Hypoxia induces changes in expression of isoforms of the divalent metal transporter (DMT1) in rat pheochromocytoma (PC12) cells

Although hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis, including transferrin and its receptor, little is known about the effect of low oxygen on formation of isoforms of the major iron transport protein, divalent metal transporter 1, DMT1. Accordingly, we examined the effects of hypoxia on expression and subcellular distribution of the different isoforms of DMT1 in rat PC12 cells. Treatment with low oxygen modestly increased expression of protein and mRNA levels for both the +IRE and -IRE species of DMT1. In contrast, expression of the exon 1A containing species of DMT1 was greatly increased by hypoxia as indicated by Western blot and real-time RT-PCR analysis. Message levels for the 1A isoforms increased approximately 60-fold after exposure of PC12 cells to 1% oxygen for 5 h. The subcellular distribution of exon 1A isoforms of DMT1 remained consistently in the cytoplasmic milieu of the cell after hypoxic exposure, as also did the distribution of +IRE species of DMT1. The -IRE species of DMT1, however, responded to hypoxia by becoming increasingly associated with the regions adjoining the outer cellular membranes, while a portion partially colocalized with an early endosomal marker (EEA). Hypoxia also caused a significant increase in the uptake of manganese in PC12 cells. In summary, these results demonstrate that hypoxia selectively increases expression of exon 1A containing species of DMT1 with lesser increases in either the +IRE or -IRE isoforms the transporter.     

Involvement of DMT1 +IRE in the transport of lead in an in vitro BBB model

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB). The BBB is particularly vulnerable to lead (Pb) insults. This study was designed to test the hypothesis that divalent metal transporter 1 (DMT1), which is a divalent cation membrane transporter, was involved in transcellular transport across the BBB. An in vitro BBB model, which was a co-culture system of human umbilical vascular endothelial cells (ECV304) and rat glioma cells (C6), was established. Transendothelial electrical resistance (TEER) and fluoresceinisothiocyanate (FITC)-dextran permeability results showed that Pb exposure at the tested concentrations had no significant effects on intercellular tightness. Pb transport displayed properties that were associated with iron response element (IRE) positive isoform of DMT1. Accordingly, Pb transport was significantly blocked by iron (Fe). Moreover, ECV304 cells that were depleted of Fe with the chelator deferoxamine (DFO) demonstrated increased Pb transport. By transfecting ECV-304 cells with a DMT1 expression vector, overexpression of DMT1 promoted an increase in Pb transport. Treatment of ECV304 cells with DMT1 antisense oligonucleotides (ASONs) MA1 significantly inhibited the transport of Pb. Our results suggest that Pb is transported in the in vitro BBB model by a transporter with biochemical properties similar to those of the DMT1 IRE-positive isoform.     

Tissue distribution of manganese in iron-sufficient or iron-deficient rats after stainless steel welding-fume exposure

Welders can be exposed to high levels of manganese through welding fumes. Although it has already been suggested that excessive manganese exposure causes neurotoxicity, called manganism, the pathway of manganese transport to the brain with welding-fume exposure remains unclear. Iron is an essential metal that maintains a homeostasis in the body. The divalent metal transporter 1 (DMT1) transports iron and other divalent metals, such as manganese, and the depletion of iron is known to upregulate DMT1 expression. Accordingly, this study investigated the tissue distribution of manganese in iron-sufficient and iron-deficient rats after welding-fume exposure. The feeding of an iron-deficient diet for 4 wk produced a depletion of body iron, such as decreased iron levels in the serum and tissues, and upregulated the DMT1 expression in the rat duodenum. The iron-sufficient and iron-deficient rats were then exposed to welding fumes generated from manual metal arc stainless steel at a concentration of 63.5 +/- 2.3 mg/m3 for 2 h per day over a 30-day period. Animals were sacrificed on days 1, 15, and 30. The level of body iron in the iron-deficient rats was restored to the control level after the welding-fume exposure. However, the tissue distributions of manganese after the welding-fume exposure showed similar patterns in both the iron-sufficient and iron-deficient groups. The concentration of manganese increased in the lungs and liver on days 15 and 30, and increased in the olfactory bulb on day 30. Slight and heterogeneous increases of manganese were observed in different brain regions. Consequently, these findings suggest that the presence of Fe in the inhaled welding fumes may not have a significant effect on the uptake of Mn into the brain. Thus, the condition of iron deficiency did not seem to have any apparent effect on the transport of Mn into the brain after the inhalation of welding fumes.     

