2005 Progress Report: Manganese, Iron, Cadmium, and Lead Transport from the Environment to Critical Organs During Gestation and Early Development in a Rat Model

EPA Grant Number: R831725C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R831725
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Harvard Center for Children’s Environmental Health and Disease Prevention Research
Center Director: Hu, Howard
Title: Manganese, Iron, Cadmium, and Lead Transport from the Environment to Critical Organs During Gestation and Early Development in a Rat Model
Investigators: Brain, Joseph D. , Molina, Ramon , Wessling-Resnick, Marianne
Institution: Harvard T.H. Chan School of Public Health
EPA Project Officer: Callan, Richard
Project Period: June 1, 2004 through May 31, 2009 (Extended to May 31, 2011)
Project Period Covered by this Report: June 1, 2005 through May 31, 2006
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2003) RFA Text |  Recipients Lists
Research Category: Health , Children's Health , Health Effects

Objective:

The objective of this research project is to explore the transport of iron (Fe), manganese (Mn), cadmium (Cd), and lead (Pb) from environments experienced by children to the blood and critical organs, such as the brain, heart, liver, and kidneys.  We seek to better understand metal exposures of children and their mothers in settings like Tar Creek by:  (1) utilizing exposures during and after pregnancy, (2) using metal ions as well as complex environmental samples from Tar Creek, and (3) comparing different routes of entry from the environment into the body.  We also will explore the role of toxic metals and iron status as they interact to influence metal absorption.

Progress Summary:

Route of Administration Determines Absorption of 59Fe in Rats

Studies on how routes of administration of 59FeCl3 influence the absorbed dose in rats were completed.  Equal doses of 59FeCl3 (10 μCi/kg) were administered by intratracheal instillation, by gavage, via intranasal route, or by intravenous injection.  Sequential blood samples were taken during a 4-hour period.  Four hours later, all rats were killed humanely, and radioactivity in various tissues was analyzed.  The blood levels of 59Fe over time and of tissues at the time of euthanasia differed among the four routes of metal administration.  59Fe accumulation in the brain was highest after intravenous injection and lowest with intranasal administration.  The results suggest that the uptake of 59Fe depends on route of entry.  It also showed that 59Fe was different from 54Mn observed previously.  These data are useful in assessing the relative risks for metal toxicity of various exposures to metals.
Figure 1. Absorption of 59Fe.  (A) For the initial period of 4 hours, 59Fe was absorbed faster and more after intratracheal instillation and gavage compared to intranasal administration.  (B) The blood levels correspond to brain levels with 59Fe accumulating most significantly in the brain of intravenously injected rats and least in intranasally administered rats.

Figure 1.   Absorption of 59Fe.  (A) For the initial period of 4 hours, 59Fe was absorbed faster and more after intratracheal instillation and gavage compared to intranasal administration.  (B) The blood levels correspond to brain levels with 59Fe accumulating most significantly in the brain of intravenously injected rats and least in intranasally administered rats.

Route of Administration and Developmental Stage Determine Absorption of Soluble 54Mn in Rats

We finished studying how routes of administration of 54MnCl2 at different stages in rat development influenced the absorbed dose in rats.  Equal doses of 54MnCl2 (2.5 μCi/kg) were administered to pregnant rats (E2) or their litters by intratracheal instillation, by gavage, via intranasal route, or by intravenous injection every 3.5 days.  The overall exposure design for each route of administration is as follows:  there were five groups designated by numbers 1 through 5 (Figure 2).

     <  Maternal Exposure >
1.  ←-----------------------→ Analysis of pups
          Gestation (21 days)   
     <                 Maternal Exposure                   >
2.  ←----------------------→←----------------------→ Analysis of pups
          Gestation (21 days)                  Lactation (21 days)

                                       <  Maternal Exposure >
3.                                  ←----------------------→ Analysis of pups
                                            Lactation (21 days)
                                                                   <  Weanling Exposure  >
4.                                                               ←-------------------------→ Analysis of pups
                                                                                        Postweaning (21 days)
5.   <                      Maternal Exposure           > <  Weanling Exposure  >
      ←------------------------------------------------ →←-------------------------→ Analysis of pups            
           Gestation (21 days)             Lactation (21 days)          Postweaning (21 days)

Figure 2.  Overall Exposure Design

This exposure design allowed us to compare tissue distribution of 54Mn in individual rat offspring at different endpoints.  The results showed that the absorbed dose of 54Mn depends on route of entry and rat developmental stage.  These data will be useful in assessing the relative risks for metal toxicity of various exposures to metals.

