Grantee Research Project Results
2000 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, 1999 through November 14, 2000
Project Amount: $832,000
RFA: Chemical Mixtures in Environmental Health (1998) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Human Health , Safer Chemicals , Land and Waste Management
Objective:
The nephrotoxicity of lead (Pb), cadmium (Cd), or arsenic (As) either alone or as mixtures is being evaluated both in vivo and in vitro.Objectives of the in vivo component comprise: (1) establishing dose-response relationships, time-course relationships, and lowest observed effect levels (LOELs) 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 and confirming previously published Pb x Cd x As interaction studies using 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 evaluation of necrosis or apoptosis.
Objectives of the in vitro component comprise the examination of dose-response patterns comparing the LOELs of Pb, Cd, and As alone, or in combination using cultures of renal proximal tubule cells from male or female rats and humans.
Progress Summary:
In Vivo Studies. Animal studies involving male Sprague-Dawley rats are currently underway to identify LOELs for Pb, Cd, and As. Once the range-finding study is completed and LOELs are identified for each metal, a second study is planned in Year 3 that involves administering mixtures of these three metals to male Sprague-Dawley rats at concentrations identified from the range-finding study. The range-finding component of the in vivo study is currently underway, with animals euthanized after 30, 90, or 180 days of test article administration. Animals administered Pb, Cd, or As for 30 days have been euthanized. Kidney, spleen, lung, liver, urine, and blood samples were collected from all euthanized animals.Extensive mortality was observed among rats fed a semipurified diet and ingesting 50 ppm As in drinking water. Within 30 days of test article administration, 13 of the 15 As 50 ppm animals were either found dead or moribund. The exact cause of death is currently being investigated through histopathological and biochemical analyses. Because of the deaths at 50 ppm As, an additional As dose group (5 ppm As) has been added to the range-finding study to accurately identify the LOEL for As toxicity.
Multiple biochemical analyses have been completed on animals euthanized after 30 days of test article administration, including enzymes involved in the heme pathway (e.g., blood and tissue ALAD), hematocrit levels, as well as indicators of kidney toxicity (e.g., urinary creatinine and protein levels). Other biochemical analyses examining changes in heme pathway enzymes and kidney toxicity are ongoing. Histology is currently being performed on kidney sections collected from animals euthanized after 30 days of metal exposure. The range finding study and the metal combination exposures will be concluded in Year 3.
In Vitro Studies. Over the past year, multiple in vitro studies were undertaken to examine differences in the induction of heat shock proteins (HSP) in NRK-52E renal tubule epithelial cells following in vitro exposures to As, Cd, and As x Cd mixtures over concentration ranges from 10-7 M to 10-4 M in Hank's Balanced Salt Solution. The expression of heat shock proteins 32kD, 60kD, 70kD, and 90kD were determined by Western blot analysis in relation to cellular toxicity as monitored by Alamar Blue metabolism and cell density by Cyquant assay. In addition, apoptosis or programmed cell death was determined utilizing electron microscopy, the TUNEL Assay, and DNA laddering. Results of Alamar Blue and Cyquant assays showed the cell line NRK-52E was more sensitive to combined As3+ and Cd2+ toxicity than a single element exposure at lower doses and at earlier time points. Statistical analysis by Hotelling's T-square test indicated a synergistic interaction between As and Cd for these parameters. Further analysis of three species of renal cell lines?rat cell line NRK-52E, hamster cell line HaK, and human cell line HK-2?have shown differences in sensitivity to As, Cd, and its combinations, with human renal cells being the most sensitive, followed by hamster and then rat. Expression of heat shock proteins 32kD, 60kD, 70kD, and 90kD varied considerably as to function element composition, dose, and duration of exposure, indicating there is no common ?switch' that turns on all heat shock proteins. Combined As3+ and Cd2+ mixtures produced greater expression of heat shock proteins at lower doses than single element exposures. Overt cell toxicity/cell death was associated with decreased expression of heat shock proteins. Western blot analysis for tyrosine-phosphorylated proteins showed higher expression with increasing doses of As, Cd, and its combinations, a likely indication of disruption of ATP production and excess Pi in the cell. Analysis by electron microscopy, the TUNEL assay, and DNA laddering of exposed NRK-52E cells indicated that a very small population of cells might undergo apoptosis. Apoptosis is not the major mechanism of cell death of As3+ and Cd2+ toxicity under these conditions. This finding is further verified by Western blot analysis for caspase-3 and HSP 25, where there was little change except for decreased expression associated with overt toxicity/cell death at the highest dose. The mechanisms by which As3+ and Cd2+ induced cellular toxicity under these in vitro conditions appear different, and it is hypothesized that As3+ produces toxicity to cellular mitochondria, while Cd2+ is operating at other sensitive cellular loci.
Future Activities:
The second phase of the in vivo study will commence once LOELs are identified from the range-finding study, and will involve administering mixtures of As, Cd, and Pb to male Sprague-Dawley rats. In vitro investigations will continue to be performed to investigate the toxicity of metal mixtures upon renal tubule epithelial cell lines.Journal Articles:
No journal articles submitted with this report: View all 21 publications for this projectSupplemental Keywords:
chemical mixtures, media, drinking water, risk assessment, mammalian, vulnerability, sensitive populations, gender, cellular, health effects, apoptosis, kidney cell toxicity, in vitro toxicity, in vivo toxicity., RFA, Health, Scientific Discipline, Water, Waste, POLLUTANTS/TOXICS, Health Risk Assessment, Risk Assessments, chemical mixtures, Biology, Environmental Chemistry, Epidemiology, Molecular Biology/Genetics, Arsenic, Biochemistry, cancer risk, Drinking Water, Water Pollutants, bioavailability, carcinogens, occupational safety and health, workplace exposure, lead, nephrotoxic chemicals, environmental toxicants, cadmium, human exposure, monitoring, complex mixtures, epidemiological studies, exposure, cell biology, chemical interactions, effects, kidney damage, 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.