The effect of manganese exposure in Atp13a2-deficient mice.

Loss of function mutations in the P5-ATPase ATP13A2 are associated with Kufor-Rakeb Syndrome and Neuronal Ceroid Lipofuscinosis. While the function of ATP13A2 is unclear, in vitro studies suggest it is a lysosomal protein that interacts with the metals manganese (Mn) and zinc and the presynaptic protein alpha-synuclein. Loss of ATP13A2 function in mice causes sensorimotor deficits, enhanced autofluorescent storage material, and accumulation of alpha-synuclein. The present study sought to determine the effect of Mn administration on these same outcomes in ATP13A2-deficient mice. Wildtype and ATP13A2-deficient mice received saline or Mn at 5-9 or 12-19 months for 45days. Sensorimotor function was assessed starting at day 30. Autofluorescence was quantified in multiple brain regions and alpha-synuclein protein levels were determined in the ventral midbrain. Brain Mn, iron, zinc, and copper concentrations were measured in 5-9 month old mice. The results show Mn enhanced sensorimotor function, increased autofluorescence in the substantia nigra, and increased insoluble alpha-synuclein in the ventral midbrain in older ATP13A2-deficient mice. In addition, the Mn regimen used increased Mn concentration in the brain and levels were higher in Mn-treated mutants than controls. These results indicate loss of ATP13A2 function leads to increased sensitivity to Mn in vivo.

Kanamycin Damages Early Postnatal, but Not Adult Spiral Ganglion Neurons.

Although aminoglycoside antibiotics such as kanamycin are widely used clinically to treat life-threatening bacterial infections, ototoxicity remains a significant dose-limiting side effect. The prevailing view is that the hair cells are the primary ototoxic target of aminoglycosides and that spiral ganglion neurons begin to degenerate weeks or months after the hair cells have died due to lack of neurotrophic support. To test the early developmental aspects of this issue, we compared kanamycin-induced hair cell and spiral ganglion pathology in rat postnatal day 3 cochlear organotypic cultures with adult whole cochlear explants. In both adult and postnatal day 3 cultures, hair cell damage began at the base of the cochleae and progressed toward the apex in a dose-dependent manner. In postnatal day 3 cultures, spiral ganglion neurons were rapidly destroyed by kanamycin prior to hair cell loss. In contrast, adult spiral ganglion neurons were resistant to kanamycin damage even at the highest concentration, consistent with in vivo models of delayed SGN degeneration. In postnatal day 3 cultures, kanamycin preferentially damaged type I spiral ganglion neurons, whereas type II neurons were resistant. Spiral ganglion degeneration of postnatal day 3 neurons was associated with upregulation of the superoxide radical and caspase-3-mediated cell death. These results show for the first time that kanamycin is toxic to postnatal day 3 spiral ganglion neurons, but not adult neurons.

Carbaryl-induced ototoxicity in rat postnatal cochlear organotypic cultures.

Carbaryl, a widely used carbamate-based insecticide, is a potent anticholinesterase known to induce delayed neurotoxicity following chronic exposure. However, its potential toxic effects on the cochlea, the sensory organ for hearing that contains cholinergic efferent neurons and acetylcholine receptors on the hair cells (HC) and spiral ganglion neurons has heretofore not been evaluated. To assess ototoxic potential of carbaryl, cochlear organotypic cultures from postnatal day 3 rats were treated with doses of carbaryl ranging from 50 to 500 µM for 48 h up to 96 h. Carbaryl damaged both the sensory HC and spiral ganglion neurons in a dose- and duration-dependent manner. HC and neuronal damage was observed at carbaryl concentrations as low as 50 µM after 96-h treatment and 100 µM after 48-h treatment. Hair cell was greatest in the high frequency basal region of the cochlea and progressively decreased towards the apex consistent with the majority of ototoxic drugs. In contrast, damage to the spiral ganglion neurons was of similar magnitude in the basal and apical regions of the cochlea. Carbaryl damage was characterized by soma shrinkage, nuclear condensation and fragmentation, and blebbing, morphological features of programmed cell death. Carbaryl upregulated the expression of executioner caspase-3 in HC and spiral ganglion neurons indicating that cellular damage occurred primarily by caspase-mediated apoptosis. These results suggest that chronic exposure to carbaryl and other carbamate anticholinesterases may be ototoxic. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 956-969, 2017.

