PharmacologyBtochemtstry&Behavtor,Vol 10, pp 505--512 Prmtedm the U S A
Behavioral Effects of Aluminum Ingestion On
Animal and Human S u b j e c t s 1"2
N O E L L E C. B O W D L E R 3, D E B B I E S. B E A S L E Y , E. C R I S T I N A F R I T Z E ,
A N N M. G O U L E T T E , J A M E S D. H A T T O N , J A M E S H E S S I O N ,
D A V I D L. O S T M A N , D A V I D J. R U G G A N D C H A R L E S J. S C H M I T T D I E L
Michigan State Universtty, Departments o f Psychology and Zoology
East Lansing, MI 48824
( R e c e i v e d 2 J u n e 1978)
BOWDLER, N C , D S BEASLEY, E C FRITZE, A M GOULETTE, J D HATTON, J HESSION, D L
OSTMAN, D J RUGG AND C J SCHMITTDIEL Behavtora/effects of aluminum mgestton on aroma/and human
subjects PHARMAC BIOCHEM BEHAV 10(4)505-512, 1979--Abnormallyhlghbrmnalummumconcentratlonshave
been detected m hemodialysls patients who &ed of an unexplained encephalopathy As a result, this study was undertaken
to examine whether the mgestaon of aluminum produces behavioral aberrations m non-dtalysed human subjects and rats
with ostensibly normal renal function Rats were fed A1CI~by mtubatlon m varying doses, and tests measunng learning
abdlty, v~sual temporal acmty, motor coordination and actlwty were administered It was found that orally ingested
alurmnum Is absorbed by rats and deposited m the brmn High brmn alurmnum levels are assocmted with rapid general
acUwty, decreased abd~ty to mamtmn roto-rod activity, and increased sensmv~ty to fl~cker Behavioral tests were also
given to elderly human subjects and performance correlated with serum aluminum level High serum levels of aluminum in
elderly humans are assocmted with impaired VlSUO-motorcoordination, poor long-term memory, and increased sensmv~ty
to flicker
Alurmnum
Behavior
Toxlc~ty
Antacids
PRIOR research has suggested that aluminum ingestion may
be causally related to various neurological and behavioral
abnormalmes. Several studies of hemo&alysls patients receiving alummum-containlng phosphate-bmdmg gels have
reported patients to develop a progressive form of dementm
which results m death [1, 8, 12]. The dementm is characterized by such clinical symptoms as paranom, confusion,
impaired ability to perform numerical tasks, and delirium
[12]. In one invesUgatlon of patient deaths preceded by
encephalopathy, unusually high levels of aluminum were detected in the patients' muscle, bone and brain tissue [1] An
analysis of cerebral grey matter revealed an aluminum level
of 25 parts per rmllion (ppm) in the dialysis patients with the
encephalopathy, as opposed to 6.5 ppm m &alysls patients
who had died of other disorders. A control group of nondialyzed subjects was found to have a level of 2.2 ppm. From
these results, the authors concluded that the encephalopathy
may have been related to aluminum intoxication.
Studies employing animal subjects found that oral and
parenteral administration of aluminum salts resulted In aluminum intoxicataon characterized by penorbital bleeding,
lethargy, anorexia and death. For both nephrectom~zed and
non-nephrectomized rats given daffy alummum salt treatments, elevated aluminum levels were reported m plasma,
hver, heart, striated muscle, brain and bone [2]. Another
study [14], had shown orally ingested aluminum to be deposited m measurable amounts in the t~ssues of rats, especmlly
when administered as aluminum hydroxide, a major component of several commercially avadable antacids for human
use.
Aluminum ingestion has also been shown to be associated
with neurological and behavioral disorders in normal animal
subjects. A team of investigators [6,7] found aluminum to
reduce neurofibrillary degeneration (NFD) when rejected
into the h~ppocampus, entorhmal cortex, and neocortex of
cats. Another study [10] reported an absence m the cat visual
cortex of neurons with spontaneous finng frequencies between 7 and 12 spikes per second 10 days after hippocampal
injections of A1CI 3 This finding was attributed as an effect of
N F D caused by elevated aluminum concentrations m the
lateral gyrus
The clinical features observed m the cats of these studies
were slmdar to those seen m patients with Alzhelmer's &sease, which ~s characterized by dementia It was noted that
some brain regions m the victims of Alzhe~mer's &sease
contmned rinsed aluminum concentrations, particularly the
regions of the cerebral cortex likely to exhibit NFD [1,9].
