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Toxicology and Applied Pharmacology 221 (2007) 131–147www.elsevier.com/locate/ytaap

Contemporary Issues in Toxicology

Manganese: Recent advances in understanding its transport and neurotoxicity

Michael Aschner a,⁎, Tomás R. Guilarte b, Jay S. Schneider c, Wei Zheng d

a Departments of Pediatrics, Pharmacology, and The Kennedy Center for Research on Human Development, B-3307 Medical Center North,Vanderbilt University, School of Medicine, Nashville, TN 37232-2495, USA

b Neurotoxicology and Molecular Imaging Laboratory, Department of Environmental Health Sciences,Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

c Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USAd School of Health Sciences, Purdue University, West Lafayette, IN, USA

Received 1 August 2006; revised 16 January 2007; accepted 2 March 2007Available online 12 March 2007

Abstract

The present review is based on presentations from the meeting of the Society of Toxicology in San Diego, CA (March 2006). It addressesrecent developments in the understanding of the transport of manganese (Mn) into the central nervous system (CNS), as well as brain imaging andneurocognitive studies in non-human primates aimed at improving our understanding of the mechanisms of Mn neurotoxicity. Finally, we discusspotential therapeutic modalities for treating Mn intoxication in humans.© 2007 Elsevier Inc. All rights reserved.

Keywords: Manganese; Parkinson's disease; Transport; Neurotoxicity

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132Manganese (Mn) transport in the central nervous system (CNS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132Positron emission tomography (PET) imaging in Mn neurotoxicity: old questions, new approaches. . . . . . . . . . . . . . . . . . . 134

Contribution of PET imaging to the understanding of Mn neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135PET studies in Mn-intoxicated monkeys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135What can be surmised from the PET studies in Mn-exposed humans and primates? . . . . . . . . . . . . . . . . . . . . . . . . . 136PET studies on Mn neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Behavioral effects of Mn intoxication on non-human primates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138Effects of Mn on motor functioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138Effects of Mn on cognitive and behavioral functioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Human studies of Mn intoxication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Diagnosis of manganism in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Search for biomarker of Mn exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

⁎ Corresponding author. Fax: +1 615 322 6541.E-mail address: [email protected] (M. Aschner).

0041-008X/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.taap.2007.03.001

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132 M. Aschner et al. / Toxicology and Applied Pharmacology 221 (2007) 131–147

Introduction

Mn exists in a number of physical and chemical forms in theEarth's crust, in water and in the atmosphere's particulatematter. Because its outer electron shell can donate up to 7electrons (USEPA, 1984), Mn can assume 11 different oxidationstates. Of environmental importance are Mn2+, Mn4+ and Mn7+.In living tissue, Mn has been found as Mn2+, Mn3+ and possiblyas Mn4+ (Archibald and Tyree, 1987). Mn5+, Mn6+, Mn7+ andother complexes of Mn at higher oxidation states are generallyunrecognized in biological materials (Keen, 1995). Mn plays anessential role as a cofactor in many enzymatic reactions inhumans, but in excess quantities, can cause damage to thenervous system. Typically found in compounds with acoordination number of 6 and lacking octahedral coordinationcomplexes, Mn tends to form very tight complexes with othersubstances (Keen, 1995). As a result, its free plasma and tissueconcentrations tend to be extremely low (Cotzias et al., 1968).

Manganese (Mn) is an essential metal found in a variety ofbiological tissues and is necessary for normal functioning of avariety of physiological processes including amino acid, lipid,protein and carbohydrate metabolism (Erikson et al., 2005). Mnalso plays an essential role in immune system functioning,regulation of cellular energy, bone and connective tissue growthand blood clotting (Erikson and Aschner, 2003). In the brain,Mn is an important cofactor for a variety of enzymes, includingthe anti-oxidant enzyme superoxide dismutase (Hurley andKeen, 1987), as well as enzymes involved in neurotransmittersynthesis and metabolism (Golub et al., 2005).

Despite its essentiality, Mn has been known to be aneurotoxicant for at least 150 years (ATSDR, 2000). Mnneurotoxicity was first identified as an extra-pyramidalsyndrome in miners exposed to high concentrations of Mnore (Barbeau et al., 1976; Barbeau, 1984; Couper, 1837;Mena et al., 1967). Exposure to excessive amounts of Mn isassociated with a variety of psychiatric and motor dis-turbances (Chia et al., 1993; Calne et al., 1994; Pal et al.,1999; Stredrick et al., 2004). The signs and symptoms fromrelatively high levels of exposure, as might occur inoccupational settings, include postural instability, mood andpsychiatric changes (i.e., depression, agitation, hallucinations)(Mena et al., 1967) and parkinsonian symptoms such asbradykinesia, rigidity, tremor, gait disturbance, posturalinstability and dystonia and/or ataxia (Josephs et al., 2005).Cognitive deficits such as memory impairment, reducedlearning capacity, decreased mental flexibility, cognitiveslowing (Josephs et al., 2005) and difficulty with visuomotorand visuospatial information processing (Bowler et al., 2003)have also been reported. It has been suggested that theearliest signs of Mn intoxication may be subtle (Bowler etal., 2006) and that psychomotor testing, in particular, may bemore sensitive than standardized neurological examinationsin detecting central nervous system (CNS) defects in exposedworkers (Roels et al., 1987, 1992).

The primary source of Mn intoxication in humans is due tooccupational exposure in miners, smelters, welders and workersin dry-cell battery factories (Bowler et al., 2006; Chandra et al.,

1981; Huang et al., 1989; Jiang et al., 2006; Myers et al., 2003;Ono et al., 2002). Significant neurological dysfunction has beenassociated with the duration of Mn exposure (Roels et al.,1987). Other epidemiological studies of industrial workers haveshown a positive correlation between neurological dysfunctionand lifetime integrated or cumulative Mn exposure (Lucchini etal., 1995, 1999; Roels et al., 1992).

The present review is based on presentations from themeeting of the Society of Toxicology in San Diego, CA (March2006). It addresses recent developments in the understanding ofthe transport of manganese (Mn) into the central nervous system(CNS), as well as brain imaging and neurocognitive studies innon-human primates aimed at improving our understanding ofthe mechanisms of Mn neurotoxicity. Finally, we discusspotential therapeutic modalities for treating Mn intoxication inhumans.

Manganese (Mn) transport in the central nervous system(CNS)

Mn enters the brain from the blood either across the cerebralcapillaries and/or the cerebrospinal fluid (CSF). At normalplasma concentrations, Mn appears to enter into the CNSprimarily across the capillary endothelium, whereas at highplasma concentrations, transport across the choroid plexusappears to predominate (Murphy et al., 1991; Rabin et al., 1993),consistent with observations on the rapid appearance andpersistent elevation of Mn in this organ (London et al., 1989).Radioactive Mn injected into the blood stream is concentrated inthe choroid plexus within 1 h after injection; 3 days post-injection, it is localized to the dentate gyrus and CA3 of thehippocampus (Takeda et al., 1994). Manganism, a conditionassociated with exposure to excessive Mn levels, is associatedwith elevated brain levels of Mn, primarily in those areas knownto contain high concentrations of non-heme iron (Fe), especiallythe caudate–putamen, globus pallidus, substantia nigra andsubthalamic nuclei.

How and in what chemical form Mn is transported across theBBB has come to light in a series of studies conducted over thelast two decades. It appears that facilitated diffusion (Rabin et al.,1993), active transport (Murphy et al., 1991; Aschner andGannon, 1994; Rabin et al., 1993), divalent metal transport 1(DMT-1; also known as DCT-1/NRAMP2)-mediated transport(Erikson et al., 2002; Garrick et al., 2003), ZIP8- and transferrin(Tf)-dependent transport (Aschner and Gannon, 1994) mechan-isms are all involved in shuttling Mn across the BBB (Fig. 1).Although non-protein-bound Mn enters the brain more rapidlythan Tf-bound Mn (Murphy et al., 1991; Rabin et al., 1993), thequestion remains as to which form represents the predominantmechanism of transport in situ. Analyses of transport mechan-isms which are based on tracer techniques employing bolusinjections of Mn into the circulation cannot be easily interpreted.While tracer studies represent a sensitive technique forquantifying Mn transport, it must be noted that the informationderived from such studies does not necessarily reflect thechemically active or functional forms in which Mn exists and istransported in vivo. This is due to the saturation of blood ligands

Fig. 1. Mechanisms of Mn transport across the blood–brain barrier (BBB) underphysiological exposure levels (physiological Mn plasma levels). Transportersassociated with Mn transport (relevant to its oxidation state) are indicated. Mnbound to albumin is excluded from passing the BBB given its size. Arrow sizedepicts the relative importance of each of the transporters in this process, bolderarrows representing more prominent transport mechanisms. Please refer to thediscussion for additional details. Since it has yet to be determined whether ZIP8functions to transport Mn across the BBB, the process has been annotated with aquestion mark.

