Extended abstract.Linda M Heath, Kathleen L Soole, Michael J McLaughlin, Gordon T McEwan, John W Edward

Edwards, J.

Heath, L.

McEwan, G.

McLaughlin, M.

Soole, K.

Adelaide Research &amp; Scholarship

Is Lead Uptake Through the Immature Intestine Related to the Number of Free Pb2+ Cations in Acidic Solution?   Linda M Heath1, Kathleen L Soole2, Michael J McLaughlin 3, Gordon T McEwan4 John W Edwards1,  1Dept Environmental Health, School of Medicine, Flinders University South Australia, AUSTRALIA Linda.Heath@flinders.edu.au 2School of Biological Sciences, Flinders University, South Australia, AUSTRALIA 3CSIRO Land and Water, Adelaide, South Australia, AUSTRALIA 4Dept Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, UK  INTRODUCTION Children absorb a greater proportion of consumed lead than adults do and this difference may be based on physiological development. The mechanism for this variation has not been adequately described, but it is possible an enhanced uptake of metal via poorly developed intercellular junctions in the immature intestinal tract and changes in the density of transporters in the enterocyte may play a significant role (Pacha 2000). In solution Pb forms complexes with organic and inorganic ligands. The inorganic forms of Pb found in soil (Pb II oxide, Pb II sulfide, Pb II chloride), all have the same biological toxic endpoint but with very different bioavailability (Ballatori 2002).  Lead species are transported into the cell through DMT1 or an H+ active process in the free Pb2+ metal state (Ballatori 2002). Geochem computational calculations indicate that the addition of NTA to 10µM Pb(NO3)2 in NO3- transport medium (pH 5.5), will complex Pb reduce the percentage of free Pb2+ cations in solution. The hypothesis is that Pb is transported through the cell monolayer exclusively in the free Pb2+ state. The complexing of Pb2+ with NTA will further reduce bioavailability.  METHODS A confluent cell culture monolayer was used as a model for the intestinal absorption of lead. The CACO-2 cell line displays a polarised morphology when grown on supporting matrices and expresses a range of functional transport processes similar to those used in the intact human intestine. Cell Culture Cells were cultured at 37 0C in DMEM supplemented with: Glucose 4500mg/L, 2mM L-glutamine, 10% FBS, 1% Pen/strep, 1% NEAA and 5% CO2/95% air atmosphere until reaching 70% confluence. Cells were then harvested using 0.25% trypsin / 0.02% EDTA solution. Harvested cells were seeded onto 0.9cm2 Falcon collagen coated inserts and fed every second day until day 21.  Transepithelial transport study The transepithelial transport experiments were performed on day 21 at 37 °C, in freshly prepared 10 µM Pb(NO3)2 complexed with NTA at concentrations from 1 µM  to 7.4 µM. The Pb/NTA complex was delivered onto the apical compartment of the monolayer at pH 5.5. The basolateral compartment contained modified krebs medium at pH 7.4. The procedure was terminated after 60 minutes by washing the monolayer with ice cold phosphate buffer solution. Cell lysate, apical and basolateral washings were analysed for Pb content using OES and MS Gas Chromatography.    RESULTS AND DISCUSSION The results of this study found (fig 1) 1. A decreasing trend in lead uptake retrieved from the cell lysate with increasing Pb/NTA complexes.  2. The transport of Pb into the basolateral compartment with increasing Pb/NTA complexes was not significantly different.  3. A decrease in the percentage of free Pb2+ cations in solution with an increase in Pb/NTA complexes was observed.  4. The presence of one or more complex Pb species appears to inhibit or impede Pb uptake into the cell and slightly impede transport into the basolateral compartment CONCLUSION A decrease in the specific active uptake of free Pb2+ cations through the CACO-2 cell monolayer was assumed in this study but not proven.. Lead may continue to enter the cell not only as free Pb2+ cations but also as a complex form slowing down the rate of uptake. In regards to bio availability of Pb species further studies are required to confirm exclusivity for free Pb2+ cation transport.   REFERENCES Pacha, J., Development of Intestinal Transport Function in Mammals. Physiological Reviews, 2000. 80(4): p. 1633-1667. Ballatori, N., Transport of Toxic Metals by Molecular Mimicry. Environmental Health Perspectives, 2002. 110 (Supplement 5): p. 689-694. NTA 0 uM NTA 1uM NTA 2.5uM NTA 5uM NTA 7.4uM0510152025BasolaterallysateF i g . 1 . Percentage of Pb retrieved from 10 µM  P b(N0 3)2 w hen com pl exed w i th NTA% Pb retrieved from total applied

Is lead uptake through the immature intestine related to the number of free Pb2+ cations in acidic solution?

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Is lead uptake through the immature intestine related to the number of free Pb2+ cations in acidic solution?

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Adelaide Research & Scholarship

Is lead uptake through the immature intestine related to the number of free Pb2+ cations in acidic solution?

Abstract

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Adelaide Research &amp; Scholarship

Adelaide Research & Scholarship