That paUents with Alzhelmer's &sease also showed an
1This research was supported by Grant No 77-05159from the National Science Foundation
2Repnnts may be requested m care of Dr James Zacks, Department of Psychology, Michigan State University, East Lansing, M148824
aproJect D~rector
C o p y r i g h t © 1979 A N K H O I n t e r n a t i o n a l Inc.--0091-3057/79/040505-08501.30/0
506
BOWDLER ET AL
alurmnum-related encephalopathy suggests that aluminum
uptake may result from sources other than dialysis therapy
One study [1] reported that an aluminum flake-powder factory worker developed an encephalopathy and was found to
have a brain aluminum level of 20 ppm Since alummum is
found in the enwronment, human diet, and in many commercial antacid preparations, ~t is possible that indiwduals
might mgest enough aluminum dunng the course of their
hves to cause behavioral or neurological impairment
The present study ~s an attempt to investigate the relationship of aluminum intake and behavioral and neurological
functionmg m both human and ammal subjects Therefore,
given the evidence that (a) aluminum is toxic to animals
when administered m large doses, (b) ingested aluminum is
absorbed into the tissues of humans and rats w~th normal
renal function, and (c) behavorlal and neurological mamfestat~ons are assocmted w~th aluminum-reduced NFD, the
present study addressed the hypothesis that: administered
either in high doses, or over considerable periods of time,
aluminum salts wdl produce behavioral manffestaUons m
non-dmlyzed subjects, such as decreased learning ability,
impaired motor coordmatlon, and v~sual aberrations
METHOD
Human Study
SubJects. Ninety-three male and female volunteers between the ages of 56 and 90 years, who were participating m
various senior citizens orgamzatlons in the south-central
Michigan area took part in the study They were ambulatory
and in good general health; none was from a nursing home
population Education levels vaned from partial completion
of grade school to four years of college
Instruments and apparatus Demographic mformatmn
was secured by means of a questionnaire which included
factors such as age, education, occupation, medical history,
antacid consumpUon and other medicines taken regularly
Dosages and lengths of usage of all medications were assessed
The Trail Making Test [19], an indicator of orgamc brain
damage, was employed to measure visual-conceptual and
wsuo-motor tracking The Serial Sevens Test [15] was used
as a test of mental tracking.
Several tests were selected from the Wechsler Adult
Intelligence Scale (WAIS) [20] including the Digit Symbol
Test to assess VlSUO-motor coordination, the Block Design
Test to measure visuo-spatial organization, and the Digit
Span Test an indicator of short term memory
Visual-temporal acuity was measured using an apparatus
for determining crmcal flicker frequency [3] The flicker
stimulus was a 0 5 cm amber light-emitting &ode (LED)
(Monsanto MB5352, with an intensity of 0.4 log foot Lamberts) driven by a square-wave generator (all measurements
of hght intensity were made with a SEI exposure photomoter
which was calibrated against a Spectra brightness standard)
The diode was situated at one end of a 45 7×45 7×40 6 cm
box The box contained a partmon, 10 cm from the face of
the apparatus Holes of diameter 7 9 cm were drilled in the
center of the partIUon and the face of the box The box was
painted fiat black to mlmmize reflection and four 10 watt
incandescent bulbs (with a combined intensity of 0.2 log foot
Lamberts) were placed symmetrically around the viewing
hole on the partition inside th~ box to uniformly ~llummate
the back of the box The frequency of flicker was varied by
means of a dial and push-button system, with fhcker rates
calibrated to range from 27 to 45 Hz
Procedure The subjects were reformed as to the nature of
the study and informed consent was obtained for the behavioral tests and collection of a blood sample The subjects
then completed the questlonnmres
Standard procedures were followed for admimstration of
the Trml Making Test, the Serial Sevens Test, and the three
tests from the Wechsler Adult Intelhgence Scale
Prior to commencing the flicker fusion test, the subjects
fixated on the stimulus for three minutes to adapt to the
amount of light inside the apparatus They were then shown
a stimulus that clearly appeared to be flickering and were
asked to turn the dml controlhng the frequency of flicker
until the light appeared to cease flickering This mmal
threshold frequency was noted and the random doublestmrcase method [5] was then used to determine the flicker
fusion threshold Staircases were commenced at the
threshold level obtained with the adjustment method Subjects made binocular discriminations for 56 trials