Figure 3 shows selected tissue (brain, heart, and liver) uptake of Mn at birth (E2-1d), at weaning (E2-21d), after exposure throughout the rat life until 42 days (E2-42d), after exposure via milk only (1d-21d), or after exposure of each pup only from 21 to 42 days (21d-42d).  The data show that 54Mn accumulated most significantly in the brain of individual rats instilled into the nose.  The data also show the significant contribution of in utero exposure and exposure through the milk of rat pups.

Dissolution and Clearance of “Chat” Particles in Rats:  The Fate of Zinc, Manganese, and Iron In Rats

Our hypothesis is that dissolution of metal-containing particles deposited in the nose or lungs is a critical step influencing their availability.  We began with collected “chat” from Tar Creek provided by Project 2 (R831725C002).  The sample was size-fractionated by molecular sieving.  The aggregate sample was less than or equal to 37 microns.  We hypothesize that dissolution of chat particles and the presence of multiple metals such as Fe, Zn, and Mn in these particles will affect their pharmacokinetics in rats.  Neutron activation of chat was conducted at the Massachusetts Institute of Technology Nuclear Laboratory using neutron flux of 50 x 1012 n/cm2sec for 120 hours.  Using a Na iodide detector, the specific radioactivities in chat sample were calculated as follows: 
                                                                       
Sample                        59Fe                             65Zn                             54Mn
Chat                             28.9  μCi/g                   827.2  μCi/g                 230.8  μCi/g

It is predicted that 54Mn will not be produced with neutron activation because 100 percent of Mn in nature is 55Mn, and will predictably yield 56Mn (a beta emitter with 2.56 hour half-life).  The 54Mn produced in the chat, however, may have originated from either (n,p) or (n,2n) reaction from 54Fe or 55Mn, respectively.  These reactions are both fast neutron reactions with very small cross sections (reaction probabilities).  Fast neutron contamination occurs in higher flux conditions such as used in this neutron activation.


Figure 3. The Effects of Different Routes of Administration and Developmental Stage on the Absorption of 54Mn Into the Blood and Tissue Uptake.Figure 3. The Effects of Different Routes of Administration and Developmental Stage on the Absorption of 54Mn Into the Blood and Tissue Uptake.
Figure 3. The Effects of Different Routes of Administration and Developmental Stage on the Absorption of 54Mn Into the Blood and Tissue Uptake.Figure 3. The Effects of Different Routes of Administration and Developmental Stage on the Absorption of 54Mn Into the Blood and Tissue Uptake.    

Figure 3.   The Effects of Different Routes of Administration and Developmental Stage on the Absorption of 54Mn Into the Blood and Tissue Uptake.  Top left graph (IN), top right (IT), bottom left (Gavage), and bottom right (IV). 

Experimental Design––Pharmacokinetic Studies in Rats.  Fifteen 23-day-old male CD/Hsd rats were obtained from Harlan Sprague Dawley.  Each rat was weighed, and the dose of each particle was calculated at 5 mg/kg for intratracheal, intranasal, and gavage administration.  Chat particles were suspended in sterile physiologic saline solution at appropriate concentrations.  The particle suspension was dispersed in a bath sonicator for 5 minutes initially, and for 1 minute before each rat administration.  At time of euthanasia (42 days), rats were killed humanely by overdose of isoflurane anesthesia and subsequent exsanguinations via abdominal aorta.  Tissue samples were obtained from various organs, weighed, and analyzed for 59Fe and 54Mn.  Radioactivity was measured in a Packard gamma counter (Cobra Quantum) (Packard Instrument, IL).  Disintegrations per minute were calculated from the counts per minute measured.  All data were expressed as μCi/g tissue.  The percentage of instilled radioactivity dose retained in each organ was calculated.  The organ and tissue weights not measured were estimated (as a percentage of total body weight) as follows:  skeletal muscle, 45 percent; bone marrow, 3 percent; and peripheral blood, 7 percent.

Selected Tissue Levels of 59Fe, 65Zn, or 54Mn After Multiple Chat Administration59Fe, 65Zn, or 54Mn from neutron-activated “chat” particles was absorbed in the blood after intratracheal, intranasal, or gavage and was retained in various tissues.  Figure 4 shows the percentage of administered 59Fe, 65Zn, or 54Mn in chat found in the brain, heart, and liver of rats after multiple (five doses) of chat.  The data show that the levels of radioactive metals from chat in these organs are higher after intratracheal administration compared with intranasal or gavage.  Unlike the results with soluble manganese described above, the brain levels of 54Mn derived from chat were not significantly higher after intranasal compared with intratracheal or gavage.  The graph below shows the relative levels of 54Mn from MnCl2 or chat.  This supports the hypothesis that bioavailability of metals like Mn depends in part on dissolution from particles.  Further analyses of data are being undertaken.