Effect of manganese and manganese plus noise on auditory function and cochlear structures.

The degenerative actions of Mn caused by persistent exposure to high atmospheric levels not only provokes irreversible damage to the CNS with symptoms comparable to that of Parkinson's disease but also may have deleterious consequences to other organs including the auditory system. The putative deleterious consequences of prolonged Mn overexposure on hearing, however, is confounded by the fact that chronically-exposed individuals often work in high noise environments where noise by itself is known to cause hearing loss. Thus, the question as to whether Mn alone is actually ototoxic and whether exposure to Mn when combined with noise increases the risk of hearing loss and cochlear pathology has never been examined. To examine whether noise effects Mn ototoxicity, we exposed rats to a moderate dose of Mn (10mg MnCl2/liter water) alone, a high level of noise (octave band noise, 8-16kHz, presented at 90dB SPL for 8h/d) alone or the combination of Mn plus noise and measured the changes in auditory function and the cochlear histopathologies. Results of these studies, based on various measures of hearing including histological examination of cochlear tissue suggest that noise alone produced significant hearing deficits whereas semi-chronic exposure to moderate levels of Mn in drinking water for 90days either in the presence or absence of noise had, at best, only a minor effect on hearing.

Ototoxicity of Divalent Metals.

Excess exposure to both essential and non-essential heavy metals can lead to a variety of adverse clinical conditions which selectively affect a variety of organs and cells in the body. The diverse, but highly specific nature of the symptoms produced by each metal indicates that they can interact with a restricted population of cellular targets ultimately resulting in unique clinical manifestations. The symptoms, which can be reversible or irreversible, often present with different patterns and outcomes depending on the net accumulated dose of any given metal. There are some common pathological conditions that result from excess exposure to heavy metals which unfortunately have not received widespread recognition and thus, have not been extensively investigated. For example, chronic exposure to several heavy metals such as Co, Mn, Cd, Pb, and Hg has the potential to affect hearing in humans and experimental animals based on previous studies including case reports and ex vivo studies. Understanding exactly how these metals induce hearing deficits is complicated by the fact that the inner ear is an extremely complex system that composed of a diverse collection of sensory, neural, and supporting cells which must act in synchrony to produce a neurophysiological signal terminating in the central auditory system. This review will focus on the anatomical, cellular, and functional changes that occur in the cochlea, the sensory organ for hearing, due to excessive exposure to manganese, cadmium, cobalt, lead, and mercury.

Quantitative PCR analysis and protein distribution of drug transporter genes in the rat cochlea.

Membrane transporters can be major determinants in the targeting and effectiveness of pharmaceutical agents. A large number of biologically important membrane transporters have been identified and localized to a variety of tissues, organs and cell types. However, little is known about the expression of key membrane transporters in the inner ear, a promising site for targeted therapeutics, as well as a region vulnerable to adverse drug reactions and environmental factors. In this study, we examined the levels of endogenous membrane transporters in rat cochlea by targeted PCR array analysis of 84 transporter genes, followed by validation and localization in tissues by immunohistochemistry. Our studies indicate that several members of the SLC, VDAC and ABC membrane transporter families show high levels of expression, both at the RNA and protein levels in the rat cochlea. Identification and characterization of these membrane transporters in the inner ear have clinical implications for both therapeutic and cytotoxic mechanisms that may aid in the preservation of auditory function.

Trimethyltin-induced cochlear degeneration in rat.

Trimethyltin (TMT) is an occupational and environmental health hazard behaving as a potent neurotoxin known to affect the central nervous system as well as the peripheral auditory system. However, the mechanisms underlying TMT-induced ototoxicity are poorly understood. To elucidate the effects of TMT on the cochlea, a single injection of 4 or 8 mg/kg TMT was administered intraperitoneally to adult rats. The compound action potential (CAP) threshold was used to assess the functional status of the cochlea and histological techniques were used to assess the condition of the hair cells and auditory nerve fibers. TMT at 4 mg/kg produced a temporary CAP threshold elevation of 25-60 dB that recovered by 28 d post-treatment. Although there was no hair cell loss with the 4 mg/kg dose, there was a noticeable loss of auditory nerve fibers particularly beneath the inner hair cells. TMT at 8 mg/kg produced a large permanent CAP threshold shift that was greatest at the high frequencies. The CAP threshold shift was associated with the loss of outer hair cells and inner hair cells in the basal, high-frequency region of the cochlea, considerable loss of auditory nerve fibers and a significant loss of spiral ganglion neurons in the basal turn. Spiral ganglion neurons showed evidence of soma shrinkage and nuclear condensation and fragmentation, morphological features of apoptotic cell death. TMT-induced damage was greatest in the high-frequency, basal region of the cochlea and the nerve fibers beneath the inner hair cells were the most vulnerable structures.