133M. Aschner et al. / Toxicology and Applied Pharmacology 221 (2007) 131–147

for Mn and the likelihood that Mn in the free form exists atconcentrations in excess to those expected under physiologicalconditions. Thus, transport kinetics under such conditions maynot necessarily mimic physiological conditions. Leak pathwaysfor Mn also likely exist, especially in areas lacking intact BBB,namely the circumventricular organs (Fig. 1).

This brief review will address experimental evidencesupporting of each of the above mentioned transporters inshuttling Mn across the BBB. No unique mammalian Mntransporters have been identified. The affinity of divalent Mn(2+) towards endogenous ligand is relatively low. Divalent Mn(2+) does not avidly complex with sulfhydryl (−SH) groups oramines, and it shows little variation in its stability constants forendogenous complexing ligands such as glycine, cysteine,riboflavin and guanosine. Within the plasma, approximately80% of Mn is bound to globulin and albumin, and a smallfraction of trivalent (3+) Mn is bound to Tf (Aisen et al.,1969). There is much theoretical and some experimentalevidence suggesting that Tf, the principal Fe-carrying proteinof the plasma, functions prominently in Mn transport acrossthe BBB. In the absence of Fe, the binding sites of Tf canaccommodate a number of other metals, raising the possibilitythat Tf functions in vivo as a transport agent for many of thesemetals. Mn binding to Tf is time-dependent (Keefer et al.,1970; Scheuhammer and Cherian, 1985; Aschner and Aschner,1990). When complexed with Tf, Mn is exclusively present inthe trivalent oxidation state, with 2 metal ions tightly bound toeach Tf molecule (Aisen et al., 1969). At normal plasma Feconcentrations (0.9–2.8 μg/ml), normal iron binding capacity(2.5–4 μg/ml) and at normal Tf concentration in plasma−3 mg/ml with 2 metal-ion-binding sites per molecule (Mr

77000) of which only 30% are occupied by Fe+3-Tf hasavailable 50 μm of unoccupied Mn3+ binding sites per liter.

Since Tf receptors are present on the surface of the cerebralcapillaries (Fishman et al., 1985; Jeffries et al., 1984; Partridgeet al., 1987) and endocytosis of Tf is known to occur in thesecapillaries (Partridge et al., 1987), it has been suggested that Mn(in the trivalent oxidation state) enters the endothelial cellscomplexed with Tf. Mn is then released from the complex in theendothelial cell interior by endosomal acidification, and theapo-Tf Tf complex is returned to the luminal surface (Morris etal., 1992a, 1992b). Mn released within the endothelial cells issubsequently transferred to the abluminal cell surface for releaseinto the extracellular fluid (Fig. 1). The endothelial Mn isdelivered to brain-derived Tf for extracellular transport andsubsequently taken up by neurons, oligodendrocytes andastrocytes for usage and storage. Support for receptor-mediatedendocytosis of a Mn-Tf complex in cultured neuroblastomacells (SHSY5Y) was recently demonstrated by Suarez andEriksson (1993). Sloot and Gramsbergen (1994) have demon-strated anterograde axonal transport of 54Mn in both nigros-triatal and striatonigral pathways. Furthermore, in vivointravenous administration of ferric-hydroxide dextran complexsignificantly inhibits Mn brain uptake, and high Fe intakereduces CNS Mn concentrations, corroborating a relationshipbetween Fe and Mn transport (Aschner and Aschner, 1990;Diez-Ewald et al., 1968).

The distribution of Tf receptors in relationship to CNS Mnaccumulation is noteworthy. The pallidum, thalamic nuclei andsubstantia nigra contain the highest concentrations of Mn(Barbeau et al., 1976). Interestingly, Fe concentrations in thesestructures are the highest as well (Hill and Switzer, 1984).Although the areas with dense Tf distribution (Hill et al., 1985)do not correspond to the distribution of Mn (or Fe), the fact thatMn-accumulating areas are efferent to areas of high Tf receptordensity suggests that these sites may accumulate Mn throughneuronal transport (Sloot andGramsbergen, 1994). For example,the Mn-rich areas of the ventral pallidum, globus pallidus andsubstantia nigra receive input from the nucleus accumbens andthe caudate–putamen (Walaas and Fonnum, 1979; Nagy et al.,1978), two areas abundantly rich in Tf receptors.

A second prominent Mn transporter is the divalent metaltransporter-1 (DMT-1; also known as NRAMP-2 and DCT-1;Fig. 1). It is best known for its role as an intestinal, interluminalprotein responsible for Fe regulation (Conrad and Umbreit,2002; Mackenzie and Hediger, 2004). Indeed, gene transcrip-tion for this protein is actually regulated by Fe concentration viaan iron-response element (IRE) located on the mRNA (Garricket al., 2003). There is growing evidence that DMT-1 is involvedin brain Mn delivery. It has been well established that DMT-1has an affinity for Mn (Roth and Garrick, 2003). In themicrocytic anemia (mk) mouse and the phenotypically similarBelgrade (b) rat (Fleming et al., 1997, 1998), orthologousmutations (glycine 185 to arginine) in the DMT-1 gene result insignificantly reduced dietary iron (Fe) absorption. The role ofthe defective DMT-1 allele in the transport of Mn across theBBB has been evaluated in homozygous Belgrade (b/b) rats thatexhibit hypochromic anemia and also in heterozygous (+/b)Belgrade rats (Chua and Morgan, 1997). Plasma clearance anduptake by the CNS after intravenous injection of radioactive


54Mn bound to Tf or mixed with serum have demonstrated thatplasma clearance of Mn-Tf was much slower than Mn-serum,but both were faster than the clearance of Fe-Tf. Uptake of 54Mnand 59Fe by the brain was less in b/b rats than in +/b rats,suggesting that the defective DMT-1 allele affects themetabolism of both metals and that Mn and Fe might shareDMT-1 transporters in the BBB (Chua and Morgan, 1997).Other in vitro work also strongly supports a role for thistransporter in Mn movement across the BBB. Interestingly,DMT-1 activity is pH-dependent and probably Fe-dependent aswell. Additionally, because metal transport via DMT-1 is anactive process, it is likely to be temperature-dependent. This isinteresting because data suggest that Mn transport across ratbrain endothelial (RBE4) cells in culture is, among other things,temperature-, pH- and Fe-dependent (Fitsanakis et al., 2005).While the mammalian data are probably more germane tohuman Mn transport, it is also known that mutations inNRAMP-2, the DMT-1 homologue in bacteria and yeast,disrupt Mn transport (Kehres and Maguire, 2003; Portnoy et al.,2002; Rosakis and Koster, 2004, 2005). However, it is clear thatDMT-1 is not the sole transporter of Mn across the BBB since inthe Belgrade rat Mn is efficiently transported into the CNS(Crossgrove and Yokel, 2004). This is not altogether surprisingsince many metals and other molecules are dependent onnumerous transporters and other proteins to facilitate theirmovement across various barrier systems. For an extensivereview on the role of DMT-1 in mammalian transport ofmultiple metals, please refer to Garrick et al. (2003).

A small fraction of Mn is found in plasma as Mn-citrate(Crossgrove et al., 2003; Yokel and Crossgrove, 2004). Inadditional studies, these authors have assessed the regulation ofMn movement across the blood–brain barrier (BBB) with the insitu brain perfusion technique. The influx rates of Mn werecompared to their predicted diffusion rates, which were determinedfrom their octanol/aqueous partitioning coefficients and molecularweights. Results from these studies have suggested that aMn citratetridentate complex with a non-coordinated central carboxylaterecognition moiety is likely a substrate for the organic aniontransporter or a monocarboxylate transporter (MCT). Candidatesfor transport ofMn citratemay includeMCTand/ormembers of theorganic anion transporter polypeptide (OATP) or ATP-bindingcassette (ABC) superfamilies. However, as pointed out by theseauthors, themost likely candidate for mediating transport of theMnacross the BBB is DMT-1.

