Grantee Research Project Results
Final Report: Aluminum in Drinking Water Induces Neuronal Apoptosis Via Endoplasmic Reticulum Stress
EPA Grant Number: R829782Title: 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 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 was 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 carried out to study how this neurotoxic effect of Al in drinking water is modulated by silicic acid and fluoride.
Summary/Accomplishments (Outputs/Outcomes):
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 Al, 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); Zn(NO3)3 (38.2 μM).
Study A: High Concentration of Aluminum in Drinking Water
Two groups of 12 rabbits each have been studied with a treatment time of 36 weeks following acclimatization.
Group 1: 12 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: 12 control New Zealand white rabbits (2-3 years of age) were administered control (low Al concentration) drinking water containing 1.5 mM NO3- as NaNO3.
Results and Discussion. It was expected that the most dramatic evidence of toxicity caused by Al treatment would be observed in the group of rabbits treated with drinking water containing a high concentration of Al. However, there is no marked evidence of neuronal death/injury by the increased concentration of Al in the drinking water. Nor is there distinct evidence of endoplasmic reticulum or mitochondrial stress. However, there is some suggestion of increased inflammation by Western blot analysis of NF-κB. This marker is an apoptosis regulatory protein but also an indicator of inflammation. NF-κB is detected as 2 bands of 65 and 50 kDa in the cytosolic fractions of both controls and Al-treated rabbits. The 50 kDa band is barely detected in the nuclear fraction of controls but is more intense in the Al-treated animals, perhaps suggesting some degree of inflammation in the brain tissue studied. However, neuropathological studies reveal no confirmatory evidence of inflammation and, therefore, any suggestion of inflammation is minimal and inconclusive.
The clinical chemistry and hematological studies are most complete in these first two groups of animals. Later groups suffered from problems of clotting of specimens causing obvious problems in the analyses. Certainly, in these groups of animals treated with high and low Al in the drinking water, there is no difference between the two groups. Glucose concentrations are higher in both groups than in normal humans, and calcium is also much higher than equivalent human reference intervals but within the range that we have established for New Zealand white rabbits. Nothing dramatic stands out in any of these plasma studies that we carried out. Some abnormal results were observed but are undoubtedly a result of aging or perhaps other pathological processes unrelated to the drinking water treatment.
No significant differences in Al concentration are found in brain, plasma, liver, kidney, bone, and spleen between the groups of animals treated with drinking water containing high (0.5 mM) and low (3.7 μM) concentrations of Al. This strongly suggests that there is no increase in body burden of Al caused by these concentrations of Al in the drinking water.
Study Group B: Aluminum and Silicic Acid Treatment
Five groups of 12 rabbits each were studied as follows:
Group 1: 12 New Zealand white rabbits (2-3 years of age) were administered drinking water containing 0.5 mM Al + 1.0 mM silicic acid treatment. This group is designated AS1.
Group 2: 12 New Zealand white rabbits (2-3 years of age) were administered drinking water containing 0.5 mM Al + 0.1 mM silicic acid treatment. This group is designated AS0.1.
Group 3: 12 New Zealand white rabbits (2-3 years of age) were administered control (low Al concentration; 3.7 μM) drinking water containing 1.0 mM silicic acid treatment. This group is designated S1.
Group 4: 12 New Zealand white rabbits (2-3 years of age) were administered drinking water containing 0.5 mM Al + 0.25 mM silicic acid treatment. This group is designated AS0.25.
Group 5: 12 control New Zealand white rabbits (2-3 years of age) were administered control (low Al concentration; 3.7 μM) drinking water containing 0.1 mM silicic acid treatment. This group is designated S0.1.