Effect of manganese treatment on the accumulation on biologically relevant metals in rat cochlea and brain by inductively coupled plasma mass spectrometry.

Manganese (Mn), iron (Fe), zinc (Zn), and copper (Cu) are essential transitions metals that are required in trace amounts, however chronic exposure to high concentrations can cause severe and irreversible neurotoxicity. Since prolonged exposure to Mn leads to manganism, a disorder exhibiting a diverse array of neurological impairments progressing to a debilitating and irreversible extrapyramidal condition symptomatically similar to Parkinson's disease, we measured the concentration of Mn as well as Fe, Zn and Cu in three region of the brain (globus pallidus, striatum and inferior colliculus) and three regions in the cochlea (stria vascularis, basilar membrane and modiolus) under normal conditions or after 30 or 60 days of oral administration of Mn (10 mg/ml ad libitum). Under normal conditions, Mn, Zn and Fe were typically higher in the cochlea than in the three brain regions whereas Cu was equal to or lower. Oral treatment with Mn for 30 or 60 days resulted in 20-75 % increases in Mn concentrations in both cochlea and brain samples, but had little effect on Cu and Fe levels. In contrast, Zn levels decreased (20-80 %) with Mn exposure. Our results show for the first time how prolonged oral Mn-ingestion affects the concentration of Mn, Cu, Zn and Fe, in the three regions of the cochlea, the inferior colliculus in auditory midbrain and the striatum and globus pallidus, two regions implicated in Parkinson's disorder. The Mn-induced changes in the concentration of Mn, Cu, Zn and Fe may provide new insights relevant to the neurotoxicity of Mn and the transport and accumulation of these metals in cochlea and brain.

Cobalt-Induced Ototoxicity in Rat Postnatal Cochlear Organotypic Cultures.

Cobalt (Co) is a required divalent metal used in the production of metal alloys, batteries, and pigments and is a component of vitamin B12. Excessive uptake of Co is neurotoxic causing temporary or permanent hearing loss; however, its ototoxic effects on the sensory hair cells, neurons, and support cells in the cochlea are poorly understood. Accordingly, we treated postnatal day 3 rat cochlear organotypic cultures with various doses and durations of CoCl2 and quantified the damage to the hair cells, peripheral auditory nerve fibers, and spiral ganglion neurons (SGN). Five-day treatment with 250 µM CoCl2 caused extensive damage to hair cells and neurons which increased with dose and treatment duration. CoCl2 caused greater damage to outer hair cells than inner hair cells; damage was greatest in the base of the cochlea and decreased towards the base. CoCl2 increased expression of superoxide radical in hair cells and SGNs and SGN loss was characterized by nuclear condensation and fragmentation, morphological features of apoptosis. CoCl2 treatment increased the expression of caspase-3 indicative of caspase-mediated programmed cell death. These results identify hair cells and spiral ganglion neurons as the main targets of Co ototoxicity in vitro and implicate the superoxide radical as a trigger of caspase-mediated ototoxicity.

Neurotoxicity of trimethyltin in rat cochlear organotypic cultures.

Trimethyltin (TMT), which has a variety of applications in industry and agricultural, is a neurotoxin that is known to affect the auditory system as well as central nervous system of humans and experimental animals. However, the mechanisms underlying TMT-induced auditory dysfunction are poorly understood. To gain insights into the neurotoxic effect of TMT on the peripheral auditory system, we treated cochlear organotypic cultures with concentrations of TMT ranging from 5 to 100 µM for 24 h. Interestingly, TMT preferentially damaged auditory nerve fibers and spiral ganglion neurons in a dose-dependent manner, but had no noticeable effects on the sensory hair cells at the doses employed. TMT-induced damage to auditory neurons was associated with significant soma shrinkage, nuclear condensation, and activation of caspase-3, biomarkers indicative of apoptotic cell death. Our findings show that TMT is exclusively neurotoxicity in rat cochlear organotypic culture and that TMT-induced auditory neuron death occurs through a caspase-mediated apoptotic pathway.

