lead to toxic effects. The utility of Mn has increased in industrial settings and products. The consequences of these industrial processes or common utility (i.e. burning of gasoline by cars) have led to increased Mn pollution. Workers dealing with Mn related products in occupational settings are at risk for developing neurotoxic symptoms. The general population, with chronic exposure to Mn-polluted air, is also at risk. We noted inconsistencies in standards of allowed occupational exposure by various agencies. Guidelines levels of air Mn exposure for the general public can also vary. These agencies for both occupational and non-occupational levels derive their guidelines on a handful of studies, some of them decades old. Updated and more thorough studies on toxic effects to occupational exposure to Mn can help further inform newer governmental guidelines for both occupational Mn exposure as well as the exposure limits for the general public.

Clinical studies of those exposed to Mn in occupational settings have informed us on the affect of Mn and have helped to establish these occupational exposure limits. A variety of animal models and cell studies have given insight into the molecular pathways that may explain how Mn causes damage and how to potentially protect against it. Neurotoxic effects of Mn, as summarized, can be due to oxidative stress, inflammatory response, transporter dysregulation, and metal imbalance. Many altered pathways and molecular changes are implicated in relation to Mn overexposure but require further investigation to clarify the picture of how Mn accumulates in the body and subsequently damages it to give rise to phenotypic changes. Further studies are also required to investigate potential therapeutic targets to tackle the consequences of Mn overexposure.

Highlights.

1. Mn pollution in the air and overexposure in subsets of the general population is likely to increase with increased industrial utility for the metal.

2. Overexposure to Mn can lead to metal dyshomeostatis in the brain.

3. Dopaminergic systems are uniquely affected by Mn and can lead to types of parkinsonisms

4. Mechanisms explaining Mn toxicity revolve around increased oxidative stress, mitochondrial dysfunction and calcium related pathways but much is left to be investigated.

Abbreviations

Mn

Manganese

MnSOD

Mn superoxide dismutase

CNS

Central nervous system

IPD

Idiopathic Parkinson’s Disease

ATSDR

Agency for Toxic Substances and Disease Registry

US

United States

EPA

Environmental Protection Agency

MMT

methylcyclopentadienyl manganese tricarbonyl

Fe

Iron

MANEB

Mn ethylene-bis-dithiocarbamate

ACGIH

American Conference of Governmental Industrial Hygienists

NIOSH

National Institute for Occupational Safety and Health

OSHA

Occupational Safety and Health Administration

MIR

Mn/Fe ratio

eMIR

Mn/Fe ratio of erythrocytes

pMIR

Mn/Fe ratio of plasma

ROS

reactive oxygen species

GSH

glutathione

GSSG

glutathione disulfide level

DMT1

Divalent metal transporter-1

Tf

Transferrin

ZIP

Zrt-/Irt-related protein

SLC39A8

solute carrier family 39 member 8

SLC39A14a

solute carrier family 39 member 14a

Cu

copper

Zn

zinc

Ca

calcium

Cd

cadmiun

Pb

lead

Co

cobalt

Ni

nickel

GPX

glutathione peroxidase

TfR

transferrin receptor

MPT

mitochondrial permeability transition pore

TNF-α

tumor necrotic factor

IL-1β

interleukin 1-beta

IL-6

Interleukin-6

COX-2

cyclooxygenase-2

iNOS

inducible nitric oxide synthase

NF-κB

nuclear factor kappa B

HO-1

heme oxygenase-1

Nrf2

nuclear factor erythroid 2-related factor 2

MAPK

mitogen activated protein kinase

LRRK2

Leucine-rich repeat kinase 2

LPS

lipopolysaccharides

PI3K

Phosphatidylinositol 3 kinase

Akt

Serine-threonine protein kinase

FOX

forkhead box transcription factors