Results and Discussion. As with the first two groups of animals treated with high and low concentrations of Al in the drinking water, in these additional groups, there is no evidence of changes in any of the tissues studied. The slight suggestion by Western blot analysis of NF-κB of inflammation in the high Al-treated group is not evident in the studies of Al and silicic acid in various combinations of concentrations. No suggestion of endoplasmic reticulum stress is found, nor is there any indication of changes in patterns of apoptosis and oxidative stress in any of the groups. The proapoptotic protein, Bax, and the anti-apoptotic protein, Bcl-2, are the same in all groups. Gadd-153, which is a protein suggestive of endoplasmic reticulum stress, is unchanged in all groups in brain tissue. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining for evidence of apoptosis failed to reveal any evidence of excessive apoptosis within any of the five groups of animals, nor was there any increase in staining for 8-hydroxyguanine, a marker of oxidative stress. The clinical chemistry parameters again show no changes within the five groups. Unfortunately, problems with clotting made the hematology studies of little value. However, such studies did not reveal any information in the earlier groups of animals treated with high and low Al concentrations in the drinking water. It is these two groups of rabbits that would have had the best chance of producing abnormal results. The only abnormalityseen is in the evidence of axonal changes in neuropathological studies in the AS0.1, S0.1 and AS0.25 groups which were more evident than in the S1 and AS1 groups. These groups of animals were treated as follows:
AS1 Group: Drinking water containing 0.5 mM Al + 1.0 mM silicic acid treatment.
AS0.1 Group: Drinking water containing 0.5 mM Al + 0.1 mM silicic acid treatment.
S1 Group: Control (low Al concentration; 3.7 μM) drinking water containing 1.0 mM silicic acid treatment.
AS0.25 Group: Drinking water containing 0.5 mM Al + 0.25 mM silicic acid treatment.
S0.1 Group: Control (low Al concentration; 3.7 μM) drinking water containing 0.1 mM silicic acid treatment.
It is of considerable significance that there is evidence of Al accumulation in organs in some treatment groups. As stated above, Al measurements revealed the following significant changes:
Bone: | AS1 > AS0.1 | ||
AS0.25 > AS.1 |
Liver: | S0.1 > S1 |
Spleen: | S0.1 > AS0.25 | |
AS0.1 > AS0.25 | ||
S0.1 > S1 |
Thus, axonal changes in the AS0.1, S0.1, and AS0.25 Groups are consistent with some evidence of Al accumulation is some tissue specimens. The exact mechanism for such Al accumulations is not clear at this time, but it is of interest that treatment with silicic acid, with or without increased Al concentrations in the drinking water, can result in some tissue accumulation of Al and also evidence of axonal changes.
Study Group C: Aluminum and Fluoride Treatment
Four groups of 12 rabbits each were studied as follows:
Group 1: 12 New Zealand white rabbits (2-3 years of age) administered drinking water containing 0.5 mM Al + 0.05 mM fluoride. This group is designated AF.05.
Group 2: 12 New Zealand white rabbits (2-3 years of age) administered control (low Al concentration; 3.7 μM) drinking water containing 0.05 mM fluoride. This group is designated F.05.
Group 3: 12 New Zealand white rabbits (2-3 years of age) administered drinking water containing 0.5 mM Al + 0.5 mM fluoride. This group is designated AF.5.
Group 4: 12 New Zealand white rabbits (2-3 years of age) administered control (low Al concentration; 3.7 μM) drinking water containing 0.5 mM fluoride. This group is designated F.5.
Results and Discussion. As with the first two studies using high and low Al concentrations in the drinking water, and later with most of the animals treated with Al and silicic acid in various combinations, there is no evidence of changes in any of the tissues studied. The slight suggestion by Western blot analysis of NF-κB of inflammation in the high Al-treated group is not evident in these studies of Al and fluoride in various combinations of concentration. No suggestion of endoplasmic reticulum stress is evident, nor is there any indication of changes in patterns of apoptosis and oxidative stress. Bax and Bcl-2 are the same in all groups. Gadd-153 is unchanged in all groups in brain tissue. TUNEL staining failed to reveal any evidence of excessive apoptosis within any of the five groups of animals, nor was there an increase in staining for 8-hydroxyguanine. The clinical chemistry parameters again show no changes within the five groups. Perhaps one change observed is a slight elevation in plasma creatinine in the F.5 and AF.5 groups, which is consistent with some impairment of renal function. No clear evidence of major pathological changes is seen in the tissue studies. Thus, any renal damage observed as a result of the relatively high concentration of fluoride is minor but should be noted. Of significance to this present project, any renal damage is not related to Al in the drinking water.
There are some significant differences between concentrations of Al in bone and kidney as follows:
Bone: | AF0.5 > AF0.05 |
Kidney: | AF0.5 > F0.05 | |
AF0.5 > F0.5 |
Thus, there is some minimal evidence of Al accumulation in tissue but nothing of profound significance that can be correlated with distinct pathological changes.