Endogenous concentrations of biologically relevant metals in rat brain and cochlea determined by inductively coupled plasma mass spectrometry.

Manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are essential nutrients which aid in the proper functioning of cells, but high concentrations of these metals can be toxic to various organs. Little is known about the endogenous concentrations of these metals in the cochlea, the auditory portion of the inner ear which is extremely small and difficult to access. To fill this gap, a trace quantitative digestion and inductively coupled plasma mass spectrometry method was developed to determine the concentrations of these metals in the stria vascularis, organ of Corti, and spiral ganglion, three critically important parts of the cochlea (≤ 1.5 mg); these values were compared to those in specific brain regions (≤ 20 mg) of rats. Rats were sacrificed and the cochlea and brain regions were carefully isolated, digested, and analyzed to determine baseline concentrations of Mn, Fe, Cu, and Zn. In the cochlea, Mn, Fe, Cu, and Zn concentrations ranged from 3.2-6, 73-300, non-detect, and 13-200 µg/g respectively. In the brain, Mn, Fe, Cu, and Zn concentrations ranged from 1.3-2.72, 21-120, 5.0-10.6, and 33-47 µg/g respectively. Significant differences (p < 0.05) were observed between the tissue types within the cochlea, and between the cochlea and brain. This validated method provides the first quantitative assessment of these metals in the three key subdivisions of the cochlea compared to the levels in the brain; Mn, Fe, and Zn levels were considerably higher in the cochlea than brain.

Ototoxicity of paclitaxel in rat cochlear organotypic cultures.

Paclitaxel (taxol) is a widely used antineoplastic drug employed alone or in combination to treat many forms of cancer. Paclitaxel blocks microtubule depolymerization thereby stabilizing microtubules and suppressing cell proliferation and other cellular processes. Previous reports indicate that paclitaxel can cause mild to moderate sensorineural hearing loss and some histopathologic changes in the mouse cochlea; however, damage to the neurons and the underlying cell death mechanisms are poorly understood. To evaluate the ototoxicity of paclitaxel in more detail, cochlear organotypic cultures from postnatal day 3 rats were treated with paclitaxel for 24 or 48 h with doses ranging from 1 to 30 µM. No obvious histopathologies were observed after 24h treatment with any of the paclitaxel doses employed, but with 48 h treatment, paclitaxel damaged cochlear hair cells in a dose-dependent manner and also damaged auditory nerve fibers and spiral ganglion neurons (SGN) near the base of the cochlea. TUNEL labeling was negative in the organ of Corti, but positive in SGN with karyorrhexis 48 h after 30 µM paclitaxel treatment. In addition, caspase-6, caspase-8 and caspase-9 labeling was present in SGN treated with 30 µM paclitaxel for 48 h. These results suggest that caspase-dependent apoptotic pathways are involved in paclitaxel-induced damage of SGN, but not hair cells in cochlea.

Correlation between the biochemical pathways altered by mutated parkinson-related genes and chronic exposure to manganese.

The studies presented in this review attempt to describe the operative properties of the genes involved in generation of early and late onset of Parkinson's disease or Parkinson-like disorders and how mutation in these genes relate to onset of manganism. These include the genes α-synuclein, parkin, PINK1, DJ-1, ATP13A2, and SLC30A10 which are associated with early-onset of Parkinson's as well as those genes linked with late onset of the disorder which include, LRRK2 and VPS35. Since mutations in these genes and excess Mn potentially disrupt similar cellular processes within the basal ganglia, it is reasonable to hypothesize that the expressed symptoms of Parkinson's disease may overlap with that of manganese (Mn) toxicity. There appears to be four common processes linking the two disorders, as mutations in genes associated with Parkinsonism initiate similar adverse biological reactions acknowledged to stimulate Mn-induced dopaminergic cell death including; (1) disruption of mitochondrial function leading to oxidative stress, (2) abnormalities in vesicle processing, (3) altered proteasomal and lysosomal protein degradation, and (4) α-synuclein aggregation The mutual neurotoxic processes provoked by mutations in these genes in concert with the biological disturbances produced by Mn, most likely, act in synchrony to contribute to the severity, characteristics and onset of both disorders.