A 2 ml sample of blood was then collected by venlpuncture. The serum was later analyzed for aluminum concentratlon by atomic emission spectrometnc techmques [16]
Rat Study
Ammals The animal study was performed m two phases
In Study 1, which was terminated after three weeks because
of deaths in the rat population, 60 rats were utilized. In Study
2, lasting four weeks, 75 rats were employed for testing and
an addmonal 30 rats were used for various control studies
All of the animals were male albino Sprague-Dawley rats and
were 60 days old upon receipt. Rats m various treatment
groups were randomly assigned to group cages The cages
contalnmg animals utihzed for testing were placed in a room
which was malntamed in constant darkness at 25°C There
now exists evidence [4] of retinal deterioration m albino rats
due to prolonged exposure to fluorescent lights Avoiding
this was seen as essential for electroretinogram testing
Apparatus
Open field measure The open field apparatus was patterned after that employed by Price and Huck [18] and conslsted of a circular plywood base, 1 8 m in diameter, with a
sheet metal wall, 46 cm high The base was divided into 24
sectors by five concentric circles of radii 18, 37, 55, 73, and
91 cm, and a senes of radial lines The interior was painted
white with black circular and radial lines The open field was
illuminated by red light, and was housed in a 2 7 by 2 7 m
room at 25°C The movements of each rat were followed on a
video monitor from an adjoining room and were recorded
using stop watches and counters The open field maze was
employed as a measure of general activity
Roto-rod The roto-rod was patterned after an apparatus
used by Overman [17]. The apparatus (length 25 8 era) was
covered with emery cloth to provide traction for the animals
The rod was driven by a 60 cycle AC synchronous motor A
burlap catch net was placed 90 cm below the roto-rod In
Study 1 a drum of diameter 8.4 cm was rotated at 12 revolutions per minute (rpm) and for Study 2 a drum of diameter 5 3
cm was rotated at 26 rpm
Electroretmogram The animals were secured in a
stereotaxlc apparatus equipped with ear pins and b~te bar
ERG's were recorded w~th cotton wick electrodes made
BEHAVIORAL E F F E C T S OF A L U M I N U M
from saline-filled glass pipettes containing coiled chlorided
silver filaments. An amber LED (Monsanto MB5352) with an
average intensity of 1.3 log foot Lamberts served as the flickerlng stimulus and was driven by a variable speed squarewave generator. The rats were monocularly tested, with the
stimulus placed 2 cm from the anmaal's right eye The rate of
flicker ranged between 4 and 40 Hz The responses were
monitored on a Tektronix Type 3A9 oscilloscope, with 3 dB
cutoffs at 0.1 Hz and 300 Hz.
Shuttle box The shuttle box was a 14×97x36 cm wooden
box with a clear Plexlglas top. One half of the reside of the
box was painted black and was covered to prevent light from
entering. The other half of the box was painted white and
illuminated from above A door which could be raised by
means of a pulley separated the black and white areas of the
shuttle box A manually operated 0.5 mamp Grason-Stadtler
shock generator was connected to a wire glad that made up
the floor of the black portion of the box. The box was located
in a darkened room at a temperature of 25°C.
Procedures
Aluminum was administered to rats 7 days a week by intubatlon, using a solution of
AICI3 6H20 m tap water. The daily dose for all rats was
divided between two feeding sessions in an attempt to mcrease absorption of aluminum. To facilitate intubation,
ether was administered p n o r to each feeding, until the rat
lost its righting reflex All rats were weighed every 72 hr and
the dose of AICI3 6H20 was based on the most recent weighing
The 60 Group 1 rats were divided into four groups of 15
which received control, low, medmm, and high doses of 0,
550, 1100, and 1650 nag AICI3-6HzO/kg body weight/day respectively, for 21 days. Each of these rats received a volume
of 28.6 ml/kg/day body weight. The osmolalitles of the solutions for Group 1 were 14, 292, 612, and 939 mOsm/kg with
pH's of 7 21, 3.40, 3.04, and 2.81 for the control, low,
medmm, and high solutions respectively
Doses for 60 of the Group 2 rats (similarly divided into
four groups) were 0, 200, 400, and 600 mg AICI~ 6HzO/kg
body weight/day for 28 days The control rats were fed a
solution with an osmolality of 14 mOsm/kg while rats receiving low, medium, and high doses were fed varying volumes
of one solution having an osmolahty of 231 mOsm/kg Both
solutions had a pH of 7 21 (All pH determinations were
made by Heath EU-302A pH-voltmeter, and osmotic concentratlons were determined by Wescor 5100 B vapor pressure osomoter ) The rats in the low group received a volume
of 11 4 ml/kg body weight/day, whereas the rats in the
medium and high groups received volumes of 22 9 and 34.4
ml/kg body weight/day respectively.
