Grantee Research Project Results

2002 Progress Report: Aluminum in Drinking Water Induces Neuronal Apoptosis Via Endoplasmic Reticulum Stress

EPA Grant Number: R829782
Title: Aluminum in Drinking Water Induces Neuronal Apoptosis Via Endoplasmic Reticulum Stress
Investigators: Savory, John , Boyd, James C. , Herman, Mary M. , Exley, Christopher , Ghribi, Othman
Institution: University of Virginia
EPA Project Officer: Page, Angela
Project Period: August 1, 2002 through July 31, 2005 (Extended to July 31, 2006)
Project Period Covered by this Report: August 1, 2002 through July 31, 2003
Project Amount: $821,249
RFA: Health Effects of Chemical Contaminants in Drinking Water (2001) RFA Text |  Recipients Lists
Research Category: Drinking Water , Human Health , Water

Objective:

The objective of this research project is to test the hypothesis that aluminum (Al) in drinking water can induce neuronal injury, eventually resulting in the death of some neurons via programmed cell death (apoptosis). It also is hypothesized that aging results in increased susceptibility to Al in drinking water. Additional experiments will be conducted to study how this neurotoxic effect of Al in drinking water is modulated by silicic acid and fluoride.

Progress Summary:

The drinking water formulation has been optimized so that precipitation of Al complexes does not occur. The final formulation has the following composition to which has been, or will be added appropriate amounts of aluminum, silicic acid or fluoride depending on the experiment: Al(NO3)3 (3.70 µM); NH4⁺PO4 (11.10 µM); BaNO3 (7.30 µM); CaCl2 (1.85 mM); Ca(NO3)2 (1.85 mM); Cu(NO3)2 (23.6 µM); Fe(NO3)3 (1.8 µM); MgCl2 (1.028 mM); KNO3 (0.154 mM); NaCl (3.260 mM); NaB2O7 (6.40 µM); and Zn(NO3)3 (38.2 mM). The first two groups of animals have been treated and sacrificed, and many of the tissue, blood, and urine analyses have been completed.

Study Group 1. High concentrations of Al were added to drinking water and administered. Two groups of 12 rabbits each have been studied with a treatment time of 36 weeks following acclimatization.

Group 1. Twelve New Zealand white rabbits (2-3 years of age) were administered drinking water containing 0.5 mM Al as Al(NO3)3 added to the optimized formulation described above.

Group 2. Twelve control New Zealand white rabbits (2-3 years of age) were administered control (low Al concentrations) drinking water containing 1.5 mM NO3^- as NaNO3.

Results

Two animals in the high Al drinking water group and one animal in the control group died during the course of the experiment. Otherwise, no adverse clinical problems were observed. The animals slowly gained some weight with no differences between high Al and control drinking water groups.

Pro-caspase-3 was detected in the cytosolic (c) fractions from both control and Al-treated rabbits. The intermediate p20, but not the active p17 or p12, was detected in the nuclear fractions, with high levels in the Al-treated rabbits. Pro-caspase-12 was detected in the endoplasmic reticulum (ER) fractions with high levels in the Al-treated animals; however, there was no presence of the active cleaved form of this indicator of apoptosis. Gadd 153 was present in the cytosolic fractions of controls less than in the Al-treated animals; however, there was no evidence of translocation into the nucleus. Therefore, there was no evidence of increased ER stress in the Al-treated animals as compared to the controls. There also was no evidence of ER-mediated apoptosis between the groups.

Cytochrome c, a mitochondrial apoptosis initiator protein, was restricted to the mitochondrial fractions and was not released into the cytoplasm in both controls and Al-treated rabbits. These results suggest that there is no apparent difference in mitochondrial stress between the two groups and that stress is minimal.

Bcl-2, an apoptosis-regulatory protein, was present in the mitochondrial, ER, and nuclear fractions in levels that are similar in both controls and Al-treated rabbits. Again, this suggests that ER and mitochondrial injury is minimal, with no differnce between the two groups of rabbits.

NF-B, an apoptosis regulatory protein and an indicator of inflammation, was detected as two bands of 65 and 50 kDa in the cytosolic fractions of both control groups and the Al-treated group. The 50 kDa band is barely detected in the nuclear fraction of controls. This 50 kDa band, however, was more intense in the Al-treated animals. This observation may indicate the presence of an increased inflammatory response in the Al-treated group. This finding is being pursued.

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling staining for DNA fragmentation, which is a marker of apoptosis, was performed on the first two groups of rabbits. No apparent differences were observed between the high-Al drinking water group and the control groups.

Al measurements have been performed on drinking water and brain tissue. The drinking water analyses were performed for quality assurance purposes to guarantee that the preparation of the water was of the highest quality. The results of these analyses confirm that the preparation of the drinking water meets the standards necessary for the purposes of the study. The brain tissue analyses did not demonstrate any significant differences between the Al-treated group and the control groups and, thus, there is no evidence of increased brain uptake of Al in the Al-enriched drinking water group.

Clinical chemistry and hematology results on the control group and the Al-enriched drinking water group demonstrate no apparent differences between the first two groups of rabbits.

Preliminary Conclusions

At this time, the first two groups of animals have been treated, sacrificed, and some of the analyses have been performed. The animals were not sacrificed until the end of July 2003, and this time lag has limited our ability to perform all of the studies. At this time, however, there is no marked evidence of neuronal death injury by the increased concentration of Al in the drinking water. There also is no distinct evidence of ER or mitochondrial stress. There is some suggestion of increased inflammation, which has been pursued.

Table 1. Timetable for Future Studies

Experimental Group
Study Group 2. Aluminum and Silicic Acid Treatment	Start	End
1. 0.5 mM Al + 1.0 mM Si treatment	3/31/03	12/8/03
2. 0.5 mM Al + 0.1 mM Si treatment	3/31/03	12/8/03
3. 1.0 mM Si treatment	3/31/03	12/8/03
4. 0.5 mM Al + 0.25 mM Si treatment	9/8/03	5/17/04
5. 0.1 mM Si treatment	9/8/03	5/17/04
Study Group 3. Aluminum and Fluoride Treatment	Start	End
1. 0.5 mM Al + 0.05 mM fluoride treatment	1/12/04	9/20/04
2. 0.5 mM Al + 0.50 mM fluoride treatment	1/12/04	9/20/04
3. 0.05 mM fluoride treatment	1/12/04	9/20/04
4. 0.50 mM fluoride treatment	1/12/04	9/20/04

Future Activities:

Future activities are to study the groups of animals included in the original proposal with the time of sacrifice (see Table 1).

Supplemental Keywords:

aluminum, Al, drinking water, neurotoxic, apoptosis, risk assessment, exposure, metals, fluoride, silicic acid, endoplasmic riticulum., RFA, Health, Scientific Discipline, Water, Waste, Risk Assessments, Environmental Chemistry, Contaminated Sediments, Environmental Microbiology, Hydrology, Drinking Water, other - exposure, aluminum, aluminum toxicokinetics, treatment, contaminated sediment, ecological risk assessment, water quality, water quality parameters, human exposure, chemical contaminants, groundwater disinfection, neurotoxicity, pathogens, monitoring, exposure, drinking water contaminants, drinking water treatment, apoptosis, water treatment, human health effects, human health risk, aquifer characteristics, drinking water distribution system

Progress and Final Reports:

Original Abstract

2003 Progress Report

2004 Progress Report

2005

Final Report