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2004 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: R831725C003Subproject: 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 School of Public Health
EPA Project Officer: Fields, Nigel
Project Period: June 1, 2004 through May 31, 2009
Project Period Covered by this Report: June 1, 2004 through May 31, 2005
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2003)
Research Category: Children's Health , Health Effects
Description:
Objective:The objective of this research project is to explore the transport of iron, manganese, cadmium, and lead from environments experienced by children to the blood and critical organs like 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 54Mn in Rats
We finished studying how routes of administration of 54MnCl2 influence the absorbed dose in rats. Equal doses of 54MnCl2 (7.5 μCi/kg) were administered by intratracheal instillation, by gavage, or via intranasal route. Sequential blood samples were taken during a 3-day period of time. At 4 and 72 hours postadministration of 54Mn, all rats were killed humanely, and the radioactivity in various tissues was analyzed. The blood levels of 54Mn over time and of tissues at time of euthanasia differed among the three routes of metal administration. 54Mn accumulation in the brain was highest at 72 hours after intranasal administration. These results suggest that the absorbed dose of 54Mn depends on route of entry and duration. They also suggest that the importance of uptake from the nose and lungs has been underappreciated. These data will be useful in assessing the relative risks for metal toxicity of various exposures to metals.
Figure 1. The Effects of Different Routes of Administration on the Absorption of 54Mn Into the Blood and Tissue Uptake. Figure 1A shows that, for the initial period of 4 hours, 54Mn was absorbed much faster after intranasal and intratracheal instillation compared to ingestion. Absorption from the nose also appeared faster than from the lungs. Figure 1B shows that, although the blood levels were identical from over 4 hours until 3 days (time of euthanasia), 54Mn accumulated most significantly in the brain of intranasally instilled rats.
Dissolution and Clearance of “Chat” Particles in Rats: The Fate of Manganese and Iron
Our hypothesis is that dissolution of metal-containing particles deposited in the lung is a critical step influencing their availability. We began with collected “chat” from piles at Tar Creek. Aliquots from 15 sites were selected randomly and combined into a single composite sample. The composite sample was pulverized using mortar and pestle. We hypothesize that dissolution of chat particles and the presence of multiple metals such as iron and manganese in these particles will affect their pharmacokinetics in rats.
Results—Irradiation of Chat at Rensselaer Polytechnic Institute. We sent the chat particle sample to Gaetnner Linear Accelerator (LINAC) Laboratory at Rensselaer Polytechnic Institute (RPI) in Troy, New York, for photonuclear irradiation. Precise gamma spectrometry was used to analyze different elements rendered radioactive-based on their different gamma energy spectra. The samples were irradiated in a Bremsstrahlung beam for 4 hours. The LINAC was run at about 50 MeV with a current of 90 mA. Measurements of the sample were taken at 2, 3, 7, and 9 days after irradiation with a high-purity Germanium gamma ray detector.
The gamma-energy emitting manganese isotope (54Mn) was produced. The level of 54Mn produced per hour of irradiation, however, was only 0.00013 ng/g of chat. It corresponds to approximately 0.00052 nCi/g chat. This specific activity for 54Mn is insufficient for the proposed experiment. Assuming a minimum dose of 1 mg/kg of chat particles, each rat will receive only approximately 0.25 mg of particles (0.25 mg x 0.00052 nCi/mg = 0.00013 nCi of 54Mn).
Results—Neutron Activation at The Massachusetts Institute of Technology. To understand the competition between manganese and iron present in the fumes during their transport from lung to the rest of the body through blood and other mechanisms, it is useful to have radioisotopes of iron (59Fe) and manganese (54Mn) in the same samples. Therefore, we originally proposed to take the same RPI-irradiated samples, containing 54Mn isotope, and neutron activate them at the Massachusetts Institute of Technology (MIT) Nuclear Reactor Laboratory, Cambridge, Massachusetts. The reactor at MIT generates thermal neutron fluxes of about 6 x 1012 n/cm2sec. Neutron irradiation of samples generates 59Fe with a half-life of 45 days and 56Mn with a half-life of 2.6 hours. The isotope 59Fe emits gamma-energies of 191, 1100, and 1290 KeV, which clearly are different from the gamma-energy emitted by isotope 54Mn (835 KeV). Therefore, we think that identification of 59Fe and 54Mn isotopes in the samples will be feasible and quantification of iron and manganese transport from the animal lungs will be possible with this approach.