In addition, 16 rats were given a solutmn of a commercial
antacid preparation containing AI(OH)~ (Wyeth-Amphojel),
which had a pH of 7 05. These rats received 195 mg
Al(OH)~/kg body weight/day in a volume of 24 3 ml to yield a
dose of elemental aluminum equivalent to that received by
the high dose animals in Group 2 This experiment was performed in an attempt to assess whether the uptake of aluminum from antacids approximates that of AICI3. These rats
were housed in a constantly lighted room at 25°C for most of
the study Behavioral tests (including the ERG) were administered to these rats after four weeks of Amphojel ingestion. Twenty-four hr before testing these animals were
placed in constant darkness
507
In an attempt to ascertain whether any of the deaths m
Group 1 were due to hyperosmolahty or acidity of the feeding solutaons, a study was initiated in which NaCl of varying
concentrations and acidity were administered to rats Seven
rats were fed a soluUon of NaCl of concentration 891
mOsm/kg and pH 7.21, and seven rats were fed NaCl of 899
mOsm/kg and pH 2 81.
Finally, fifteen rats were added to the high AICl~ dose
group of Group 2 m anticipation of an elevated mortality rate
among these rats
Blood and brain samples Baseline blood samples were
obtained from Group 1 rats by heart puncture At the end of
the stt~dy both Group 1 and Group 2 rats were anesthetized
with ether and decapitated. Blood samples were obtained at
this time The blood was centrifuged and the serum frozen
for later analysis Upon sacrifice, the brains of all rats were
removed and frozen for subsequent analysis of aluminum
content by Atomic Emission Spectrophotometry [16] The
rate of uptake of aluminum into the brain was calculated
according to the equation"
r
Admtmstratlon of alummum
B1
-
where
r=rate of uptake
-
B,,
-
t
B~=mean brain level of aluminum for group i upon sacrifice
B,,=mean control group brain level of aluminum upon sacrlflce
t = t h e length of time which the rats had been fed aluminum
(In days)
Behavtoral tests All behavioral tests except the Shuttlebox test were administered the day before AICI) 6H_,O doslng was commenced, and two and four weeks after the beginning of dosing On the days of testing, the mornmg dosing
of aluminum was eliminated The Open Field and Roto-rod
tests were given on the same day, whereas the Electroretmogram test was administered the following morning.
The Shuttle-box test was administered only once, on the
afternoon of the day before sacrifice
Open field maze The rats were transferred directly from
their home cages to the test apparatus in a 15× 10x 15 cm
handling box which had a shdlng floor The box was placed
in the center of the open field and the shdmg floor removed
After a two minute acclimation period the handhng box was
raised from an adjoining room by a pulley The following
data were recorded over a ten minute period
1. Start time the time taken for the rat to initially move
from the innermost circle after the handhng box was
raised
2 Radial movement" the number of sectors entered m each
of circles two, three, and four (the outer circle)
3 Time, Circle 4" the total time spent in the sectors of the
outermost c~rcle
4 Side-center crossings the number of times the rat moved
between individual circles
5 Inactw~ty the total durauon of periods m which the rat
remained in one sector longer than three seconds.