Figure 4. Percentage of Administered 59Fe, 65Zn, or 54Mn in Chat Found in the Brain, Heart, and Liver of Rats.Figure 4. Percentage of Administered 59Fe, 65Zn, or 54Mn in Chat Found in the Brain, Heart, and Liver of Rats.

Figure 4.  Percentage of Administered 59Fe, 65Zn, or 54Mn in Chat Found in the Brain, Heart, and Liver of Rats 

Mn and Fe Absorption From the Lungs and the Influence of Iron Status

Mn transport into the blood can occur via inhalation of metal-containing particles.  Intestinal manganese uptake is mediated by divalent metal transporter 1 (DMT1) and is upregulated by Fe deficiency.  Because iron status varies significantly within the population, and DMT1 may mediate metal absorption from the lungs, we tested the hypothesis that Fe status may alter pulmonary transport of Mn and Fe and explored the potential role of known iron transport proteins in lung metal absorption.  Our studies have established that Mn and Fe are absorbed by the lungs through different mechanistic pathways.  We also have provided the first detailed analysis of the expression pattern and regulation of proteins involved in Fe transport in the lungs.  In-situ analysis detected DMT1 mRNA in airway epithelium, airway macrophages, and bronchus-associated lymphatic tissue (BALT); however, mRNA levels did not change in rats made Fe deficient by diet or by phlebotomy.  In Fe2O3-exposed rats, local increases in DMT1 mRNA transcript were seen in Fe2O3 particle-containing macrophages and adjacent epithelial cells.  These data are consistent with the model that DMT1 levels are regulated differentially in the lungs in response to Fe-containing particles to enhance clearance of the metal from the lungs.  This pattern of regulation was unexpected because upregulation of intestinal DMT1 is promoted by Fe deficiency.

Furtheranalysis of lung DMT1 levels showed that BALT staining was reduced in rats that were fed a high Fe diet.  In-situ analysis of transferrin receptor mRNA also showed staining in BALT, supporting the idea that BALT is an Fe-responsive region that might be responsible for the clearance of metals.  Transferrin mRNA was detected in focal regions of BALT as well; in addition, staining was observed in bronchial epithelium, type II alveolar cells, and macrophages. Because transferrin levels in lung and bronchoalveolar lavage fluid did not change in Fe deficient rats despite increased plasma levels, results of these studies revealed for the first time that the transferrin content in pulmonary fluid is controlled locally by lung transferrin synthesis and that it is regulated in a manner independent of body Fe status.  Consistent with this model, lung Fe2O3 exposure upregulated transferrin mRNA in bronchial and alveolar epithelium, macrophages, and BALT, and a trend toward increased protein was detected in whole lung lysate. The sum of the data are consistent with the view that Fe transport proteins that normally are upregulated by Fe deficiency to enhance intestinal absorption are upregulated in the lungs in response to Fe exposure to promote pulmonary Fe absorption as mechanism for detoxification.

Pharmacokinetic studies have defined the influence of body Fe status on Mn absorption by the lungs.  Uptake of intratracheally instilled 54Mn across the air-blood barrier was characterized in rats made anemic by phlebotomy and diet as well as rats with Fe loading induced by  Fe2O3 exposure and diet.  After instillation, blood levels of 54Mn were higher in anemic animals, and this effect was identified to be attributable to the accumulation of the radioisotope in circulating red blood cells of Fe deficient animals rather than enhanced absorption from the lungs.  In the case of  Fe2O3 exposure, uptake of 54Mn from the lungs was reduced, possibly caused by competitive interactions with ambient Fe released from the oxide particles.  Systemic Fe loading (by diet), however, also reduced manganese uptake from the lungs.  These studies showed that unlike 59Fe, instilled 54Mn is taken up rapidly by the lungs and does not associate with pulmonary fluid transferrin.  Fe loading reduced pulmonary transport of 54Mn from the lungs to the blood, but Fe deficiency had no effect.  To assess the role of DMT1 in lung Mn absorption directly, instillation studies also were carried out with Belgrade rats, an animal model of defective DMT1 function.  The pharmacokinetics of 54Mn absorption by homozygous Belgrade rats and heterozygote littermate controls were identical.  Thus, a major role for DMT1 in pulmonary manganese absorption appears unlikely, although our evidence does support its potential function in the pathway of lung Fe absorption.  At the molecular level, pharmacological studies of 54Mn uptake by A549 type II alveolar epithelial cells indicate that manganese uptake may be associated with activities of both L-type Ca2+ channels and TRPM7, a member of the Transient Receptor Potential Melastatin subfamily.  Thus, our results have delineated mechanistic distinctions between Mn and Fe absorption by the lungs and have revealed a potential role for nonselective Ca channels in lung manganese clearance.