Cellular localization and developmental changes of Zip8, Zip14 and transferrin receptor 1 in the inner ear of rats.

Prior studies have demonstrated that the inner ear can accumulate a variety of essential and potentially toxic heavy metals including manganese, lead, cobalt and cadmium. Metal accumulation is regulated in part by the functionality and affinity of these metals for the different transport systems responsible for uptake across the blood-cochlea barrier and their subsequent uptake into the different cells within the inner ear. Transport of these metals across cell membranes occurs by many of the same transport systems which include DMT1, Zip8 and Zip14. All three metal transporters have been identified in the cochlea based on quantitative PCR analysis. Prior studies in our laboratory examined the localization and developmental changes of DMT1 in rat cochlea and since the two Zip proteins are also likely to contribute to the transport of essential and non-essential divalent cations, we performed immunolabeling experiments in postnatal day three rat pups and adult rats. For comparison, we also immunolabeled the specimens with antibody against transferrin receptor 1 (TfR1) which is important in DMT1-mediated transport of Fe and Mn. Results presented in this paper demonstrate that the cellular and subcellular distribution of both Zip8 and Zip14 within the different components of the inner ear are distinct from that of DMT1. Nuclear localization for both Zip transporters as well as TfR1 was observed. The findings also reveal that the selective distribution of the three proteins was altered during development presumably to meet the changing needs of the cells to maintain normal and functional levels of iron and other essential metals.

Cadmium-induced ototoxicity in rat cochlear organotypic cultures.

Cadmium (Cd), a widely used industrial metal, is extremely nephrotoxic and neurotoxic; however, its effects on the peripheral auditory system are poorly understood. To evaluate the ototoxicity of Cd, we treated cochlear organotypic cultures from postnatal day 3 rats with Cd concentrations from 10 to 500 µM for 24 or 48 h. Afterward, we evaluated the degree of damage to hair cells, auditory nerve fibers, and spiral ganglion neurons. Damage to the hair cells, auditory nerve fibers, and spiral ganglion neurons systematically increased in a dose and time-dependent manner. Exposure to Cd concentrations of 10 µM for 24 and 48 h resulted in minor inner and outer hair cell loss in the basal third of the cochlea. As Cd concentrations increased, toxicity spread toward the apex, also in a time-dependent manner. Treatment with 100 µM Cd for 48 h resulted in substantial (>30 %) hair cell loss over the entire cochlea. Cd was also toxic to auditory nerve fibers and spiral ganglion neurons; 100 µM of Cd for 24 h or 10 µM of Cd for 48 h resulted in considerable damage to auditory nerve fibers and spiral ganglion neurons. These findings are the first to demonstrate that Cd can cause significant lesions to peripheral auditory nerve fibers, spiral ganglion neurons, and sensory hair cells in organotypic cultures from postnatal cochleae.

Nicotinamide adenine dinucleotide prevents neuroaxonal degeneration induced by manganese in cochlear organotypic cultures.

Manganese (Mn) is an essential trace mineral for normal growth and development. Persistent exposures to high atmospheric levels of Mn have deleterious effects on CNS and peripheral nerves including those associated with the auditory system. Nicotinamide adenine dinucleotide (NAD) is a coenzyme which functions in the electron transfer system within the mitochondria. One of the most notable protective functions of NAD is to delay axonal degenerations caused by various neurodegenerative injuries. We hypothesized that NAD might also protect auditory nerve fibers (ANF) and SGN from Mn injury. To test this hypothesis, cochlear organotypic cultures were treated with different doses of Mn (0.5-3.0 mM) alone or combined with 20 mM NAD. Results demonstrate that the percentage of hair cells, ANF and SGN decreased with increasing Mn concentration. The addition of 20 mM NAD did not significantly reduce hair cells loss in the presence of Mn, whereas the density of ANF and SGN increased significantly in the presence of NAD. NAD suppressed Mn-induced TUNEL staining and caspase activation suggesting it prevents apoptotic cell death. These results suggest that excess Mn has ototoxic and neurotoxic effects on the auditory system and that NAD may prevent Mn-induced axonal degeneration and avoid or delay hearing loss caused by excess Mn exposure.