Overall Impressions and Conclusions
We previously evaluated the long-term oral administration via drinking water of 5 mg/L Al citrate to rabbits (Wills, et al., 1993a; Wills, et al., 1993b; Hewitt, et al., 1992). Calcium and magnesium was added to reproduce hard and soft water and the diet was varied between low and normal calcium content. Animals were sacrificed at 3, 6, 9, and 12 months. Although decreased weight gain was seen, no significant histological changes were found in the central or peripheral nervous systems nor were Al concentrations in brain noted to be elevated by bulk analysis (Wills, et al., 1993b). There was some renal accumulation of Al and occasional hepatic changes were detected (Wills, et al., 1993a), as well as decreases in hematocrit and hemoglobin levels and in red blood cell counts (Hewitt, et al., 1992). It should be emphasized that these experiments were carried out on young adult rabbits with markers of neurotoxicity, which we now know are relatively insensitive. The present study used older (middle-aged) rabbits with more sensitive markers of overall toxicity and lower, more relevant, concentrations of Al in the drinking water.
It was expected that the first two groups of rabbits studied, namely the high drinking water Al and the control, would induce the greatest toxic changes. However, nothing of significance was observed other than perhaps some indication of mild inflammation. No evidence of an increased body burden of Al was observed, and there was no indication of pathological changes that could be attributable to the increased Al in the drinking water. Thus, drinking water with Al concentrations encountered in some municipal water supplies did not cause toxicity in mature rabbits of the middle aged group.
The incorporation of silicic acid into the drinking water did result in an unexplained accumulation of Al into some tissues. This uptake was correlated in some cases with the observation of axonal changes in brain tissue that were similar to what is observed in acute Al toxicity in rabbits (Forbes, et al., 2002). These observations on the effects of silicic acid suggest that further studies are warranted to confirm the findings and to explore the mechanisms of the effects.
The final groups of animals, which were treated with Al and fluoride in various combinations, also failed to demonstrate specific changes that could be attributable to Al. There was some indication of mild inflammation in a few rabbits but no suggestion of endoplasmic reticulum stress. There were some significant differences between concentrations of Al in bone and kidney but nothing of profound significance that could be correlated with distinct pathological changes.
Overall, the present extensive study did not provide any definitive data that Al treatment in drinking water, with and without supplementation with silicic acid or fluoride, induced toxic effects. Admittedly, the duration of the study was 36 weeks and should not be construed in any way as providing firm evidence that Al in drinking water is safe for humans to drink for a lifetime. Thus, for short term exposure, there is no suggestion that Al in drinking water at the concentrations used in many municipal supplies is toxic.
No publications or presentations have been forthcoming so far as a direct result of these studies, primarily because of the long-term nature of the studies. A manuscript focusing on this investigation is in preparation.
References:
Wills MR, Hewitt CD, Sturgill BC, Savory J, Herman MM. Long-term oral or intravenous aluminum administration in rabbits. I. Renal and hepatic changes. Annals of Clinical Laboratory Science 1993a;23:1-16.
Wills MR, Hewitt CD, Savory J, Herman MM. Long-term oral aluminum administration in rabbits. II. Brain and other organs. Annals of Clinical Laboratory Science 1993b;23:17-23.
Hewitt CD, Innes DJ, Herman MM, Savory J, Wills MR. Hematological changes after long-term aluminum administration to normal adult rabbits. Annals of Clinical Laboratory Science 1992;22:85-94.
Forbes MS, Ghribi O, Herman MM, Savory J. Aluminum-induced dendritic pathology revisited: Cytochemical and electron microscopic studies of rabbit cortical pyramidal neurons. Annals of Clinical Laboratory Science 2002;32:75-86.
Supplemental Keywords:
drinking water, risk assessment, exposure, metals, aluminum, fluoride, silicic acid, apoptosis, endoplasmic reticulum,, RFA, Health, Scientific Discipline, Waste, Water, Hydrology, Contaminated Sediments, Environmental Chemistry, Risk Assessments, Environmental Microbiology, Drinking Water, other - exposure, groundwater disinfection, pathogens, monitoring, ecological risk assessment, aquifer characteristics, human health effects, water quality parameters, exposure and effects, exposure, contaminated sediment, aluminum, chemical contaminants, neurotoxicity, drinking water distribution system, treatment, human exposure, apoptosis, water quality, drinking water contaminants, drinking water treatment, water treatment, aluminum toxicokineticsProgress 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.