6 Defecation: the number of fecal boluses deposited during
the 10 minute testing period
7 Total distance total distance traveled in the open field
maze was calculated using the equation
4
4
D = ~ , d~Sl + ~d,c~
I=1
J=l
508
BOWDLER ET AL
where
D=total distance
d,=the arc length connecting the midpoints of the straight
sides of a sector in a given circle
s,=the number of sectors crossed c~rcumferentmlly m a
given c~rcle
dj=the distance connecting the midpoints of the curved sides
of a sector m a given circle
c,=the number of sectors crossed radmlly m a given orcle
Roto-rod The roto-rod test was admimstered after testing
for open field behavior. The rats were transferred to m&vidual cages for the duration of the testing to facdltate handhng
On the first test date, the rats were trained on the rotatmg
rod they were repeatedly placed on the rod untd they had
accumulated one minute of walking time. After training,
actual testing began, with the st~pulaUon that a trial last a
minimum of five sec and a maximum of 15 mm. The length of
t~me until the rat fell from the rotating rod into the catch net
was recorded All ammals were tested sequentmlly before
starting a new trial, and three trmls were averaged
Electrorettnogram After a mmtmum of five hr dark adaptation, each rat was anesthetized w~th sodmm pentobarbltol
(Nembutal, 40 mg/kg) The portion of the pinna occluding the
au&tory meatus was treated with Lldocalne (XylocaaneHCI) and then chpped; ear pros were |nserted, and the rat
was secured m a stereotaxic apparatus Atropine was administered topically to the cornea to dilate the pupil and the
anesthetic Proparicalne HCI (0 5%) (Opthalne) was apphed
to both eyes. The upper eyehds were then sutured and held
open with weights. The recordmg electrode was placed m
one eye and a reference electrode was placed m the other
eye The fhcker rate was increased untd the response was
m&stmgmshable from the 60 cycle background hum Two
addmonal measures were lmmedmtely taken and the average
of the three was recorded After this, the animal was ldent~fied and placed m a recovery cage. Three such trials were
made in the course of the study
Shuttle-box Prior to the first trial, each rat was placed m
the white portion of the box for 45 sec The rat was then
placed m the black end of the box with the base of its tad
touching the end wall. The partmon separating the ends of
the box was rinsed After 10 sec, a shock was delivered to
the grid under the black porUon of the box for 15 sec. If the
rat remained m the black section of the box he was then
placed in the white half for 45 sec with the partmon between
the halves of the box closed If the base of the rat's tail had
crossed into the white end of the box before or dunng the
administration of the shock, the partition was lmmedmtely
lowered and the rat was left m the white end for 45 sec
The rats were tested for 15 trials m a rotation of six rats
The total time which the rat remained in the black portion of
the box was recorded, as was the number of trials undertaken before the rat crossed to the white portion of the box
before being shocked
RESULTS
Human Study
To perform statlsUcal analyses on the data obtained from
the human subjects, groups were formed on the bas~s of
serum aluminum level after all testing had been completed
The 93 subjects were rank-ordered by serum aluminum level,
the 25 highest were chosen as the "high alurmnum" group,
and the 25 lowest were selected as the "low aluminum"
group The mean serum alunnnum level for the high group
was 504 ng/ml, which was signdicantly greater than the low
group mean of 387 ng/ml (p<0 001 by Student's t-test). The
subject's verbal reports of antacid consumption did not correlate with actual serum aluminum levels. Compansons of
age, sex, race, urban versus rural background, education
level, disease, or medicauons taken other than antacids
yielded no significant differences between the two groups
(throughout this report, "not significant" will imply
p >0.05)
Of the tests administered to the human subjects, three
failed to produce s|gnlficant differences between the groups
the Digit Span, Trials A, and Block Design tests. Each of the
other tests successfully differentiated between the groups
the "low aluminum" group performed significantly better on
the Digit Symbol, Trails B, Serial Sevens--Pauses and Serial
Sevens---Errors tests, and demonstrated a lower sensitw~ty
to fl|cker, than did the "bJgh alummum" group. Results of
these tests are summarized in Table 1 Complete data was
not obtained from all subjects. This is reflected in the varying
group numbers hsted in Table 1.
Rat Study-Group 1
Drawing basehne blood samples contributed to the high
rate of rat mortality m this group (see Fig. 1). (The dose
levels included on Figures 1 to 5 refer to the brmn levels
hsted m Table 2 Those figures which hst " C " as the first
point refer to results based on dose groups, whereas those
which hst " V L " refer to comparison groups.) However,
enough rats survived to determine that there were no s|gnificant differences between groups with respect to basehne
serum aluminum level. For the purpose of comparing behavioral test data for these rats, the animals were divided into
four groups by brain aluminum level Therefore, terminal
brain and serum levels of aluminum are shown both by dose
and by analysis group m Table 2 Correlations were found
between terminal serum aluminum levels and dose (r=0 581,
TABLE 1
SIGNIHCANTHUMANTEST RESULTS
Test
*tDlglt Symbol
~) -+ SE
15 05 _+ 0 579 points
13 48 _+0600
r,
p
21
21
<0 05
$Senal Sevens---Pauses
0 43 --- 0 173
2 57 _+ 0 761
14
14
<0 01
§Serial Sevens---Errors
1 375 _ 0 301
2 500 ___0 652
16
15
<0 01
34 80 _+ 0 762 Hz
37 16 + 0616
24
25
<0 01
107 75 -+ 8 390 sec
12500 _+ 8959
20
20
<0 05
tCntlcal Flicker Frequency
tTralls B
*Mean score for "low AI+~'' group given first m each pair
~Analysls by Student's t-test
¢Analysls by Mann-Whitney U-test
§Analysis by Lohrdmg's Test of Two Means
r,=the number of subjects per group
B E H A V I O R A L E F F E C T S OF A L U M I N U M
12-
TABLE 2
TERMINAL BRAINAND SERUMALUMINUMLEVELS
I
1I
.