Tissue Distribution of Manganese in Iron-Sufficient or Iron-Deficient Rats After Stainless Steel Welding-Fume Exposure

Welders can be exposed to high levels of manganese through welding fumes. Although it has already been suggested that excessive manganese exposure causes neurotoxicity, called manganism, the pathway of manganese transport to the brain with welding-fume exposure remains unclear. Iron is an essential metal that maintains a homeostasis in the body. The divalent metal transporter 1 (DMT1) transports iron and other divalent metals, such as manganese, and the depletion of iron is known to upregulate DMT1 expression. Accordingly, this study investigated the tissue distribution of manganese in iron-sufficient and iron-deficient rats after welding-fume exposure. The feeding of an iron-deficient diet for 4 wk produced a depletion of body iron, such as decreased iron levels in the serum and tissues, and upregulated the DMT1 expression in the rat duodenum. The iron-sufficient and iron-deficient rats were then exposed to welding fumes generated from manual metal arc stainless steel at a concentration of 63.5 ± 2.3 mg/m³ for 2 h per day over a 30-day period. Animals were sacrificed on days 1, 15, and 30. The level of body iron in the iron-deficient rats was restored to the control level after the welding-fume exposure. However, the tissue distributions of manganese after the welding-fume exposure showed similar patterns in both the iron-sufficient and iron-deficient groups. The concentration of manganese increased in the lungs and liver on days 15 and 30, and increased in the olfactory bulb on day 30. Slight and heterogeneous increases of manganese were observed in different brain regions. Consequently, these findings suggest that the presence of Fe in the inhaled welding fumes may not have a significant effect on the uptake of Mn into the brain. Thus, the condition of iron deficiency did not seem to have any apparent effect on the transport of Mn into the brain after the inhalation of welding fumes.     

Intracellular localization and subsequent redistribution of metal transporters in a rat choroid plexus model following exposure to manganese or iron

Confocal microscopy was used to investigate the effects of manganese (Mn) and iron (Fe) exposure on the subcellular distribution of metal transporting proteins, i.e., divalent metal transporter 1 (DMT1), metal transporter protein 1 (MTP1), and transferrin receptor (TfR), in the rat intact choroid plexus which comprises the blood-cerebrospinal fluid barrier. In control tissue, DMT1 was concentrated below the apical epithelial membrane, MTP1 was diffuse within the cytosol, and TfR was distributed in vesicles around nuclei. Following Mn or Fe treatment (1 and 10 microM), the distribution of DMT1 was not affected. However, MTP1 and TfR moved markedly toward the apical pole of the cells. These shifts were abolished when microtubules were disrupted. Quantitative RT-PCR and Western blot analyses revealed a significant increase in mRNA and protein levels of TfR but not DMT1 and MTP1 after Mn exposure. These results suggest that early events in the tissue response to Mn or Fe exposure involve microtubule-dependent, intracellular trafficking of MTP1 and TfR. The intracellular trafficking of metal transporters in the choroid plexus following Mn exposure may partially contribute to Mn-induced disruption in Fe homeostasis in the cerebrospinal fluid (CSF) following Mn exposure.     

Intestinal absorption of cadmium is associated with divalent metal transporter 1 in rats.