Finally, recent evidence implies a role for the ZIP transporterproteins in Mn transport; although it has yet to be determinedwhether these proteins are functional at the BBB. The ZIPtransporter proteins, members of the solute-carrier-39 (SLC39)metal-transporter family, have 14 members – highly conservedorthologs – between mouse and human (Eide, 2004). In plants,several ZIP proteins have been implicated in divalent metaltransport, including zinc (Zn), Fe and Mn. A recent in vitrostudy (He et al., 2006) in mouse fetal fibroblast culturesestablished that ZIP8 has a high affinity for Mn. The Km of2.2 μM for Mn2+ is close to physiological concentrations andwithin the same range determined in many cell lines or tissues.However, like many of the previously mentioned studies, one

must be cautious in extrapolating into the intact animal becausethe ability of this protein to transport a particular cation does notnecessarily imply that this protein is physiologically relevant.Thus, whether Slc39 and organic transporters function in Mntransport remains to be tested under physiologically relevantconditions. To date, the strongest evidence-based studies on Mnimply a physiological role for the transport of Mn both by Tfreceptors and DMT-1. Definitive studies to assess other proteinfunctions (Slc39, MCT) in physiological roles have yet to becarried out.

Positron emission tomography (PET) imaging in Mnneurotoxicity: old questions, new approaches

Mn is an essential element (see above), but exposure toexcessive concentrations increases brain Mn levels resulting ina parkinsonian-like syndrome. Parkinson's disease (PD) is thesecond most prevalent neurodegenerative disease and itshallmark neuropathology is the loss of nigrostriatal dopamine(DA) neurons in the substantia nigra pars compacta (SNpc). Theprogressive loss of DA neurons leads to the classical clinicalsymptoms of PD including resting tremor, hypoactivity, loss ofbalance and bradykinesia. Etiological and pathological datasuggest that there are both genetic and environmentalcomponents that cause and contribute to dopaminergicdegeneration (Jenner, 1998). Epidemiological studies alsosuggest that long-term Mn2+ exposure may be a risk factorfor PD (see above).

Early work examining miners exposed to Mn described aconstellation of extra-pyramidal symptoms with features similar tothose exhibited by patients with idiopathic PD (Calne et al., 1994;Lee, 2000; Olanow, 2004; Pal et al., 1999). These features includedbradykinesia, rigidity and gait abnormalities compounded byimpaired dexterity and clumsiness (Calne et al., 1994; Olanow,2004; Pal et al., 1999). Individuals with Mn-induced parkinsonismalso have more dystonia and fewer tremors than patients withidiopathic PD (Calne et al., 1994; Olanow, 2004; Pal et al., 1999).Furthermore, while L-dopa therapy is an effective means toameliorate the clinical expression of PD, the benefits of L-dopatherapy have been described as having mixed results in Mn-exposed workers. Some reports describe the beneficial effects of L-dopa in the early treatment of Mn-induced parkinsonism (Huang etal., 1989), while other reports describe no benefit (Lu et al., 1994).Therefore, both similarities and differences in the clinicalexpression of Mn-induced parkinsonism and idiopathic PD exist.

From a neuropathological perspective, there are a verylimited number of human studies in which autopsy samples ofMn-exposed brains have been examined (Bernheimer et al.,1973; Yamada et al., 1986, and references within). Furthermore,many of these brain autopsy examinations were performed inthe early 1900s upon individuals working in heavily con-taminated environments. Nevertheless, even from this limitednumber of human brain samples, there is clear evidence thatbasal ganglia nuclei are primary targets for Mn neurotoxicity.These early studies described significant neuropathologicalchanges such as cell loss and gliosis in the globus pallidus,caudate, putamen and subthalamic nucleus (Bernheimer et al.,


1973; Yamada et al., 1986, and references within). For the mostpart, the substantia nigra pars compacta has been described asbeing intact (Yamada et al., 1986), although one case indicatedpathological involvement, loss of DA and the presence of Lewybodies (Bernheimer et al., 1973). These neuropathologicalfindings appear different from the consistent and markeddegeneration of DAergic neurons of the substantia nigra parscompacta documented in idiopathic PD (Bernheimer et al.,1973).

The clinical expression and neuropathology of Mn-inducedparkinsonism has been duplicated to a large extent in studiesusing non-human primates (Chandra et al., 1979; Eriksson et al.,1987; Neff et al., 1969; Olanow et al., 1996; Pentschew et al.,1963). The early studies used extremely high doses of Mn inattempting to mimic human exposure (Pentschew et al., 1963;Neff et al., 1969). More recent studies have used lower dosingprotocols for longer times of exposure (Chandra et al., 1979;Eriksson et al., 1987; Olanow et al., 1996). In general, thebehavioral, neurochemical and neuropathological findings inprimates agree strongly with findings documented in the humanliterature. Similar to human studies, non-human, primate studieshave demonstrated motor function abnormalities following highdoses of Mn exposure, including rigidity, hypoactivity, posturalinstability and action tremor (Neff et al., 1969; Eriksson et al.,1987, 1992a; Shinotoh et al., 1995). From a neuropathologicaland neurochemical perspective, non-human primates exposedto high doses of Mn also exhibit alterations in dopamine levelsas well as cell loss and gliosis in basal ganglia structures,especially in the globus pallidus (Pentschew et al., 1963;Chandra et al., 1979; Bird et al., 1984; Eriksson et al., 1987).

Contribution of PET imaging to the understanding of Mnneurotoxicity

The advent of new technologies such as positron emissiontomography (PET) and single photon emission computedtomography (SPECT) in the early 1980s provided scientiststhe tools necessary to visualize and quantify the chemistry of theliving brain. PET and SPECT are nuclear medicine imagingtechniques that produce an image or map of functionalprocesses in the body. These new technologies provided theadvantage of being able to assess brain chemistry in humanbeings and in experimental animals in both healthy states andduring active states of disease. Today, brain imaging iscommonly used in many hospitals and research institutionsthroughout the United States and throughout the world. The firstapplication of PET imaging to study Mn-induced neurologicaldysfunction was performed by a group led by Dr. Donald Calne(Wolters et al., 1989). In this study, the investigators performedPET imaging in 4 patients with Mn-induced parkinsonism whohad been working for more than 2 years in the Mn smeltingindustry in Taiwan. These individuals expressed bradykinesiaand rigidity with masked facial expression, clumsiness,impaired dexterity and gait abnormalities. [18F]-fluorodopaPET studies were performed in these Mn-exposed individuals,and the results were compared to 21 normal, healthy controlsand 13 patients with idiopathic PD. [18F]-fluorodopa PET

provides an index of the ability of dopamine neurons tosynthesize dopamine from L-dopa and represents an indirectmeasure of nigrostriatal dopaminergic neuronal integrity. [18F]-fluorodopa PET has been shown to be significantly decreased inpatients with idiopathic PD (Brooks et al., 1990; Vingerhoets etal., 1994). As expected, in the study byWolters et al. (1989), PDpatients exhibited a dramatic decrease in the [18F]-fluorodopaactivity in the striatum relative to controls. On the other hand,normal activity was measured in Mn-exposed workers thatexhibited clinical signs of parkinsonism and elevated levels ofMn in the blood and hair. Based on these findings, the authorsconcluded that Mn-exposed workers have an intact nigrostriataldopaminergic system and that the parkinsonian symptoms mustbe associated with the disturbance of post-synaptic striatal orpallidal neurons.

In a subsequent study by the same group of investigatorspublished 8 years later (Shinotoh et al., 1997), they performed afollow up investigation in which they repeated [18F]-fluorodopaPET and also added [11C]-raclopride PET, a radioligand thatbinds to D2-dopamine receptors. They found that whileparkinsonian symptoms had progressed since their previousstudy, [18F]-fluorodopa PET was still normal in all of thepatients, thereby corroborating their previous findings. [11C]-raclopride PET revealed a small (12%) but significant decreasein caudate/occipital ratios, for which the authors inferred thatthere was no major change in striatal D2 receptors.

More recently, SPECT studies with dopamine transporterradioligands have suggested that dopamine transporters in thestriatum are decreased in parkinsonian patients with a history ofMn exposure (Huang et al., 2003; Kim et al., 2002). Dopaminetransporter levels in the striatum have been used as markers ofdopamine terminal integrity in the diagnosis of PD (Pirker et al.,2002). These findings suggest that dopamine terminals in thestriatum are degenerating in individuals with a history of Mnexposure. One of the confounding factors in this study is that itremains unknown whether the individuals with Mn exposurewere already in the “pre-clinical” stages of PD and had merelyexperienced coincidental Mn exposure. Similarly, a case reportby Racette et al. (2005a) of a female with a history of alcoholiccirrhosis with parkinsonian symptoms showed MRI findingsconsistent with elevated Mn accumulation in the basal ganglia.The increase in brain Mn levels observed in this patient was dueto the fact that, in liver disease Mn biliary excretion is impaired.The patient exhibited reductions in [18F]-fluorodopa in thestriatum as compared to normal, age-matched controls, but notto the same degree as PD patients. This study is also confoundedby the fact that this woman could have had PD with Mnaccumulation in the basal ganglia due to her liver cirrhosis.However, the possibility that Mn exposure accelerates theprogression of idiopathic PD cannot be ruled out. Therefore, therelationship between Mn exposure and PD remains a con-troversial topic in the human literature.