6
u')
"1I-<~
LLI
509
5
H
C
I
L
M
"~
*Brain AI+
(by dose)
Brmn AI+
(by comparison
group)
Serum AI÷
(by dose)
12• = DISTENTIONS
0
Z
9"
Serum AI+
(by comparison
group)
.
3-
y ± SE
r,~
p
Group 1
H
LL
o
Test
control
low
medmm
Mgh
690
794
929
921
± 18 ng/gt
___ 14
± 24
_ 27
9
9
7
7
<0 001
very low
low
medmm
Mgh
653
758
863
967
±
±
±
±
17 ng/g
5
16
9
6
6
6
6
<0 001
control
low
medium
Mgh
725
734
833
850
±
±
±
±
19 ng/ml
29
47
26
10
7
4
7
<0 005
very low
low
medmm
high
717
728
786
917
~
±
±
±
31 ng/ml
30
23
7
6
6
5
3
<0 005
control
low
medmm
Mgh
688
700
818
820
±
±
_
±
16 ng/g
14
22
31
11
11
9
7
<0 005
very low
low
medmm
Mgh
645
708
768
888
_+
±
±
±
4 ng/g
4
5
7
10
I0
10
10
<0 005
Group 2
Brmn AI+
(by dose)
C
L
M
H
GROUP
FIG 1 Rat mortahty rates prior to sacrifice m studies I and 2, by
dose groups The term "dtstentmns" refers to the number of rats
from Group 2 which dmd and m which distended stomachs were
dtscovered upon autopsy D~stentmns were not recorded for Group
1 rats
p < 0 0 0 1 ) , between serum and brain aluminum levels
(r=0 628, p<0.001), and between brain level and dose
(r=0.829, p < 0 001).
Analysis of the behavmral test data for Group 1 showed
no s~gnfficant differences between groups with respect to
baseline or final administratmn of each test, using one-way
analysis of variance t-tests.
Rat Study--Group 2
Basehne serum aluminum levels were not determined for
Group 2 due to the high rate of mortality resultmg from this
procedure m Group 1. However, there were also deaths in
the Group 2 test population (see Fig. 1). As the only abnormal finding upon autopsy of Group 1 rats dying for reasons
other than heart puncture was the presence of d~stended
stomachs, distensions were noted for Group 2 rats which
dmd Terminal brain aluminum levels are shown by dose and
analysis group in Table 2. In forming the analysis groups, 17
shifts were made from one dose group to another. These are
Brain AI÷
(by comparison
group)
*Analysis by Bonferrom t-test (one-way ANOVA)
tBram aluminum values correspond to wet weight of tissue
er,=the number of subjects per group
evenly distributed between control and low movements, and
medium and high movements. The eight Amphojel-fed rats
included m the analysis groups were also distnbuted relatively evenly across groups. Serum aluminum levels were
not s~gmficantly different across groups when compared by
dose or by analys~s group. Furthermore, terminal serum
aluminum levels were not correlated w~th dose or wtth brain
aluminum levels However, brain aluminum levels were
found to be correlated with dose (r=0 649, p < 0 001).
Analysis of baseline data for all behavioral tests showed
no sigmficant differences between analysis groups for any
test except the ERG (refer to Discussion)
Analysis of final test data using one-way A N O V A Bonferroni t-tests showed no significant differences between
groups on the majority of the tests, including blood
hematocnt and weight gain over the testing period. However, significant differences were seen with respect to the
roto-rod (p<0 001, see Fig 3), and total distance traveled in
510
BOWDLER ET AL
.
,81
C)
= VERY
LOW
ROTO-ROD
= LOW
[]
= MEDIUM
(~)
= HIGH
Z
.
v
47 -I
LU
46LI..
t,
I'-
45-
L)
44.(.9
O
._j
VL
LO
MED
BRAIN
45-
HI
LEVEL
FIG 3 The amount ot time roto-rod activity was maintained m the
last test before sacrifice, by analysts group (mean _+_SE)
4?.-
TOTAL
58-
41
DISTANCE
5440
0
I
?.