The intestinal absorption of cadmium (Cd) increases when the body iron (Fe) stores are depleted. The depletion of Fe upregulates the expression of divalent metal transporter 1 (DMT1), which is located at the apical membrane of enterocytes lining the small intestine. DMT1 has been shown to transport Fe and other divalent metal ions in vitro. However, it is not known whether DMT1 mediates the intestinal absorption of Cd. To investigate DMT1 involvement in Cd absorption, rats were fed a diet for 4 weeks either deficient in Fe (FeD diet, 2-6 mg Fe/kg) or supplemented with Fe (FeS diet, 120 mg Fe/kg), followed by a single oral administration of 109 CdCl2. Body Fe status, hemoglobin, and tissue Cd concentration were determined at 48 h after Cd administration. Also, DMT1 mRNA levels were quantified in duodenum, kidney, and liver by the branched DNA signal amplification method. Animals fed the FeD diet exhibited a reduced body weight gain, depletion of body Fe, and Fe deficiency anemia. Tissue Cd concentration was significantly higher in FeD than in FeS diet-fed rats, especially in the duodenum. The amount of Cd retained in the body was 10-fold higher in rats fed the FeD diet than in those fed the FeS diet. DMT1 mRNA was highly expressed in duodenum and was 15-fold higher in the FeD diet group. The levels of DMT1 mRNA were significantly lower in kidney and liver than in duodenum, but were 30 and 40% higher, respectively, in rats fed the FeD diet than in rats fed the FeS diet. These findings suggest that functional DMT1 protein is likely upregulated in the small intestine at the mRNA level by body iron depletion and increases Cd uptake from the gastrointestinal tract with subsequent transfer of Cd to the circulation and body tissues. Furthermore, the data from this study may indicate that DMT1 is a nonspecific metal transporter, which can transport not only Fe, but probably the toxic metal as well.     

Transport pathways for cadmium in the intestine and kidney proximal tubule: Focus on the interaction with essential metals

Cadmium (Cd) is a toxic metal with a propensity to accumulate in the proximal tubules cells (PTC) of the kidney where it can lead to tubular dysfunction and eventually renal failure. Although Cd²⁺-induced nephrotoxicity has been well described there is still uncertainty about how this metal gains entry into these cells to induce toxicity. As a non-essential metal, specific transport proteins for Cd are unlikely to exist. Rather transport proteins/channels used by essential metals (iron, zinc, calcium) are thought to be responsible. When these dietary essential metals are in short supply and deficiencies develop, Cd absorption and toxicity are enhanced. This is primarily due to increased expression of essential metal transport proteins such as divalent metal transporter 1 (DMT1) which can transport Cd in the intestine and enhance toxicity in the kidney. The zinc/bicarbonate sympoters ZIP8 and 14 are expressed at the apical membrane of enterocytes and PTC, and can transport Cd into cells. TRPV5 and 6 are major transporters for calcium in intestine and kidney and may be involved in Cd transport in these locations. Cd in the circulation is bound to proteins such as metallothioneins (MT) which are readily filtered. Two multiligand receptors, megalin and cubulin, reabsorb filtered proteins including albumin and MT by the process of receptor-mediated endocytosis. This review summarises the transport pathways for Cd in the intestine and kidney proximal tubule focusing in particular at how Cd uses essential metal transport processes to gain entry to the circulation and the kidney.     

Relative contribution of CTR1 and DMT1 in copper transport by the blood-CSF barrier: implication in manganese-induced neurotoxicity

