Grantee Research Project Results
1999 Progress Report: Mechanisms of Pb,Cd, As Interactions
EPA Grant Number: R827161Title: Mechanisms of Pb,Cd, As Interactions
Investigators: Fowler, Bruce A.
Institution: University of Maryland - Baltimore
Current Institution: University of Maryland - College Park
EPA Project Officer: Hahn, Intaek
Project Period: November 15, 1998 through November 14, 2001 (Extended to November 14, 2003)
Project Period Covered by this Report: November 15, 1998 through November 14, 1999
Project Amount: $832,000
RFA: Chemical Mixtures in Environmental Health (1998) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Human Health , Land and Waste Management , Safer Chemicals
Objective:
In vivo studies are aimed at: (1) establishing dose-reponse relationships, time-course relationships, and lowest observed effect level (LOEL) values for the development of Pb, Cd, and As porphyrinuria patterns and proteinuria patterns in rats in relation to in vivo studies of renal tubule cell injury; (2) repeating the previously published Pb x Cd x As interaction studies using the established LOEL dose levels and newer analytical methods for speciating the intracellular availability of Pb, Cd, and As and new biomarkers of cell injury; (3) measuring the effects of Pb, Cd, and As on heme pathway enzyme activities in the kidney at sacrifice; and (4) conducting histopathology and electron microscopy studies of kidneys taken at sacrifice for necrosis and apoptosis.In vitro studies are aimed at examining dose-response patterns comparing the LOELs of Pb, Cd, and As alone or in combination on primary cultures of renal proximal tubule cells from rats and humans using cells derived from both males and females.
Progress Summary:
The in vivo studies are scheduled to be initiated in Year 2 and will be reported in the next progress report. An extensive number of in vitro studies have been conducted to characterize and select optimal cell culture/metal exposure systems for establishing LOELs and examining interactions between Pb, Cd, and As when present as mixtures.The toxicity of the metals lead, cadmium, and arsenic was initially evaluated in a rat kidney cell line (NRK-52E) to refine technical parameters. The solutions of metals were made using L-15 medium and Hank's Basic Salt Solution (HBSS). The toxicity was measured over a wide concentration range using the Alamar Blue assay. Two different methods of making lead solutions as lead acetate and as lead glutamate were tested. No toxicity was observed at any concentration of lead tested with either solution. Cadmium was toxic to the cells at 10-4 M, and arsenic was toxic at 4 x 10-5 M. Lead glutamate was demonstrated to increase cell proliferation over 10 days in a dose-dependent manner. There was increased cell proliferation at 10-7, 10-6, and 10-5 M, but no increase at 10-4 M. Rat NRK-52E cells, primary cultures, and human kidney epithelial cells were exposed to Pb (as Pb-glutamate), Cd2+ and As3+ over concentration ranges of 10-7 M to 10-4 M for each element and time periods of 3, 5, 16, and 24 hours. Exposures were conducted in both HBSS and L-15 cell culture media. Cell injury was monitored by morphology, the ALAMAR Blue assay for cellular metabolism, and the cyquant assay for cell density. The TUNEL assay was utilized for assessment of apoptosis. Results of these studies indicated that, using HBSS media, 10-4 M concentrations produced the LOEL with maximal effects for Cd and As at 24 hours, but that the mixture of these two elements produced similar levels of toxicity at 10-6 M indicating an additive/synergistic interaction for these two elements using the ALAMAR Blue and cyquant assays. Replicate studies using L-15 media showed attenuated effects. The TUNEL assay conducted on kidney cells from all three species exposed to Cd2+ and As3+ in HBSS showed that both elements produced apoptosis in all three species, but that As3+ also produced necrosis.
Future Activities:
During the subsequent reporting period, the in vivo animal exposure studies will be initiated, and the in vitro multi-element exposure systems utilizing both male and female derived renal tubule cells will continue to be refined.Journal Articles:
No journal articles submitted with this report: View all 21 publications for this projectSupplemental Keywords:
media, drinking water, risk assessment, mammalian, vulnerability, sensitive populations, gender, cellular, health effects, chemical mixtures, apoptosis, kidney cell toxicity, in vitro toxicity, in vivo toxicity., RFA, Health, Scientific Discipline, Waste, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Health Risk Assessment, Arsenic, Epidemiology, Risk Assessments, chemical mixtures, Biochemistry, Molecular Biology/Genetics, Water Pollutants, Biology, Drinking Water, cancer risk, complex mixtures, chemical interactions, monitoring, kidney damage, exposure and effects, cell biology, bioavailability, lead, exposure, occupational safety and health, nephrotoxic chemicals, effects, carcinogens, human exposure, environmental toxicants, epidemiological studies, cadmium, arsenic exposureProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.