TRIAL
FIG 2 Log of cnttcal fhcker frequency (CFF) over time, by
analysis group (mean ± SE) The mterval between trials ts two
weeks The numbers in parentheses refer to the number of rats per
analysts group for a given trial
50-
O3
LU
I-LU
464258
54
the open field maze (p<0 025, see Fig 4). Differences were
also seen with respect to the ERG test between the very low
comparison group and the other three comparison groups
(p<0 05, see the final trial of Fig. 2) Further analysis yielded
a figure showing trends over time for the comparison groups
(The light-housed animals were excluded from these groups
due to lack of data Th~s resulted in basically no change m
the mean brain aluminum concentration of the comparison
groups.) This graph clearly demonstrates the existence of
unexpected significant dLfferences between the very low/low
and the medmm/hlgh group performances m the baseline and
first trmls
Possible explanations for the basehne differences m the
flicker fusion rates of the Group 2 rats were considered One
possibility d~scussed was the age of the rats at the ume of
imtial testing" all rats were 60 days old when recewed from
the supplier, but basehne testing (and the commencement of
AICI~ administration) was staggered over a period of 2
weeks. The mean performance of the rats tested on each day
was compared and no differences were found In addition,
no differences were found between the performance of the
rats tested dunng the first weeks as compared with those
tested dunng the second week This pehnomenon was therefore considered to be a statistical anomaly
The mean brain level for the 15 rats which were mtubated
with Wyeth-Amphojel was 761 ng/g, which was not significantly different from the brain aluminum levels of the
:50.
t
VL
LO
MED
HI
GROUP
FIG 4 Total distance traveled m the open field maze m the last test
before sacnfice, by analysts group (mean -+ SE)
medmm and high dose A1Clrfed rats Likewise, their mean
serum aluminum level, 702 ng/ml was not different from the
serum aluminum levels of all the AICI cfed rats Also, levels
of aluminum in serum and brain were here found to be correlated (r=0 728, p < 0 001). Their performance on the behavioral tests, after four weeks of aluminum lngesUon, did not
differ from that of the AICI 3-fed rats of comparable brmn
aluminum level (It may be unwise to directly compare the
test results for the Amphojel-fed rats with those of the other
test animals as the groups were housed under d~fferent hghtmg condiUons However, the lack of significant differences
on the ERG test between Amphojel-fed ammals and AICI r
fed ammals of comparable brain aluminum levels, despite
these hghtmg differences, seems to support the comparability of the results of the other tests ) There were no
deaths in the Amphojel-fed group during the four weeks of
the study
BEHAVIORAL E F F E C T S OF A L U M I N U M
TABLE 3
RATES OF AL÷aUPTAKEINTO THE BRAIN
Rates
By Dose
By Comparison Group
0
ng/g/day*
49
I14
11 0
0
ng/g/day
32
82
132
Group 1 (t=21d)
Control
Low
Medium
High
Group 2 (t=26d)
Control
Low
Medmm
High
0
0 46
5 00
5 04
H~gh (t= 13d)
5.85
Amphojel (t=26d)
2 81
0
0 77
3 08
7 69
*These values cannot be considered exactly zero as even the control rats were receiving some aluminum in the|r feed and dnnklng
water
Similarly, there were no deaths among the rats fed NaCI
of varying osmolality and pH. It is therefore probable that
neither of these two parameters was the sole cause of rat
mortahty in Group 1.
Finally, analysis of the rates of uptake of aluminum into
the brain for all groups indicates that such uptake is dependent upon the dose adrmnistered and not upon the duration
of admimstration (see Table 3) It is noteworthy that although lndiwdual rates of aluminum uptake mto the brains of
Amphojel-fed rats v a n e d considerably, the mean rate was
lntermedmte between the mean rates for the low and medium
AIClz dose groups
DISCUSSION
The results obtained from the human subject data suggest
that high serum levels of aluminum are associated with impaired complex visuo-motor coordination, poor long-term
memory, and increased sensitivity to flicker. Some of these
findings were anticipated on the basis of previous animal
research, as noted m the introduction. Visuo-spatial organization, simple visuo-motor coordination, and short-term
memory do not appear to be correlated with serum aluminum
level This might be biased by the difficulty of the tests of the
latter parameters, however the Block Demgn test was a difficult task for most of the subjects, resulting m a preponderance of low scores, conversely, the Trails A test was relaavely simple, with the result that most of the subjects
achieved high scores Neither test could therefore be regarded as a good discrimmator
It Is of considerable interest that serum aluminum levels
did not appear to correlate closely with reported ingestion of
ahimlnum-contaming antactds. While it is possible that the
verbal reports of antacid consumpaon are not accurate, ~t
may well be that there are significant alternative sources of
aluminum which have confounded the correlation.