PET studies in Mn-intoxicated monkeys

There are several PET imaging studies in non-humanprimates, providing additional insight into potential


mechanisms of Mn-induced neurotoxicity. Shinotoh et al.(1995), were the first to use PET imaging in Mn-exposedprimates. They exposed 3 male, adult rhesus monkeys tocumulative MnCl2 (single i.v. injections from 10 to 14 mg/kg)doses ranging from 71 to 87 mg Mn/kg of body weight andperformed [18F]-fluorodopa PET to assess dopamine synthesis,[11C]-raclopride PET to examine D2-dopamine receptors and[18F]-fluoro-deoxyglucose PET to evaluate glucose metabo-lism. These PET studies were performed at baseline (prior to Mnadministration) and at various time points after the initiation ofMn exposure. During Mn administration, two of the animalsexhibited hypoactivity and weakness of the limbs; one animaldid not express any degree of clinical abnormality. The latterresult suggests that individual animals or human beings mayhave differential susceptibilities to Mn intoxication. At thispoint, a cautionary note must be considered, namely thatindividual susceptibility to Mn in single animals may not bediscriminated from experimental variation about a threshold foran effect. To ascertain this possibility, additional studies withlarger animals will have to be carried out. The studies showedno significant changes from baseline for any of the PET studiesduring the course of Mn administration despite the fact that 2out of the 3 animals exhibited clinical symptoms of parkinson-ism with significant accumulation of Mn in the basal ganglia,which was confirmed by MRI (Shinotoh et al., 1995).

Studies by Eriksson and colleagues using PET also showedthat Mn-exposed monkeys expressed normal [11C]- L-dopaturnover, but [11C]-nomifensin uptake (a measurement ofdopamine transporter levels) declined progressively to reach a60% reduction from baseline in the striatum, therebysuggesting dopaminergic terminal loss (Eriksson et al.,1992a). [11C]-raclopride was transiently decreased in theearly stages of Mn intoxication, but levels normalized by theend of the studies. These animals were administered very highdoses of Mn (400 mg MnO2 per injection on 12 differentoccasions for a cumulative dose of 4.8 grams of MnO2 orapproximately 960 mg MnO2/kg body weight) and this veryhigh dose paradigm may have caused the effect on dopaminetransporter levels. They developed an unsteady gait withclumsiness of the hands and feet as well as hypoactivity. Thesestudies suggested two possibilities: (1) the number ofdopaminergic terminals in the striatum was constant with areduced amount of dopamine transporter protein or (2) therewas dopaminergic terminal loss with no change or with acompensatory increase in L-dopa activity. In either case, noconclusive statement could be made, and no postmortemexamination of the brain was performed.

Postmortem studies of Mn-intoxicated primates have alsoprovided conflicting information. For example, three reportshave described decreased levels of dopamine in the striatum(Neff et al., 1969; Chandra et al., 1979; Eriksson et al., 1987)following relatively high doses of Mn exposure, while onereport indicates no change in striatal dopamine levels and anormally appearing substantia nigra (Olanow et al., 1996). Allprimate studies noted prominent neuropathological changes(i.e., gliosis and cell loss) in the globus pallidus with somedegree of involvement in the caudate, putamen and subthalamic

nucleus (Neff et al., 1969; Chandra et al., 1979; Eriksson et al.,1987; Olanow et al., 1996; Pentschew et al., 1963). Finally, areport by Eriksson et al. (1992b) using receptor autoradiographyindicated a significant decrease in striatal [3H]-SCH23390binding to D1-dopamine receptors with no effect on D2-dopamine receptors and a significant decrease in [3H]-mazindolbinding to dopamine transporters in the caudate and putamen.The latter study seems to confirm the PET findings by the sameinvestigators (Eriksson et al., 1992a), namely that Mn-intoxicated monkeys exhibit decreased levels of dopaminetransporters in the striatum.

What can be surmised from the PET studies in Mn-exposedhumans and primates?

As discussed above, Mn exposure likely alter differentdopaminergic neuron markers or processes. Nevertheless, thereis evidence to suggest that [18F]-fluorodopa PET is not affectedby Mn exposure (Shinotoh et al., 1995; Wolters et al., 1989),consistent with the idea that Mn intoxication does not alter theintegrity of nigrostriatal dopaminergic neurons. However, usingradioligands that measure dopamine transporters, the datasuggest that chronic exposure to high levels of Mn significantlydecreases the level of this protein. Furthermore, there is evidenceto suggest that dopamine transporters are targets for Mn sinceacute Mn administration has been shown to transiently increasedopamine transporter levels in the striatum as measured by PET(Chen et al., 2006). The question that remains is whether, in thecase of chronic Mn intoxication, the loss of dopaminetransporters indicates the loss of dopaminergic terminals or themodulation of the protein. Taken together, the data suggest thatthe latter may be the case since PET studies assessing theconversion of L-dopa to dopamine are consistently found to benormal. Alternatively, the idea that Mn intoxication causesdopamine terminal loss without altering [18F]-fluorodopa PETcannot be dismissed. For example, inWilson's disease, a geneticdisorder of copper deposition in the basal ganglia, there are casereports of patients in which PET studies to measure dopaminetransporter levels demonstrate significant loss in the striatumwith no evidence of a change in [18F]-L-dopa PET (Westermarket al., 1995). Importantly, similar to Mn intoxication, parkinso-nian symptoms and dystonia are prominent clinical features inWilson's disease (Jeon et al., 1998). In both conditions, metalhomeostasis in the caudate and putamen is disrupted. Interest-ingly, recent studies have documented an increase in the copperconcentration in the globus pallidus, caudate and putamen ofMn-exposed primates (Guilarte et al., 2006).

PET studies on Mn neurotoxicity

The molecular mechanisms by which Mn produces extra-pyramidal symptoms and other central nervous system effectsare not well understood. To this end, Guilarte et al. (2006)have studied non-human primates chronically exposed to Mn.These animals received behavioral training for a variety oftests of cognitive function as well as monitoring of motorfunction and activity (See Schneider in this review). A


comprehensive approach was undertaken, in which threeimportant elements involved in dopaminergic neurotransmis-sion – dopamine transporters, dopamine receptors anddopamine release – were measured using PET. Following

Fig. 2. Effect of chronic Mn exposure on [11C]-raclopride BP and AMPH-inducedraclopride binding to D2-DAR in the striatum of one animal at baseline and at theMn-1represent low levels of [11C]-raclopride binding to D2-DAR. Note the progressive lackfrom baseline to Mn-2. (B) [11C]-raclopride time–activity curves in the striatum and ccorresponds to the adjacent images in panel A. At baseline (top graph in B), thereadministration. Increasing Mn exposure reduces the effectiveness of AMPH-inducedgraph in B). (C) Quantitative data on [11C]-raclopride BP and AMPH-induced in vivothe mean percent change from baseline. Each value is the mean±SEMof 4Mn-exposeand subtle motor deficits in manganese-exposed non-human primates, Experimental

baseline training of the animals for cognitive and motorassessment, PET studies were performed on the same day forD2-dopamine receptors ([11C]-raclopride), dopamine transpor-ters ([11C]-methylphenidate) and in vivo dopamine release

dopamine release. (A) Representative pseudocolor trans-axial images of [11C]-andMn-2 time points. Red areas represent high levels of binding and green areasof a change in [11C]-raclopride levels in the striatum after AMPH administrationerebellum before and after AMPH in the same animal as in panel A. Each graphis a dramatic decrease in [11C]-raclopride levels in the striatum after AMPH

DA released to displace [11C]-raclopride from the striatum (see middle and lowerDA release for all animals. For dopamine release, the numbers in parenthesis ared animals. *p<0.05. (Reprinted fromNigrostriatal dopamine system dysfunctionNeurology 202:381–390, 2006, with permission from Elsevier.)


([11C]-raclopride with amphetamine challenge). These PETstudies were performed at baseline and during chronic Mnexposure (Guilarte et al., 2006). While no significant changesin [11C]-raclopride and [11C]-methylphenidate binding poten-tial were present in any of the animals during Mn exposure, asignificant and progressive decrease of in vivo dopaminerelease was measured in all of the animals (Fig. 2) and(Guilarte et al., 2006). The lack of effect of Mn exposure on[11C]-methylphenidate binding despite observations ofdecreased transporter binding by other investigators (Huanget al., 2003; Kim et al., 2002) may be attributable to themagnitude and/or duration of Mn exposure, account for thedifferences between these studies.

