Published ahead of print on December 9, 2005, doi:10.1165/rcmb.2005-0101OC
© 2006 American Thoracic Society DOI: 10.1165/rcmb.2005-0101OC Effects of Iron Status on Transpulmonary Transport and Tissue Distribution of Mn and FeDepartment of Environmental Health, and Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts; and Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida Correspondence and requests for reprints should be addressed to Ramon M. Molina, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. E-mail: rmolina{at}hsph.harvard.edu
Manganese transport into the blood can result from inhaling metal-containing particles. Intestinal manganese and iron absorption is mediated by divalent metal transporter 1 (DMT1) and is upregulated in iron deficiency. Since iron status alters absorption of Fe and Mn in the gut, we tested the hypothesis that iron status may alter pulmonary transport of these metals. DMT1 expression in the lungs was evaluated to explore its role in metal transport. The pharmacokinetics of intratracheally instilled 54Mn or 59Fe in repeatedly bled or iron oxide–exposed rats were compared with controls. Iron oxide exposure caused a reduction in pulmonary transport of 54Mn and 59Fe, and decreased uptake in other major organs. Low iron status from repeated bleeding also reduced pulmonary transport of iron but not of manganese. However, uptake of manganese in the brain and of iron in the spleen increased in bled rats. DMT1 transcripts were detected in airway epithelium, alveolar macrophages, and bronchial-associated lymphoid tissue in all rats. Focal increases were seen in particle-containing macrophages and adjacent epithelial cells, but no change was observed in bled rats. Although lung DMT1 expression did not correlate with iron status, differences in pharmacokinetics of instilled metals suggest that their potential toxicity can be modified by iron status.
Key Words: anemia • DMT1 • iron status: manganese • transport
Breathing mixtures of metals is common. Ambient metal-containing particles from fuel combustion and other industrial sources can be significant sources for inhalation. Some occupations, such as welding, have greater inhaled metal exposure. Extended exposures to high concentrations of metal-containing particles can produce toxic effects. In particular, inhalation exposure to manganese-containing materials can result in manganism, a Parkinson's-like neurological disorder (1, 2). Understanding the fate of metals contained in inhaled particles that deposit in the lungs is essential in evaluating health effects of metals in the environment. Divalent metals such as Fe, Mn, Pb, Ni, Cu, and Co may share common transport systems such as divalent metal transporter 1 (DMT1) (3). Different metallic ions may interact and thus their absorption through the lungs may be altered. Iron absorption from the gut is a regulated mechanism, mainly influenced by iron stores in the body. In iron deficiency anemia, iron and manganese absorption in the duodenum is enhanced primarily by upregulation of DMT1 (3–5). Varying the levels of iron in oral dosages has been shown to influence the percent of manganese absorbed (6). Moreover, manganese uptake into the brain is increased during iron deficiency (7, 8). In contrast, dietary iron overload diminishes manganese accumulation in the brain. Iron status varies significantly among the population (e.g., severely anemic individuals versus welders who chronically breathe iron). Thus, relative risks of toxic effects from pulmonary absorption of manganese may differ within the population. Two hypotheses are that (1) iron and manganese share common carrier transport systems in the respiratory and gastrointestinal tracts, and (2) these systems may be downregulated in individuals who consume an iron-rich diet or have high iron levels in their lungs. Iron and manganese may share ion transport system(s) in the respiratory system as in the gastrointestinal tract (9, 10). To examine whether iron status affects transport of manganese through the lungs, we manipulated iron status in a rodent model by repeated bleeding or by introducing iron oxide particles into the lungs. We then examined the fate of instilled radioactive manganese and iron as well as the expression of the metal transporter protein, DMT1.