Significance

The results of the 54MnCl2 and 59FeCl3 administration studies suggest that the absorbed dose of metals depends on route of entry and duration and indicate that the importance of uptake from the nose and lungs may be underappreciated.  These data will be useful in assessing the relative risks for metal toxicity of various exposures to metals.  Other preliminary results on fate and transport of Mn and Fe show differences in absorption, vascular kinetics, and tissue retention of 59Fe, 65Zn or 54Mn from irradiated chat administered via different routes in normal rats.  Data from these studies will be used to assist in estimating the relative risks of metal toxicities from different exposures (e.g., eating contaminated food and water, inhaling airborne chat particles, or children playing in contaminated playgrounds).  Our studies on molecular mechanisms of metal transport in both normal and mutant Belgrade rats are beginning to elucidate mechanistic differences between Mn and Fe absorption in the lungs and to explore other potential mechanisms in metal metabolism.

Future Activities:

We are benefiting from the interactions within the Center projects.  Our data will be correlated with outcomes in both animal (Project 4, R831725C004) and human studies (Project 1, R831725C001).  When data from this project are combined with exposure assessment in Project 2 (R831725C002), we will be able to better identify which routes of exposure result in the most significant body burdens of toxic metals.  From this knowledge, we should be able to craft optimal strategies in Tar Creek to reduce the doses of toxic metals to mothers and children and thus better respond to the environmental concerns of the citizens in Tar Creek, Oklahoma.


Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other subproject views: All 12 publications 12 publications in selected types All 12 journal articles
Other center views: All 35 publications 26 publications in selected types All 25 journal articles
Type Citation Sub Project Document Sources
Journal Article Brain JD, Heilig E, Donaghey TC, Knutson MD, Wessling-Resnick M, Molina RM. Effects of iron status on transpulmonary transport and tissue distribution of Mn and Fe. American Journal of Respiratory Cell and Molecular Biology 2006;34(3):330-337. R831725 (2005)
R831725 (2007)
R831725 (2009)
R831725C001 (2007)
R831725C003 (2005)
R831725C003 (2007)
R831725C003 (2008)
R831725C004 (2007)
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  • Journal Article Heilig EA, Thompson KJ, Molina RM, Ivanov AR, Brain JD, Wessling-Resnick M. Manganese and iron transport across pulmonary epithelium. American Journal of Physiology–Lung Cellular and Molecular Physiology 2006;290(6):L1247-L1259. R831725 (2005)
    R831725 (2007)
    R831725 (2009)
    R831725C001 (2007)
    R831725C003 (2005)
    R831725C003 (2007)
    R831725C003 (2008)
    R831725C004 (2007)
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  • Journal Article Heilig E, Molina R, Donaghey T, Brain JD, Wessling-Resnick M. Pharmacokinetics of pulmonary manganese absorption: evidence for increased susceptibility to manganese loading in iron-deficient rats. American Journal of Physiology–Lung Cellular and Molecular Physiology 2005;288(5):L887-L893. R831725 (2007)
    R831725 (2009)
    R831725C001 (2007)
    R831725C003 (2005)
    R831725C003 (2007)
    R831725C004 (2007)
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  • Journal Article Thompson K, Molina R, Donaghey T, Brain JD, Wessling-Resnick M. The influence of high iron diet on rat lung manganese absorption. Toxicology and Applied Pharmacology 2006;210(1-2):17-23. R831725 (2005)
    R831725 (2007)
    R831725 (2009)
    R831725C001 (2007)
    R831725C003 (2005)
    R831725C003 (2007)
    R831725C003 (2008)
    R831725C004 (2007)
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  • Supplemental Keywords:

    children, Native American, tribal, mixtures, lead, PBPK, community, Superfund, intervention, environmental management,, RFA, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, INTERNATIONAL COOPERATION, Waste, Environmental Chemistry, Health Risk Assessment, Biochemistry, Hazardous Waste, Children's Health, Hazardous, Risk Assessment, community-based intervention, fate and transport , developmental toxicity, animal model, lead, Human Health Risk Assessment, neurodevelopmental toxicity, iron, manganese, children's environmental health, cadmium, mining waste, metal wastes, metals, human health risk, metal contamination

    Relevant Websites:

    http://www.hsph.harvard.edu/niehs/children Exit

    Progress and Final Reports:

    Original Abstract
  • 2004 Progress Report
  • 2006
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009
  • Final

  • Main Center Abstract and Reports:

    R831725    Harvard Center for Children’s Environmental Health and Disease Prevention Research

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R831725C001 Metals, Nutrition, and Stress in Child Development
    R831725C002 Exposure Assessment of Children and Metals in Mining Waste: Composition, Environmental Transport, and Exposure Patterns
    R831725C003 Manganese, Iron, Cadmium, and Lead Transport from the Environment to Critical Organs During Gestation and Early Development in a Rat Model
    R831725C004 Metals Neurotoxicity Research Project