The decrease of in vivo dopamine release was mostpronounced at a time in which the Mn-exposed animalsexpressed deficits in fine motor dexterity and activity (Guilarteet al., 2006). The marked effect of Mn exposure on in vivodopamine release measured by PET has not been described inearlier studies, and it represents a new mechanism by which Mnexposure may affect basal ganglia function. These results showthat nigrostriatal dopamine system dysfunction is possible in theabsence of a change in a marker of dopamine terminal integrity.These findings may help explain previous observations that L-dopa therapy is not an effective means of ameliorating theclinical symptoms in Mn-exposed humans or primates since theproblem may not be one of dopamine synthesis or loss ofdopamine terminals but one of dopamine release. Furthermore,these findings provide a new avenue for investigating the effectsof Mn on the nigrostriatal dopaminergic system.

Notably, in a previous study in which acute high dose Mnwas given to 2 non-human primates, a transient but reproducibleincrease in striatal DAT levels was ascertained by PET (Chen etal., 2006). In this study by Chen et al. (2006), it was also shownthat Mn inhibits the binding of the cocaine analog [3H]-WIN35,428 to rat striatal membranes and the uptake of [3H]-dopamine to rat striatal synaptosomes. The acute effect of Mnon dopamine transporter levels measured by PET is differentfrom the lack of a significant effect of chronic Mn exposure onstriatal dopamine transporter levels described by Guilarte et al.(2006). It is likely that this variation in effect is based onwhether Mn is given acutely or chronically. The consistentfinding with both studies is that dopamine transporters are directtargets for Mn-induced effects. As demonstrated in the work byChen et al. (2006), acute Mn increased DAT levels. On the otherhand, in the chronic study (Guilarte et al., 2006); Mn abrogatesthe stimulatory effect of amphetamines on dopamine release,which is presumed to be due to a Mn-induced inhibition ofdopamine transporters. In summary, these studies clearly pointout that molecular imaging with PET, SPECT and othermodalities will play an increasingly important role in definingthe complex neurotoxic effects of Mn on the living brain.

Behavioral effects of Mn intoxication on non-humanprimates

As discussed above, much has been learned about the basictoxicology of Mn from studies in rodents, and especially from

the relatively few studies on the effects of Mn exposure in non-human primates, a species whose behavioral repertoire moreclosely resembles that generated by the human neurobehavioralsystem. Because Mn affects dopaminergic systems and itsneuropathology closely resembles PD (see above), studies onthe behavioral consequences of Mn exposure in non-humanprimates are extremely valuable.

To date, most studies have examined outcomes followingrelatively high level acute or chronic exposure to Mn. Less isknown about the effects of chronic exposure to lower levels ofMn or about the threshold exposures sufficient for alteringcognitive and motor function. This is of primary importance asconcerns about possible adverse effects from long-termexposure to increasing ambient levels of Mn in the environmentcontinue to mount (Aschner et al., 2005). To begin to addressthis issue, this section of the review will also briefly discuss databeing collected as part of an on-going, multi-disciplinary studyassessing the behavioral, neuroimaging and neuropathologicalconsequences of chronic exposure to different levels of Mn innon-human primates.

Effects of Mn on motor functioning

A number of studies have described the effects of Mnexposure on motor functioning in non-human primates.Although these studies used different levels of cumulativeexposure and different forms of Mn, the types of motordysfunctions noted (mostly associated with increasing cumula-tive exposure) include rigidity and bradykinesia (Olanow et al.,1996), intention tremor (Newland and Weiss, 1992; Suzuki etal., 1975), uncoordination (Neff et al., 1969; Suzuki et al.,1975), hyperactivity (Eriksson et al., 1992) and unsteady gaitand clumsiness (Eriksson et al., 1992).

In a study of 3 rhesus monkeys, Olanow et al. (1996)described the development of marked generalized bradykinesia,rigidity and abnormal extensor posturing of the hindlimbs afteronly 2 intravenous injections of 10 mg/kg each of Mn chloride.Movements suggestive of facial dystonia were also noted. Asecond animal also developed moderately severe bradykinesia,rigidity, extensor posturing and grimacing suggestive of facialdystonia after 2 injections of Mn chloride (total 23 mg/kg).Interestingly, a third animal did not develop any neurologicalsigns after receiving 87 mg/kg of Mn over a 43-day period(Olanow et al., 1996). The motor dysfunction in the 2symptomatic animals did not respond to L-dopa therapy.

In contrast to the rapid development of motor symptomsdescribed above, other investigators have reported much moreslowly evolving motor function deficits following chronicexposure to Mn. Mella (1923) injected monkeys intramuscu-larly with 5 mg of Mn chloride every other day for 18 monthsand reported that the animals developed choreoathetoid move-ments, tremor and rigidity. Gupta et al. (1980) described thedevelopment of muscular weakness and rigidity in the lowerlimbs of rhesus monkeys after 18 months of daily exposure toMn (25 mg/kg Mn chloride, p.o.). Eriksson et al. (1987)exposed each of 4 monkeys to a cumulative dose of 8 g of Mnoxide by repeated subcutaneous injections of 0.2 g of Mn per


injection over a 5-month period. All animals developedhyperactive behaviors after about 2 months of Mn administra-tion and then became hypoactive with an unsteady gait andsome action tremors by approximately 5 months of Mnexposure. The animals developed increasing weakness andclumsiness throughout the latter part of the study period. Inanother study, Eriksson et al. (1992) administered Mn oxide to 2monkeys on 11 occasions over a 4-month period, with 0.4 g ofMn oxide injected subcutaneously on each occasion. Bothanimals developed an unsteady gait, minor clumsiness of thehands and feet and hypoactivity after 4–5 months. Eriksson etal. (1992a) also administered Mn oxide by subcutaneousinjection to 3 monkeys on 13 occasions (0.2 g Mn per injection)over a 26-month period. The authors reported that the monkeysin this study did not show any obvious symptoms (although oneanimal, however, did show a time-dependent decrease inlocomotor activity) from this “low dose Mn intoxication” ascompared to the animals in the previous study which receivedlarger amounts of Mn during a shorter period of time. Onereport on the behavioral outcome of rhesus monkeys exposed toMn by inhalation (exposure to Mn dust particles, less than 5 μmin diameter at a concentration in air of 30 mg/m3 with a total of4 g of Mn dust blown through the inhalation chamber in a 6-hexposure period each day) (Bird et al., 1984) claimed that theanimals showed neither abnormal movements nor abnormalneurological signs during the 2 years of exposure. It isnoteworthy that generalizing on the effect of exposure durationon neurotoxicity is somewhat difficult, given the differentspecies and Mn salts used in various experimental paradigms.

One study has recently examined the neurobehavioraldevelopment of rhesus monkeys raised on cow's milk formula(containing 50 μg Mn/L), soy formula (containing 300 μg Mn/L) or soy formula supplemented with Mn chloride (containing1000 μg Mn/L) through 4 months of age (Golub et al., 2005).Motor endpoints thought to be potentially reflective of Mn-induced developmental neurotoxicity were more consistentlypresent during observation sessions (compared to animals thatdid not receive Mn), and an increase in overall spontaneousactivity was witnessed (Golub et al., 2005).

As part of an on-going multi-disciplinary study (Schneiderand Guilarte labs) assessing the behavioral, neuroimaging andneuropathological consequences of chronic exposure to differ-ent levels of Mn sulfate in non-human primates, recent studieshave examined several aspects of motor functioning in monkeysin “low,” “intermediate” and “high” exposure groups. Motorassessments included automated activity measurements, motorratings and assessment of fine motor skills (Schneider and Pope-Coleman, 1995). Animals were randomly assigned to treatmentgroups and received intravenous injections of Mn sulfate(MnSO4, doses calculated as the salt) at either 10 mg/kg/weekfor 5 weeks and then 15 mg/kg/week for the remainder of thestudy period (“low” dose group), 15 mg/kg/week for 5 weeksand then 20 mg/kg/week for the remainder of the study period(“intermediate” dose group) or 20 mg/kg/week for 5 weeks andthen 25 mg/kg/week for the remainder of the study period(“high” dose group). The cumulative amount of Mn adminis-tered to the “low” dose group was 156.7±9.5 mg/kg of body

weight over 272±17 days. Animals in the “intermediate” dosegroup received 210.4±1.3 mgMn/kg of body weight over 271±3 days, and animals in the “high” dose group received 97.5±21.9 mg Mn/kg of body weight over 68.3±18.2 days.