The MIT Nuclear Reactor Laboratory used neutron flux of 50 x 1012 n/cm2sec for 12 hours. They waited 12 hours for decay of short half-life radioisotopes before spectral analyses. The results of Neutron Activation Analysis showed that chat particles have an iron concentration of 0.4547 mg/g.
The prospect of neutron activation at MIT as a tool for particle preparation for rat studies was more encouraging than photonuclear activation. Thus, chat particles were neutron activated for 5 days (122.5 hours) with thermal neutron flux of 50 x 1013 cm2/sec. After neutron activation, the samples were allowed to decay for another 5 days before analysis and shipment to the Harvard School of Public Health (HSPH). MIT gamma counting of samples for shipment showed that, in addition to 59Fe and other isotopes, 54Mn also was detectable in the samples although we predicted that 54Mn would not be produced with neutron activation because 100 percent of Mn in nature is 55Mn and predictably will yield 56Mn (a beta emitter with 2.56 hour half-life). The 54Mn produced in the samples may have originated from either (n,p) 54Fe or (n,2n) reaction from 55Mn. 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. Our analyses showed that specific activities of 59Fe and 54Mn may be suitable for rat studies.
Calculations. Assuming a dose of 5 mg/kg of all particles, each rat will receive approximately 1.25 mg of chat particles (1.25 mg x 0.09238 μCi/mg = 0.116 μCi of 59Fe and 0.032 μCi of 54Mn).
Experimental Design—Pharmacokinetic Studies in Rats. Twenty-four 8-week 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 or intranasal administration and 50 mg/kg for gavage. 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. After each rat administration, blood samples were taken sequentially from the tail artery under isoflurane anesthesia. Blood samples were obtained at 5, 15, 30, and 60 minutes after each instillation, and later at 24 hours, 1 week, 2 weeks, 3 weeks, and 1 month. Urine and fecal samples were collected weekly from rats placed in metabolism cages for 24 hours. The rats were killed humanely at various time points after instillation. Two rats for each particle were euthanized at 1 hour, 1 day, 1 week, and 1 month. Additional rats were administered particles four more times over a period of 1 month. At time of euthanasia, 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 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.
RESULTS
Blood Levels of 59Fe or 54Mn from Chat Particles
Figure 2. 59Fe From Neutron-Activated “Chat” Particles was Absorbed in the Blood After Intratracheal (IT), Intranasal (IN), or Gavage. Blood levels of 59Fe were lower than previously observed after IT instillation of soluble 59Fe. Measurable amounts of 59Fe accumulate in various tissues over time. Similar results were observed with 54Mn.
Significance
The results of the 54MnCl2 administration studies suggest that the absorbed dose of 54Mn 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. Our other preliminary results on fate and transport of manganese and iron show differences in absorption, vascular kinetics, and tissue retention of 59Fe 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).
Future Activities:We are benefiting from the interactions within the program. Our data will be correlated with outcomes in both animal (Project 4) and human studies (Project 1). When data from Project 3 are combined with exposure assessment in Project 2, 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:No journal articles submitted with this report: View all 4 publications for this subproject
Supplemental Keywords:children, Native American, tribal, mixtures, lead, PBPK, community, Superfund, intervention, environmental management,
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ENVIRONMENTAL MANAGEMENT, INTERNATIONAL COOPERATION, Scientific Discipline, Waste, Health, RFA, Risk Assessment, Health Risk Assessment, Children's Health, Hazardous Waste, Biochemistry, Environmental Chemistry, Hazardous, iron, neurodevelopmental toxicity, developmental toxicity, fate and transport , children's environmental health, lead, mining wastes, cadmium, human health risk, mining waste, community-based intervention, metal contamination, metal wastes, manganese, metals, Human Health Risk Assessment
Relevant Websites:
http://www.hsph.harvard.edu/niehs/children
Progress and Final Reports:
Original Abstract
2005 Progress Report
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