The homeostasis of copper (Cu) in the cerebrospinal fluid (CSF) is partially regulated by the Cu transporter-1 (CTR1) and divalent metal transporter-1 (DMT1) at the blood-CSF barrier (BCB) in the choroid plexus. Data from human and animal studies suggest an increased Cu concentration in blood, CSF, and brains following in vivo manganese (Mn) exposure. This study was designed to investigate the relative role of CTR1 and DMT1 in Cu transport under normal or Mn-exposed conditions using an immortalized choroidal Z310 cell line. Mn exposure in vitro resulted in an increased cellular ⁶⁴Cu uptake and the up-regulation of both CTR1 and DMT1. Knocking down CTR1 by siRNA counteracted the Mn-induced increase of ⁶⁴Cu uptake, while knocking down DMT1 siRNA resulted in an increased cellular ⁶⁴Cu uptake in Mn-exposed cells. To distinguish the roles of CTR1 and DMT1 in Cu transport, the Z310 cell-based tetracycline (Tet)-inducible CTR1 and DMT1 expression cell lines were developed, namely iZCTR1 and iZDMT1 cells, respectively. In iZCTR1 cells, Tet induction led to a robust increase (25 fold) of ⁶⁴Cu uptake with the time course corresponding to the increased CTR1. Induction of DMT1 by Tet in iZDMT1 cells, however, resulted in only a slight increase of ⁶⁴Cu uptake in contrast to a substantial increase in DMT1 mRNA and protein expression. These data indicate that CTR1, but not DMT1, plays an essential role in transporting Cu by the BCB in the choroid plexus. Mn-induced cellular overload of Cu at the BCB is due, primarily, to Mn-induced over-expression of CTR1.     

The influence of high iron diet on rat lung manganese absorption

Individuals chronically exposed to manganese are at high risk for neurotoxic effects of this metal. A primary route of exposure is through respiration, although little is known about pulmonary uptake of metals or factors that modify this process. High dietary iron levels inversely affect intestinal uptake of manganese, and a major goal of this study was to determine if dietary iron loading could increase lung non-heme iron levels and alter manganese absorption. Rats were fed a high iron (1% carbonyl iron) or control diet for 4 weeks. Lung non-heme iron levels increased approximately 2-fold in rats fed the high iron diet. To determine if iron-loading affected manganese uptake, 54Mn was administered by intratracheal (it) instillation or intravenous (iv) injection for pharmacokinetic studies. 54Mn absorption from the lungs to the blood was lower in it-instilled rats fed the 1% carbonyl iron diet. Pharmacokinetics of iv-injected 54Mn revealed that the isotope was cleared more rapidly from the blood of iron-loaded rats. In situ analysis of divalent metal transporter-1 (DMT1) expression in lung detected mRNA in airway epithelium and bronchus-associated lymphatic tissue (BALT). Staining of the latter was significantly reduced in rats fed the high iron diet. In situ analysis of transferrin receptor (TfR) mRNA showed staining in BALT alone. These data demonstrate that manganese absorption from the lungs to the blood can be modified by iron status and the route of administration.     

A manganese-enhanced diet alters brain metals and transporters in the developing rat.

Manganese (Mn) neurotoxicity in adults can result in psychological and neurological disturbances similar to Parkinson's disease, including extrapyramidal motor system defects and altered behaviors. However, virtually nothing is known regarding excess Mn accumulation during central nervous system development. Developing rats were exposed to a diet high in Mn via maternal milk during lactation (PN4-21). The high Mn diet resulted in changes in hematological parameters similar to those seen with iron (Fe) deficiency in dams (decreased plasma Fe; increased plasma transferrin [Tf]) and pups (decreased hemoglobin [Hb] and plasma Fe; increased plasma Tf and total iron binding capacity). Mn-exposed pups showed an increase in brain Mn, chromium, and zinc concurrent with a decrease in brain Fe. In conjunction with the altered transport and distribution of essential metals within the brain, there was enhanced protein expression of the divalent metal transporter-1 (DMT-1) and transferrin receptor (TfR) overall in the brain; there was a general increase in each region analyzed (cerebellum, cortex, hippocampus, midbrain, and striatum). Neurochemical changes were observed as an increase in gamma-aminobutyric acid (GABA) and the ratio of GABA to glutamate, indicating enhanced inhibitory transmission in the brain. The results of this study demonstrate that developing rats undergo alterations in the transport and distribution of essential metals translating to neurochemical perturbations after maternal exposure to a diet supplemented with excess levels of Mn.