Experimental Animals Male virus-antigen–free (VAF) CD rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). They were acclimatized for a few days before the experiments. All procedures were approved by Harvard Institutional Animal Care and Use Committee. The rats were randomly divided into three groups: (1) normal control, (2) repeatedly bled, and (3) iron oxide aerosol exposed or instilled rats. To reduce iron status, rats were repeatedly bled by removal of 0.1 ml blood per kg body weight (15% of estimated blood volume) three times before the pharmacokinetic studies. The protocol consisted of bleeding via the retro-orbital sinus at 7, 5, and 3 d before the experiment. To increase pulmonary content of iron, rats for 54Mn pharmacokinetic study were exposed to iron oxide fumes (AMMD of 0.68 µm) generated by the combustion of iron pentacarbonyl vapor (11). The animals were exposed to the fumes, approximately 100 mg/m3, inside a Plexiglas chamber for 4 h, five times over a 2-wk period before the metal uptake experiments. Alternatively, rats for 59Fe pharmacokinetic study were intratracheally instilled with a suspension of iron oxide particles at a dose of 7.5 mg/kg every 3 d for a total of five instillations (12). Briefly, rats were placed on a slanted platform while anesthetized with vaporized Halothane (Halocarbons Lab, Inc., North Augusta, SC) and supported by an elastic band placed under the upper incisors. The iron oxide particle suspension was delivered to the lungs via a blunt 18-gauge needle inserted between the vocal chords and into the trachea. Transillumination of the larynx was provided by a microscope lamp shining on the neck (12). Since the rats were exposed to iron oxide by either aerosol exposure or iron oxide particle instillation, the total deposited dose and its distribution might be different. Indeed, the distribution of aerosol-delivered particles is more uniform than instilled particles (12). The deposited doses were likely to be similar from estimates of deposited dose based on aerosol exposure and ventilation parameters. Nonheme iron measurements (Table 1) confirmed that retained iron was within approximately 40% for both inhalation (1.13 mg/g lung) and instillation (1.95 mg/gm). At the end of each procedure to modify iron status, rats from each group were studied to (1) determine nonheme iron concentration in lungs, liver, and brain; (2) determine lung DMT1 expression by performing in situ hybridization or RT-PCR; (3) determine the toxic and inflammatory effects of each treatment; and (4) determine the pulmonary absorption and tissue distribution of intratracheally instilled 54Mn or 59Fe.
Nonheme Iron Determination Frozen lung, liver, and brain samples were thawed and aliquots were weighed. Samples (50–100 µg) were acid hydrolyzed in 2 ml of a mixture of equal volumes of 6-N-hydrochloric acid and 20% trichloroacetic acid at 65°C for 20 h. After cooling to room temperature, the clear yellow solution was transferred to a test tube and a color reagent (0.1% sulphonated bathophenanthroline mixed with 1% thioglycolic acid and distilled water at 1:25:25 ratio) was added. After a 10-min incubation, the optical density was measured at 540 nm. A standard curve was prepared using an iron standard solution (VWR). Nonheme iron in tissue was calculated based on the standard curve, and expressed as µg/g wet tissue. This procedure is described in detail elsewhere (13).
Bronchoalveolar Lavage and Analysis
54Mn or 59Fe Pharmacokinetics
In Situ Hybridization
RT-PCR
Dot Blot Analysis
Statistical Analysis
Characteristics of Bled and Iron Oxide–Exposed Rats The control, bled, and iron oxide–exposed rats used in the study are described in Table 1. The body weights were not significantly different among the three groups. The hematocrits of bled rats and their liver nonheme iron levels were significantly lower than controls. However, iron oxide exposure did not affect hematocrit values. Lung nonheme iron levels were significantly increased by multiple exposures of rats to an aerosol of iron oxide particles or by intratracheal instillations, but nonheme iron levels in other tissues examined did not change.
Pulmonary Effects of Repeated Bleeding or Iron Oxide Exposure
Pharmacokinetics of Intratracheally Instilled 54Mn And 59Fe The increased levels of iron in the lungs affected the rate of transport of 54Mn or 59Fe from the airways and alveoli to the blood (Figures 1 and 2). In iron oxide–exposed rats, 54Mn levels in the blood during the first 4 h were significantly lower than in untreated controls. Changes in the same direction were seen with 59Fe. Likewise, Fe2O3-instilled animals had significantly lower blood levels of 59Fe compared with controls. In contrast, rats made anemic by repeated phlebotomy had significantly higher blood levels of both 54Mn and 59Fe than control rats. Levels of radioactive metals measured in the blood reflect not only rates of transport into the blood, but also their rate of removal from the blood by organs and tissues. The amount of radioisotope remaining in the lungs at times of killing reflects the extent of metal clearance from the lungs. Significantly more 54Mn was retained in the lungs of Fe2O3 aerosol-exposed rats at 4 and 72 h than controls, confirming that pulmonary transport was attenuated by higher local iron levels (Tables 3 and 4). The same change was observed with 59Fe in Fe2O3-instilled rats. However, bled rats also retained significantly more 59Fe in the lungs despite higher levels in the blood (Table 5), showing that these higher levels reflect decreased vascular clearance rather than enhanced pulmonary transport.
In addition, the extent of absorption of 54Mn versus 59Fe from the lungs was very different. Sustained blood levels after absorption of 59Fe through the lungs were greater than that for 54Mn on a % of instilled dose basis (< 1% instilled dose for 54Mn compared with 2–12% instilled dose for 59Fe). The time course of metal transport as reflected in the shape of the blood level curves also differed. 54Mn blood levels were highest in all rats for the first time point. For 59Fe, there was a delay of  15 min in control and bled rats before reaching maximal values. Once a plateau was reached at 30 min, blood levels of 59Fe remained unchanged over the next 3.5 h.