Cellular localization and developmental changes of the different isoforms of divalent metal transporter 1 (DMT1) in the inner ear of rats.

Divalent metal transporter 1 (DMT1) is generally considered to be the major transmembrane protein responsible for the uptake of a variety of divalent cations. Four isoforms of DMT1 have been identified in mammalian cells encoded by a single gene that differ both in their N- and C-terminal sequences with two mRNA isoforms possessing an iron response element (IRE) motif downstream from the stop codon on the message. Two distinct promoter sites regulate production of the 1A or 1B isoforms (translation starts at exon 2) for both the +IRE or -IRE species of the transporter resulting in the generation of four distinct configurations of this protein. Prior studies from our laboratory using cochlear organotypic cultures isolated from postnatal day three rats (P3) have demonstrated that Mn causes significant and selective damage to sensory hair cells and auditory nerve fibers and spiral ganglion neurons in a time and concentration dependent manner. Since DMT1 plays a critical role in controlling the uptake of a variety of essential and toxic metals into the cochlea, we compared the distribution and developmental changes of the 1A, +IRE and -IRE isoforms in rat inner ear. Results reveal that all three isoforms of DMT1 are selectively expressed in different cell populations within the cochlea and, additionally, demonstrate their cellular and subcellular distribution changes with development.

Down-regulation of LRRK2 in control and DAT transfected HEK cells increases manganese-induced oxidative stress and cell toxicity.

The extra-pyramidal symptoms associated with manganism often overlap with that seen in Parkinsonism suggesting a common link between the two disorders. Since wide deviations are observed in susceptibility and characteristics of the symptoms observed in manganism, these differences may be due to underlying genetic variability. Genes linked to early onset of Parkinsonism which includes ATP13A2 and parkin have already been suggested to promote development of Mn toxicity. Of the other Parkinson-linked genes, mutations in LRRK2, an autosomal dominant gene, represent another likely candidate involved in the development of manganism. In this paper the effect of shRNA LRRK2 knock-down on Mn toxicity was examined in control and DAT transfected HEK293 cells. Results demonstrate that LRRK2 down-regulation potentiates Mn toxicity in both control and DAT-transfected cell as well as potentiates DA toxicity. Combined treatment of Mn and DA further augments cell toxicity, ROS production and JNK phosphorylation in LRRK2 deficient cells compared to controls. Consistent with studies demonstrating that LRRK2 plays a role in the phosphorylation of p38, our results similarly demonstrate a decrease in p38 activation in LRRK2 knock-down cells. Our findings suggest that null mutations in LRRK2 which cause Parkinsonism potentiate Mn toxicity and increase susceptibility to develop manganism.

Manganese homeostasis and transport.

The review addresses issues pertinent to Mn accumulation and its mechanisms of transport, its neurotoxicity and mechanisms of neurodegeneration. The role of mitochondria and glia in this process is emphasized. We also discuss gene x environment interactions, focusing on the interplay between genes linked to Parkinson's disease (PD) and sensitivity to Mn.

The effect of manganese on dopamine toxicity and dopamine transporter (DAT) in control and DAT transfected HEK cells.

Chronic exposure to Mn results in the development of a neurological disorder known as manganism characterized by neurological deficits resembling that seen in Parkinsonism. Although dopaminergic neurons within the nigrostriatal pathway appear intact, Mn-induced irregularities in DA transmission have been observed including decreased amphetamine-induced DA release and loss of the dopamine transporter (DAT). Results of studies to evaluate the effect of Mn and DA on cell viability in control and DAT-transfected HEK cells reveal that Mn is equally toxic to both cell lines whereas DA was only toxic to cells containing DAT. DA toxicity was saturable suggesting that transport may be rate limiting. When Mn and DA were added simultaneously to the media, cell toxicity was similar to that produced by Mn alone suggesting that Mn may suppress DA uptake in the DAT containing cells. Preincubation of DA prior to the addition of Mn resulted in cell death which was essentially additive with that produced independently by the two agents. Mn was also shown to decrease DA uptake and amphetamine-induced DA efflux in DAT containing cells. Time-lapsed confocal microscopy indicates that Mn can promote trafficking of cell surface DAT into intracellular compartments which may account for the decrease in DA uptake and DA efflux in these cells. Mn-induced internalization of DAT may provide an explanation for disruption in DA transmission previously reported in the striatum.