In the “low” dose group, motor rating scores, althoughsignificantly different from baseline by the end of the studyperiod, did not indicate the presence of Parkinson-like or otherclinically significant gross motor disorders. No abnormalmovements (i.e., dystonia or dyskinesia) were observed inany of these animals during the study. However, the animals diddevelop considerable difficulty with fine motor skills by the endof the study period. Fine motor skills were assessed using 2boards made of clear Plexiglas that contained 16 wells of small(14 mm) or large diameter (22 mm), all of which were 17 mmdeep (Schneider and Pope-Coleman, 1995). An “easy” versionof the board consisted of 12 large and 4 small wells while a“difficult” version of the board consisted of 12 small and 4 largewells. Animals were required to retrieve a standard rewardpellet from each well as quickly as possible. The primarymeasure obtained was the number of errors made per well (i.e.,the number of attempts to remove a pellet from a well and/or thenumber of times a pellet was dropped). On this test of fine motorskills, animals made 0.98±0.80 errors/well on the “easy” boardand 2.45±0.91 errors per well on the “difficult” board duringpre-Mn baseline testing. Over the course of the manganeseexposure period, performance on the “easy” board did notsignificantly change whereas animals made significantly moreerrors on the “difficult” version of the task at the end of the Mnexposure period (6.25±0.35 errors) (Schneider et al., 2006).

Overall activity during 2–3 consecutive 24 h periods wasrecorded prior to manganese exposure (1–3 separate sessions)and following manganese exposure (1 session approximatelyevery 2–4 weeks) using a personal activity monitor (Actitract;IM Systems, Inc.) placed into a pocket on the back of a jacketworn by the animals during the evaluation period. Overallactivity levels in the “low” dose group were significantlydecreased from baseline by the end of the study period(normalized activity levels were 100±4.4; in the pre-Mnbaseline period at 41.2±3.9 at the end of the Mn exposureperiod) (Guilarte et al., 2006). Studies with the “intermediate”and “high” dose groups are ongoing. Although there is not yetenough data to comment on the results of the studies on“intermediate” dose animals, total activity initially decreased inall “high” dose animals and then increased significantly abovebaseline levels, coinciding with the development of hyperkin-esis (a medical condition resulting in uncontrolled musclemovement, akin to spasms). All “high dose” animals developeddystonia as well as spontaneous choreiform dyskinesias(involuntary movement with jerky displacements of shortduration affecting the limbs and the face) that appeared atapproximately 7–12 weeks of Mn exposure. Some animals inthis group also developed significant ataxia. Due to thesignificant motor disturbances exhibited by these animals,they were euthanized much sooner after initiation of Mnexposure than animals that received lower amounts of Mn perinjection (“low” and “intermediate” exposure groups) and thushad shorter durations of exposure and received lower


cumulative amounts of Mn. However, that these “high” doseanimals developed significant motor problems with shorterdurations of exposure and lower cumulative amounts of Mnthan the other Mn-exposed animals suggests that it is not just thecumulative exposure that is important but the amount perexposure that plays a significant role in determining outcomefrom Mn exposure.

Effects of Mn on cognitive and behavioral functioning

Compared to the number of studies examining the motoreffects of Mn exposure, there have been considerably fewerstudies exploring the cognitive and behavioral outcomes of Mnexposure in non-human primates. Newland and Weiss (1992)investigated the effects of Mn on effortful responding andschedule-controlled behavior in 3 Cebus monkeys in an effort toevaluate the effects of Mn on motor properties and responsepatterns on operant tasks requiring different levels of physicalexertion. Monkeys were required to push against a spring-loaded device that resisted extension with a force thatapproximated their body weight and had to move the devicethrough an angle of about 45° (an arc length of 10 cm) afterwhich it returned to the starting position. This behavior wasmaintained using a multiple fixed-ratio, fixed-interval scheduleof performance. During the fixed-ratio schedule, the reinforce-ment cycle was triggered after 20 responses were completed.During the fixed-interval 90-s schedule, the reinforcement cyclewas initiated by the first response to occur after 90 s hadelapsed. Lights situated in front of the animals signaled whichreinforcement schedule was in effect. Mn chloride (5 or 10 mg/kg, calculated as the salt) was administered to the animals byintravenous injections separated by at least 1 week. After only 2administrations of 5 mg Mn/kg, a disruption of effortfulresponding (an increase in the number of incomplete responses)was observed, although only under the fixed-ratio schedule ofreinforcement that maintained a high rate of response. Onlyacute administration of the 10 mgMn/kg dose caused a transientincrease in incomplete responding under the fixed-intervalschedule. Overall response rates and patterns were not affectedby Mn exposure despite an increase in response failures(Newland and Weiss, 1992; Newland, 1999). Action tremorappeared in all monkeys after a cumulative dose of 40 mg/kg.The apparent lack of effect of Mn toxicity on motivation (i.e., noeffects on rate of responding) and the disruption of only abehavior that required considerable exertion on the part of theanimal suggests a subtle effect of relatively lowMn exposure onmotor performance, but not upon cognitive/behavioral perfor-mance (Newland and Weiss, 1992).

Golub et al. (2005), in their study of the neurobehavioraldevelopment of rhesus monkeys raised on cow's milk formula,soy formula or soy formula supplemented with Mn chloride,examined performance with a number of behavioral parameters.Group differences were not apparent on the relative perfor-mances of specific cognitive tasks (i.e., delayed non-match tosample, object discrimination, position discrimination andreversal and continuous performance test) that assessedlearning, memory and attention. Overall, infant animals (up to

18 months of age) were noted to be poorly engaged in thecognitive function tests regardless of the treatment group,suggesting that additional testing at more mature ages isnecessary. However, the soy group infants were described ashaving poorer participation across tasks and slower attainmentof performance criteria on some tasks (Golub et al., 2005). Inaddition, significant differences between cow's milk and soygroups were found on the duration of the daily sleep period(longer in soy groups), frequency of cling behaviors (increasedin soy groups), play behaviors (decreased in soy groups),premature response during a reward delay test, failure toimprove performance with experience and greater responserates during early continuous performance testing in the soy+Mn group. These results may indicate an increase inimpulsivity or a lack of normal wariness (Golub et al., 2005).Despite limitations in interpreting behavioral data obtainedfrom infant monkeys, these results suggest that developmentalMn exposure may negatively influence certain aspects ofneurobehavioral development.

In an on-going study of the consequences of chronicexposure to different levels of Mn sulfate in non-humanprimates, several aspects of cognitive and behavioral function-ing in monkeys in “low,” “intermediate” and “high” exposuregroups were examined. In the “low” dose group, the effect ofMn exposure on spatial working memory performance(variable delayed response task) varied among the animalsand thus, group analyses of these behavioral data showedinconsistent changes in each group's performance across taskdelays during Mn exposure. However, there was a trendtoward decreased performance at short and intermediatedelays (i.e., 2–10 s delays), and chronic Mn exposureinterfered with the expression of learning effects such asthose observed in normal animals during long-term taskperformance. Visual discrimination performance (referencememory) was not affected in these animals, and no consistentchanges were observed in the cognitive component of anobject retrieval task (executive functioning) (Schneider andPope-Coleman, 1995). In contrast, certain stereotypicalbehaviors such as grooming and the licking/biting of fingerssignificantly increased in frequency during the Mn exposureperiod.

As with the “low” dose exposure group, variable effects of“intermediate” dose Mn exposure on spatial working memoryperformance have been observed to date, with one animalshowing a consistent and reliable decrease in performance atshort and intermediate delays. Neither visual discrimination norobject retrieval performance has been affected by the “inter-mediate” dose of Mn exposure. Preliminary analyses ofbehavioral data also suggest an increased frequency of certainstereotypical behaviors as seen with the “low” dose exposuregroup.

To date, animals in the “high” dose Mn group have shown nochanges in performance of the spatial working memory task, butall animals have developed early appearing deficits in a non-spatial working memory task (delayed matching to sample).There was no deterioration in performance of the referencememory task or on the object retrieval task.


In summary, there have been a number of studies examiningMn toxicity in non-human primates. However, most havefocused on imaging parameters, postmortem pathologicalchanges and effects on gross motor behavior (Olanow et al.,1996; Eriksson et al., 1987, 1992a). Most studies have usedeither Mn chloride or Mn oxide and a variety of exposure routesincluding oral administration, inhalation and subcutaneous orintravenous injection. In ongoing studies in our lab (Guilarte etal., 2006), Mn sulfate was chosen as the experimental choice fortreatment, since it is one of the main combustion products ofmethyl-cyclopentadienyl Mn tricarbonyl (MMT) (Ressler et al.,1999; Zayed et al., 1999), an antiknock additive to unleadedfuel that contributes to environmental deposition of Mn(Aschner et al., 2005). Although the neurobehavioral toxicityof Mn in rodents has been shown to differ by the form of Mnadministered (Gianutsos and Murray, 1982; Komura andSakamoto, 1992; Zheng et al., 2000) as well as by the routeof administration, these parameters have not been system-atically manipulated in non-human primate studies. Thus, theeffects of different exposure types and forms of Mn mayinfluence outcomes, but this cannot be stated with any certaintyat this time.