Tissue Uptake of 54Mn and 59Fe Although bled rats retained the same amount of 54Mn in the lungs, they accumulated significantly more radioisotope in the brain. Significantly more 59Fe remained in the lungs of bled rats. Higher levels of 59Fe in the lungs at 4 h, and elevated blood levels (Figure 2), suggest that these animals absorbed significantly less 59Fe through the lungs, and at the same time cleared 59Fe more slowly from the blood. Significantly less 59Fe accumulated in the liver while increased levels were observed in the spleen of bled rats, consistent with mobilization of iron stores for enhanced erythropoiesis (19).
DMT1 Is Abundantly Expressed in Human Lungs
DMT1 Expression in Rat Lungs In situ hybridization showed that DMT1 is expressed in normal rat airway and alveolar epithelium, especially type II cells (Figures 4A and 5A). No significant overall change in DMT1 expression was observed in lungs from bled rats (Figure 4C). However, iron oxide exposure is associated with local up-regulation of DMT1 in areas where particle-containing macrophages were found (Figure 4B). In those areas, we saw significantly more DMT1-related staining in the instilled animals compared with control animals. That the presence of iron oxide is causally related to increased DMT1 mRNA expression is also suggested by Figure 5. Because intratracheal instillation of particles leads to nonuniform distribution (12), some regions of the lungs have few or no particles while other areas have significant numbers. In regions of the lung where few particles are seen, we detected very little DMT1 mRNA (Figure 5B). In contrast, areas of the same lung where many particles are deposited demonstrate local recruitment of macrophages to that area, and a significant upregulation of DMT1 mRNA in those cells and in adjacent epithelial regions (Figures 5C and 5D).
Blood vessels generally showed little DMT1 expression (Figure 6A). In contrast, large airways showed extensive staining (Figures 6A and 6B). DMT1 mRNA expression was also observed in bronchus-associated lymphoid tissue (BALT) adjacent to large airways. However, in large airways and BALT there was no discernable difference between control and iron oxide–exposed rats (Figures 6C and 6D). DMT1 expression in bled rat lungs as determined by in situ hybridization did not differ from that of controls. In lung tissues, two mRNA splice variants of DMT1 transcribed from the same gene are expressed (21). The splice variants differ only in their 3' untranslated regions; one contains an iron-responsive element (IRE) while the other does not (21). By RT-PCR, we found no difference in expression level of either isoform in lungs of bled rats (Figure 7).
Our pharmacokinetic experiments on the clearance of soluble iron and manganese in the lungs examined the effects of local iron loading of the lungs and of systemic iron deficiency induced by repeated phlebotomy. At 4 h, transport of both 59Fe and 54Mn from the lungs was suppressed in iron oxide–exposed rats. This observation might be explained by the presence of large amounts of soluble iron cations that compete with instilled radioisotope for a finite amount of transport protein, such as DMT1. Other metal transport systems such as the transferrin–transferrin receptor system would also be susceptible to such competitive inhibition. The dramatic increases in nonheme iron observed in the lungs suggest that maximum transport of iron and manganese ions may have been approached or perhaps even attained at some sites. An alternative possibility is the downregulation of transport systems by increased iron. However, we observed local upregulation of DMT1 mRNA in iron oxide–instilled rats and, although our attempt to detect DMT1 protein expression using commercially available antisera was unsuccessful, Wang and coworkers (20) have shown that non-IRE DMT1 protein is upregulated in rat lung epithelium after instillation of ferric ammonium citrate. Therefore, we favor the idea that competitive inhibition is the dominant mechanism involved in the suppression of pulmonary transport. Decreased pulmonary transport of 54Mn in iron oxide–exposed rats resulted in reduced uptake by other organs, including heart, brain, spleen, kidneys, skeletal muscle, and bone marrow. Likewise, significantly less 59Fe was found in blood, liver, spleen, stomach, and small intestine of iron oxide–exposed rats. Thus, inhalation of iron oxide particles correlated with decreased accumulation of the intratracheally instilled metals in other critical organs. In contrast, phlebotomy-induced anemia was associated with higher blood levels of both 54Mn and 59Fe. This effect was not due to enhanced pulmonary transport of metals, since levels of 54Mn remaining in the lungs of control and bled rats at 4 h were the same. In fact, the lung level of 59Fe was significantly higher in bled than in control rats. The elevated levels of circulating 54Mn and 59Fe observed in bled rats, therefore, appear to be due to decreased clearance of these metals from the blood. Increased blood levels of instilled 54Mn in anemic bled rats resulted in significantly more brain uptake of the metal compared with control animals. This trend increased over time (72 h versus 4 h). Our finding that phlebotomy-induced anemia enhances brain manganese uptake is consistent with previous studies demonstrating increased brain manganese uptake after intravenous injection of rats made iron-deficient by diet (22, 23). In the case of 59Fe, the elevated blood levels in anemic rats resulted in increased retention in the spleen but reduced liver levels, consistent with increased mobilization of iron stores for erythropoiesis (19). These results indicate that anemia due to chronic