Grantee Research Project Results
2001 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 - 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, 2000 through November 14, 2001
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:
The nephrotoxicity of lead (Pb), cadmium (Cd), and arsenic (As), either alone or in mixtures, is being evaluated both in vivo and in vitro.
In Vivo Studies. 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 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 enzymes in the kidney at sacrifice; and (4) conducting histopathology and electron microscopy studies of kidneys taken at sacrifice for evaluation of necrosis or apoptosis.
In Vitro Studies. 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. The 30-, 90-, and 180-day dose response studies for Pb, Cd, and As (as arsenite) on an individual basis in drinking water of rats have been completed, and the data were statistically analyzed. The results of these studies, based on a number of biological endpoints, yielded a 25 ppm LOEL for Pb, a 10 ppm LOEL for Cd, and a 5 ppm LOEL for As.
Major findings for Pb were dose-related decreases in the activity of a major renal heme pathway enzyme and proteinuria. For Cd, decreased hematocrit values, proteinuria, and interstitial inflammation were the major findings. For As, the LOEL value was determined based on statistically significant decreases in red blood cell zinc protoporphyrin values at the 180-day time point. On the basis of these data, the 30-day Pb x Cd x As interaction study was conducted using the identified individual LOEL dose levels for Pb, Cd, and As, respectively. Data and urinary porphyrin analyses by HPLC for the 30-day Pb x Cd x As study are currently in progress, and rats have been ordered to initiate the 90-day interaction study in January 2002.
In Vitro Studies. Studies conducted during the past year were focused on further examining the in vitro effects of Pb, Cd, and As on alterations in the stress protein response and the relationship of this protective mechanism to cellular toxicity and regulation of the apoptotic pathway. These studies were conducted in both rat NRK-52E and human renal cell lines exposed to As and Cd over a concentration range of 10-7 M to10-4 M and Pb at 10-7 M for 3, 5, 16, or 24 hours. Cellular toxicity was monitored by both the Alamar Blue and Cyquant assays in relation to Western Blot expression studies of the HSP 25/27, HSP 32, HSP 60, HSP70, and HSP 90 stress protein families. Protein tyrosine phosphorylation also was monitored by Western Blot analysis. Apoptosis was monitored in relation to caspase 3 expression, DNA laddering, and the TUNEL assay. Statistically significant synergistic toxic interactions were observed between As and Cd via Hotellings t-square test and regression analyses for the Alamar Blue and Cyquant assays with only a minority of the cells undergoing death via apoptosis. Overall, the various stress protein families exhibited differential responses with either marked induction (HSP 32) or induction followed by inhibition at the highest dose levels (HSP25/27,HSP 60, HSP 70, HSP 90) at the 3- and/or 5-hour time points. Caspase 3 expression was not altered at lower dose levels but inhibited at the highest dose levels, but protein tyrosine phosphorylation was increased at the higher dose levels. These general effects on altered stress protein expression patterns were observed at lower dose levels by combinations of As + Cd and further enhanced in the As x Cd x Pb mixture combination. Overall, results of these studies indicate that combinations of these elements alter the normal stress protein response patterns for a single element as a function of dose and time. These combinations also appear to inhibit the apoptotic pathway, thus leading to cell death from necrosis as a primary mechanism.
Future Activities:
The major effort of our research is now focused on completing the 90- and 180-day Pb x Cd x As interaction studies in rats and completing chemical, biochemical, and statistical analyses on frozen archived tissue and urine samples. In vitro studies will be conducted to the extent possible to examine gender differences in renal tubule cell responses to Pb, Cd, and As.Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 21 publications | 1 publications in selected types | All 1 journal articles |
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Madden EF, Akkerman M, Fowler BA. A comparison of 60, 70, and 90 kDa stress protein expression in normal rat NRK-52 and human HK-2 kidney cell lines following in vitro exposure to arsenite and cadmium alone or in combination. Journal of Biochemistry and Molecular Toxicology 2002;16(1):24-32. |
R827161 (2001) |
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Supplemental Keywords:
chemical mixtures, elemental interactions, 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, 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.