While it is relatively easy to detect motor dysfunction inmonkeys resulting from Mn exposure, it is much more difficultto detect and quantify subtle cognitive and behavioralabnormalities that might arise, particularly when these abnorm-alities may precede other overt manifestations of toxicity.Variation in the appearance of these more subtle signs, as weand others have observed in monkeys, reflects a variation inindividual susceptibility to the behavioral effects of Mnexposure. These variations have also been described in Mn-exposed humans (Iregren, 1990). It is possible that moreconsistent cognitive deficits might be observed with moreprolonged Mn exposure, more frequent dosing or examinationof performance on tasks that tap into different cognitivedomains than we have studied or on tasks with greater demandson the processes already explored. Additional work is clearlynecessary to better understand the long-term effects of differentforms, doses and dosing regimens of Mn on cognitive andmotor functioning in both adult and developing Mn-exposednon-human primates.

Human studies of Mn intoxication

While information exists on Mn transport and mechanismsof Mn-induced neurotoxicity in a variety of species,significant challenges remain in extrapolating the informationto humans. Several crucial issues in human Mn toxicityremain unresolved, three of which will be detailed below: (1)the lack of a clearly defined clinical standard to distinguishmanganism from idiopathic Parkinson's disease (IPD); (2) thelack of reliable biological markers to assess the internal doseof Mn and its relationship to environmental exposure; and (3)the lack of a successful clinical therapeutic strategy fortreatment. This section will briefly discuss each of theseissues, providing a critical review and directions for futureresearch.

Diagnosis of manganism in humans

As mentioned above, typical signs and symptoms shown bymanganism patients resemble those of IPD, including tremor,rigidity, bradykinesia and posture instability (Barbeau et al.,1976; Calne et al., 1994; Inoue and Makita, 1996; Mena et al.,1967). The patients may initially exhibit palpitations, headache,memory loss, hand tremor, lower limb myalgia and hyper-myotonia. Without treatment, symptoms usually progress.Giddiness, myasthenia, frequent tetany and numbness in thearms and legs may occur. In severe cases, the patient maydisplay tremors at the angle of the lips and at the tip of thetongue, the tongue bitten while speaking, profound trembling ofthe upper extremities while writing or carrying objects andabnormal nose-pointing function. More typically, the patient'shandwriting becomes characteristically irregular, with particulardifficulty drawing circles, and letters become progressivelysmaller and smaller. Additionally, a distinct, festinating gaitmay develop (Jiang et al., 2006). Patients may also displayneuropsychological difficulties that include apathy and evenpsychosis. After withdrawal from the occupational exposure,Mn concentrations in the patient's blood, urine or hair mayreturn to normal after several months. Some symptoms maystabilize or improve to a certain extent; however, manysymptoms that are associated with extrapyramidal damagepersist. Severely intoxicated patients have difficulty simplycoping with daily life.

Basic research suggests that the sites of Mn-inducedneurological lesions are different from those observed inidiopathic Parkinson's disease (IPD). The brain regions primarytargeted in manganism are the globus pallidus and the striatumof the basal ganglia, whereas the neurodegeneration in IPDoccurs mainly in the substantia nigra (Calne et al., 1994;Cersosimo and Koller, 2006; Inoue and Makita, 1996; Olanowet al., 1996; Yamada et al., 1986; Walter et al., 2003). As such, itis argued that the differences in the sites of pathologic lesionwould determine the clinical manifestation of manganism orIPD. Consequently, the lack of the response of manganismpatients to L-dopa therapy, a criterion to differentiate mangan-ism from IPD that is commonly acknowledged amongclinicians, may mirror the specific lesion sites in the brain formanganism or IPD, leading to different responses to L-dopa.

Recent studies on human subjects, however, indicate thatchronic exposure to Mn in certain occupations may increase therisk of acquiring or accelerating PD (Gorell et al., 1997; Racetteet al., 2001, 2005a, 2005b). Racette and colleagues (2005a)screened 1,423 welders in Alabama who were referred formedical evaluation for PD. By using the Unified Parkinson'sDisease Rating Scale (UPDRS), these investigators reportedthat the estimated prevalence of parkinsonism among activemale welders ages 40–69 was about 0.98–1.3%, significantlyhigher than that of the age-standardized general population(Racette et al., 2005a). In addition, the clinical symptoms andthe response of welders to L-dopa treatment are not strikinglydifferent from those of IPD patients (Racette et al., 2001). Anearlier community-based study has suggested that Mn neuro-toxicity may be a continuum of dysfunction with early, subtle


changes at lower levels of exposure (Mergler et al., 1999).However, a more recent study using a nationwide cohort ofSwedish welders has suggested that there is no relationshipbetween welding and PD or any other movement disorders(Fored et al., 2006). Thus, no conclusive judgment can be madeat present regarding the association between career weldingpractice and the etiology of idiopathic PD.

Despite these arguments, the critical point on which theentire debate rests is the credible diagnosis of manganism. Sincethere is no reliable biomarker available for clinical assessmentof the degree of Mn neurotoxicity, the diagnosis must then takea patient's occupational history into consideration. Based onexperience gained from Chinese workers (Crossgrove andZheng, 2004; Jiang et al., 2006), the diagnosis of manganism isusually less ambiguous for active working patients than forpatients who have long ago left the job, and more consistent forsmelters than for welders, likely due to welders' day-to-day jobvariation and inconsistent environmental exposure. Clearly,establishing standard criteria for diagnosing manganism is ofparamount importance in research on Mn neurotoxicity inhumans.

Search for biomarker of Mn exposure

It should be emphasized that the symptoms of Mnintoxication, once established, usually become progressive andirreversible, reflecting to some extent the permanent damage ofneuronal structures. Thus, searching for a reliable biologicalindicator or biomarker for early Mn exposure has become adaunting task in the clinical investigation of Mn neurotoxicity.

Because of the intracellular distribution and relatively shorthalf life of Mn in the blood compartment (Zheng et al., 2000),blood Mn in general does not serve as a reliable indictor of thetotal body burden of Mn or of the overall disease status. A studyon 97 career welders revealed that serum Mn concentrations donot correlate with welders' professional years. However, thesewelders indeed showed significantly higher serum levels of bothMn and iron (Fe) than those of control subjects (Lu et al., 2005).Thus, on a group comparison basis, serum Mn levels may servereasonably well as an indicator of recent Mn exposure (e.g.,welders vs. normal controls). However, a relatively largevariation in blood Mn among individuals, either due to diet orother environmental influences, disqualifies blood Mn forclinical uses. Additionally, blood Mn is not suitable for theestimation of the historical accumulation of Mn in the bodyfollowing long-term, low-level Mn exposure. Nevertheless, aconsistently high level of blood Mn in any individual above thelocal surveillance range should unequivocally suggest recentexposure to Mn.

Compared to blood Mn, urine Mn is even less likely to be aclinical indicator for Mn toxicity because the primary route ofMn excretion (>95%) is via the bile to feces (Klaassen, 1974).Yet, the limited Mn urinary excretion as a background mayprove to be valuable for the EDTA challenge test. Of eightreported Chinese patients who received EDTA treatment(Crossgrove and Zheng, 2004, Jiang et al., 2006), five hadurinary Mn levels nearly 2-fold higher than before EDTA

treatment. Thus, some clinicians have recommended EDTA as achallenge agent to test Mn exposure since a significant increasein urinary Mn levels after EDTA treatment may suggest theaccumulation of Mn in the body. However, caution must betaken, for it remains unclear whether EDTA mobilizesphysiological Mn in normal subjects, to what extent theelevated urinary Mn level is considered a confident reflectionof Mn body overload and how the EDTA dose regimen shouldbe designed to facilitate a reasonable assessment. Therefore, it isnecessary to build the database on Mn excretion by EDTAamong normal subjects in order to evaluate the potential uses ofEDTA for Mn diagnosis more comprehensively.