blood loss can dramatically change the tissue distribution and deposition of inhaled metals, and may predispose an individual to manganese neurotoxicity. Lung injury and inflammation might also influence manganese or iron transport from the airways and alveoli to the blood. To examine this possibility, we determined the effects of repeated exposure to iron oxide aerosol or repeated bleeding on lung injury and/or inflammation. Our data indicate that repeated inhalation of Fe2O3 caused modest increases in macrophages and neutrophils (Table 2). Deposition of increased particles in the lungs, which are cleared via phagocytic mechanisms, would promote increased numbers of macrophages. During infection or increased numbers of deposited particles, macrophages can be supplemented by migration of neutrophils from the blood to the alveolar spaces. In comparison to instilled iron oxide, however, other particles such as silica elicit many more neutrophils (18). Also in contrast to many other toxic dusts that promote 10- to 20-fold increases in LDH (18), a significant change in LDH levels with iron oxide exposure was not observed. Finally, it seems reasonable that significant inflammation might increase removal of 54Mn and 59Fe from the lungs; in fact, we observed a decrease. Thus, it is unlikely that our pharmacokinetic data reflect strong influences due to lung injury or inflammation. DMT1 expression has been shown to increase in bronchial epithelial cells in response to inflammatory stimuli (24), but this effect should enhance rather than attenuate metal absorption. While local upregulation of DMT1 mRNA was observed in iron oxide–instilled rats, overall transport of 54Mn or 59Fe across the air–blood barrier was not enhanced. In bled rats, DMT1 expression in lungs was not different from that in controls, and differences in the clearance of 54Mn and 59Fe from circulation appear to account for the increased blood levels observed in our study. Thus, DMT1 does not seem to be rate limiting for pulmonary clearance of these metals, although it is possible that this transport protein contributes to other lung-associated functions. For example, DMT1 in macrophages could operate at the level of the macrophage phagolysosome. As described by Kreyling and colleagues (25), export of solubilized metal from phagolysosomes might maintain a concentration gradient for dissolution of particles. Thus, increased levels of DMT1 may be related to optimizing particle dissolution rates within phagocytic cells, but have little to do with metal transport across the air–blood barrier. The presence of DMT1 could also help promote iron storage in the pulmonary epithelium. It has been shown that iron oxide inhalation is followed by significant increases in ferritin and hemosiderin within epithelial cells (26). The strong staining of DMT1 in BALT suggests that metals accumulate in this region as well. In summary, iron oxide exposure significantly reduces pulmonary transport of manganese and iron, resulting in decreased uptake by other critical organs. Phlebotomy-induced anemia is also associated with reduced transport of iron from the lungs to the blood, but manganese absorption is unaffected. However, blood clearance of both metals is reduced in phlebotomized animals, leading to increased tissue exposure and greater retention of manganese in the brain and iron in the spleen. Although DMT1 is present in rat and human lungs, it does not appear to be associated with the observed changes in pulmonary absorption of iron and manganese in iron oxide–exposed or bled rats. Since environmental sources of metals are usually found in complex particles, and since chronic exposure to high levels of manganese can lead to neurological disorders, increased knowledge about iron status and the cellular and molecular basis of metal transport may suggest preventive and therapeutic approaches to reduce the toxicity of metals.
The authors thank Krishna Murthy for help in exposure of rats to aerosol of iron oxide particles.
This work was supported by NIH grants ES-000002, (J.D.B), DK60528 (M.W.-R.), DK09998 (M.D.K.), and a gift from the American Welding Society (J.D.B.). The sponsors of this research were not involved in study design, execution, or interpretation of results and were not involved in the decision to submit this paper for publication. Additional support for the interpretation of results and authorship of this publication was made possible by P01 ES012874 from the National Institute of Environmental Health (NIEHS/NIH), and from a STAR Research Assistance Agreement No. RD-83172501 awarded by the U.S. Environmental Protection Agency (EPA). It has not been formally reviewed by either the NIEHS or EPA. The views expressed in this document are solely those of the authors and do not necessarily reflect those of either the NIEHS or the EPA. Neither the NIEHS nor the EPA endorses any products or commercial services mentioned in this publication. Originally Published in Press as DOI: 10.1165/rcmb.2005-0101OC December 9, 2005 Conflict of Interest Statement: J.D.B. received $70,361.50 between 1999 and 2002 from the American Welding Society in the form of a gift to Harvard University. E.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.C.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.D.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.W.-R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 11, 2005 Accepted in final form October 28, 2005
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Abstract
Introduction
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-tubulin, phospholipase A2, ribosomal protein S9, transferrin receptor, and hypoxanthine guanine phosphoribosyl transferase). 