A body of evidence suggests that Mn-induced neurotoxi-cities appear to be associated with altered Fe metabolism at bothsystemic and cellular levels (Chua and Morgan, 1996; Li et al.,2004; Malecki et al., 1999; Zheng and Zhao, 2001; Zheng et al.,1999). Among career welders, serum concentrations of ferritinand Tf are higher, while serum Tf receptor levels are lower thanthose of controls. Interestingly, serum Tf receptor levels declineas serum Mn concentration increases. These findings suggestthat exposure to welding fumes among welders disturbs theserum homeostasis of Fe and the proteins associated with Femetabolism (Li et al., 2004; Lu et al., 2005). In vivo Mnexposure in animals appears to facilitate the influx of Fe fromthe blood to the cerebral spinal fluid (CSF) (Zheng et al., 1999).Moreover, in vitro treatment of cultured cells with Mn promotescellular Fe overload (Li et al., 2005, in press; Malecki et al.,1999; Zheng and Zhao, 2001). In humans, a dysfunction in Femetabolism has been seen in IPD patients. High levels of totalFe, decreased ferritin, Fe-associated oxidative stress andabnormal mitochondrial complex-I have been repeatedlyreported in the postmortem substantia nigra of IPD patients(Dexter et al., 1991; Griffiths and Crossman, 1993; Loeffler etal., 1995; Sofic et al., 1991). Thus, accumulation of Fe inselected brain areas may induce the generation of Fe-mediatedreactive oxygen species; the ensuing oxidative stress may thenlead to neuronal cell death (Connor, 1997; Youdim et al., 1993).Since Fe and related metabolizing proteins are readily assayedin serum, this theory must be tested.

The life brain scan by MRI is another promising techniquewith potential uses for diagnosing Mn neurotoxicity (see alsoabove). An increased T1-weighted (the time associated with thereturn of longitudinal magnetization to its equilibrium conditionis termed the spin lattice relaxation time, T1, and reflects thestrength and nature of magnetic interactions between the spinsand their atomic neighborhood).Mn signal in the globus pallidusarea has been found among workers occupationally exposed toMn (Dietz et al., 2001; Jiang et al., 2006; Kim et al., 1999a;Lucchini et al., 2000; Nelson et al., 1993), in patients receivinglong-term total parenteral nutrition (Alves et al., 1997;Dickerson, 2001; Ejima et al., 1992; Fell et al., 1996; Fredstromet al., 1995; Iwase et al., 2000; Nagatomo et al., 1999;Quaghebeur et al., 1996) and in clinical cases of hepatic failure(Butterworth et al., 1995; Chetri and Choudhuri, 2003; Hauser etal., 1994; Hazell and Butterworth, 1999; McKinney et al., 2004;Spahr et al., 2002). Kim and his colleagues (1991b) havereported that a welder with more than 10 years of Mn exposure


whose clinical manifestations included a masked face, asym-metric resting tremor and bradykinesia had symmetrical highsignal intensities in the globus pallidus on the T1-weightedimage. The intensity, however, nearly completely disappearedsix months after he discontinued the welding practice. This hasled the authors to conclude that MRI, similar to blood Mnmeasurements, may be useful for detecting recent Mn exposure.

Therapy

Another challenging issue in the clinical aspect of the studyof Mn toxicity is the lack of an effective therapeutic strategy forMn-induced neurological impairment. Without treatment, theseverity of symptoms usually increases, and the chance ofrecovery decreases. Treatment with L-dopa has been the firstchoice in most clinics for two reasons. First, a neurologist tendsto control or alleviate the devastating symptoms associated withextrapyramidal injury. Clinical cases have shown that replace-ment of the lost dopamine could initially improve extrapyr-amidal symptoms (Lee, 2000; Mena et al., 1970; Rosenstock etal., 1971). Second, a neurologist tends to use L-dopa as adiagnostic tool to distinguish manganism from IPD. There arebodies of evidence supporting the view that L-dopa therapygenerally has limited benefits in improving clinical symptomsamong manganism patients, which contrasts to the responsefrom IPD patients. A five-year follow-up study indicates thatthe response to L-dopa treatment decreases after 2 or 3 years(Huang et al., 1993). The same group of investigators furtherindicates that, 10 years after the cessation of Mn exposure, thesame patients continued to show progression in the severity ofsymptoms (Huang et al., 1993, 1998).

In severe cases of Mn poisoning, chelation therapy has beenrecommended in order to reduce the body burden of Mn. EDTA(ethylene diamine tetra acetic acid) is a commonly usedchelating agent for treatment of a variety of metal-inducedtoxicities (e.g. lead), and it has been shown to increase toxinelimination, reducing the body burden of Mn and thereforeproving useful in treating manganism patients at early stages ofdisease development (Hernandez et al., 2006). Among sevenChinese welders with manganism, EDTA treatment increasedMn excretion in urine and decreased Mn concentrations in blood(Crossgrove and Zheng, 2004). Although the clinical symptomswere not alleviated significantly among these patients, EDTAchelation therapy appears to be helpful in reducing blood Mnlevels in acutely poisoned patients. A similar conclusion usingEDTA therapy has also been reported by other investigators,although they questioned the efficacy of this treatment forreducing neurological symptoms (Calne et al., 1994; Discalzi etal., 2000; Ono et al., 2002). This limited therapeutic benefit ofEDTA is expected. The four carboxyl groups in the EDTAstructure, while essential to its chelating property, render themolecule poorly lipophilic and thus prevent it from effectivelycrossing the blood–brain barrier (Fenstermacher and Kaye,1988; Schlageter et al., 1987; Von Holst et al., 1989). EDTAlikely chelates mainly the extracellular Mn ions.

Recently, an antibacterial drug, sodium para-aminosalicylicacid (PAS), has emerged as a potentially beneficial drug for

treating severe manganism. PAS is an FDA-approved drug usedto treat tuberculosis (TB). Clinically, the drug has never beenconsidered as the chelating drug for metal poisoning, althoughthe carboxyl group, along with the hydroxyl and amine groups,in PAS's structure do appear to provide an ideal chelatingmoiety for metals. Recent evidence has pointed out thatchelation of Fe in individuals with an excessive Fe burdenmay reduce TB viability and replication (Cronje et al., 2005),which may partly contribute to PAS's effectiveness in treatingTB.

In an animal study published in 1975, Tandon et al. (1975)showed that PAS was capable of removing Mn from the liverand testes of Mn-exposed rats. The human cases in which PASwas used successfully in the treatment of manganism were firstintroduced to English language literature by Drs. Ky and Hu ofGuangxi Medical University (Ky et al., 1992). In 2004, thisgroup of investigators conducted a follow-up study in one of thetwo cases reported in 1992 (Jiang et al., 2007). Combined with85 other cases receiving PAS treatment, most of which are inChinese literature, the clinical data appear to support theeffectiveness of PAS for treating manganism (Jiang et al., 2007).Following intravenous PAS infusion, most – if not all – signsand symptoms are reportedly significantly alleviated. Remark-ably, not only does PAS reverse extrapyramidal syndromesimmediately after therapy, but it also been shown to ensure astable prognosis long after therapy (Jiang et al., 2007),suggesting the possible repair of damaged neurons. The exactmechanism(s) of PAS therapy remain unknown. Based on thecharacteristics of its chemical structure and better lipophilicity,PAS may penetrate the blood–brain barrier more readily thanEDTA. Whether PAS chelates Mn from its brain deposit site orcombats Mn-induced inflammatory processes remainsunknown and warrants further investigation.

Conclusions

Mn is an essential mineral that is found at low levels invirtually all diets. Mn ingestion represents the principal route ofhuman exposure, although inhalation also occurs, predomi-nantly in occupational cohorts. Regardless of intake, animalsgenerally maintain stable tissue Mn levels as a result ofhomeostatic mechanisms that tightly regulate the absorption andexcretion of this metal. However, high dose exposures areassociated with increased tissue Mn levels, resulting in adverseneurological, reproductive, and respiratory effects. In humans,Mn-induced neurotoxicity, commonly referred to as manganismor Parkinson's disease-like syndrome, is of paramount concernand is considered to be one of the most sensitive endpoints. Mnneurotoxicity is associated with motor dysfunction syndromethat is recognized as a form of parkinsonism. While much hasbeen learned about transport mechanisms of Mn into the CNS,as well as its neurological effects, many challenges remain.More rigorous quantitative risk assessments that wouldextrapolate data obtained in rodents and non-human primatesto humans will require biologically based dosimetry modelswith better appreciation for neuronal transfer rates within thebrain, mechanisms of manganese-induced neurotoxicity, and


the relevance of olfactory transport to brain manganese deliveryin primates. This review, and recent studies described herein(Guilarte et al., 2006; Schneider et al., 2006) also establish thatindividuals in the general population are exposed to Mn in theirliving environment and exhibit blood Mn levels within therange of those achieved in experimental animals. This suggeststhat the behavioral and neuroimaging changes observed in the innon-human primates are likely to occur in individuals underspecific living conditions. Finally, it is clear that additionalstudies are necessary in order to identify potential efficacioustreatments for Mn intoxication along with reliable biomarkers ofexposure, representing fruitful areas for future research.

Acknowledgments

This work was supported in part by NIEHS 10563 and DoDW81XWH-05-1-0239 (MA), NIEHS 010975 (TRG),NIEHS10975 (JSS) and NIEHS 008146 (WZ).

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