It was found that aluminum, ingested orally in the form of
A1Cla or AI(OH)a, is absorbed by rats with ostensibly normal
511
renal function and deposited in the brain. This supports previous work [2] contradicting the traditional behef that orally
ingested aluminum is almost totally unabsorbed from the gut
[13] Since serum levels of aluminum were correlated with
brain levels of aluminum for Group 1 and Amphojel-fed rats
but not correlated for Group 2 rats, there zs some uncertainty
as to whether serum aluminum concentration is a reliable
indicator of brmn aluminum concentration However, the
fact that there were signLficant positive correlations between
serum and brain aluminum levels m two of the three groups
tends to support the use of serum concentrations of aluminum for the purpose of comparing the behavioral test data of
the human subjects in this study
While it could be argued that the results of the behavioral
tests might be biased by general illness imparted by
stomach-loading aluminum chloride, the lack of significant
differences between analysis groups with respect to weight
gain, hematocnt at sacrifice, and the majonty of the measures of general actlwty indicate that thls is not so It might
also be suggested that these animals suffered from phosphate
depletion, the usual explanation for symptoms assocmted
w~th ahimmum ingestion However, gross indicators of this
condmon, such as low weight gain, rough and greasy coats,
lack of movement, walking with a "cnppled gmt," and watery
eyes [11] were not evident m our test population
The results from the shock-avoidance task suggest that
learning ahihty is not 1raptured by alummum However, the
one-way shock avoidance measure may have been too s~mpie a task to make discriminations between test groups
The results of the open field maze suggest raised excitability due to ahimmum intoxication the ammals with the
higher brain alurmnum levels covered a greater d~stance than
&d the lowest brain aluminum analysis group whde starting
at the same t~me and spending the same amount of time m the
outer sectors This means that the ammals with higher aluminum levels were traveling faster It would be difficult,
therefore, to conclude that gross motor ablhty is directly
lmpmred by aluminum ingestion Assuming that it Is not, the
results of the roto-rod test are also compatible with an excltabdity model greater dlstractabdlty, an oft-mentioned
symptom of hyperactivity in chddren, could easily account
for the decreased abihty of the higher aluminum level rats to
mmntam roto-rod activity
Analysis of the electroretlnogram results from the final
test date revealed slgmficant differences in performance between the very low analysis group and the other three
analysis groups. However, further analysis m&cated that for
this test the basehne performance of the medmm and high
analysis groups differed significantly from that of the low and
very low analysis groups. The discrepancies between the
very low/low and medmm/high group performances for both
the baseline and first test are significant at p < 0 05
We are proposing a model to explain the results obtamed
from the ERG test Figure 5 is a hypothetical extension of
obtained data graphically depicting the model. Th~s model
supposes that aluminum accumulates to a cnUcal level, at
which point it causes C F F to mcrease to an age-determmed
maximum Once the ceding value is attained, the C F F begins
to decrease in accordance with the age trend shown by the
very low group As can be seen m Figure 5, the low brain
aluminum comparison group demonstrates this hypothes~s
very well, as it shifts direction with increasing alummum
concentration Finally, the essentially parallel dechnes of the
high, medium, and very low groups after Trial 1 are also
noteworthy
512
BOWDLER ET AL
0
= VERY
LOW
= LOW
48
47'
[]
= MEDIUM
O
" HIGH
\
\
\
46-
LL
(.3
/
45'
)
44
CO
0
._I
////
The fact that both rat and human subjects wRh high aluminum levels demonstrated increased sensitivity to flicker
strongly indicates that this parameter is affected by aluminum ingestion. In view of this, and prewous research indlcatmg that injections of AICI,~result in neurofibnllary degeneration [ 10], it appears that aluminum lmpmrs aspects of central
nervous system function.
In conclusion, a model supposing that aluminum mcreases excitabdity m rats will account for the results of the
animal portion of this study The human subject results support the work performed on cats and other small mammals m
which neurofibrillary degeneration was reduced. It is especially interesting that both the human subjects with high
serum concentrations of aluminum, and the rats which had
been fed aluminum demonstrated an increased sensitivity to
tinker.
/
ACKNOWLEDGMENT
43
I
J
We would like to acknowledge J L Zacks for his adwce
throughout the course of the study, G I Hatton for providing laboratory facflmes, R W. Hill for his helpful comments about the
study, and G H. Mayor and his laboratory staff for their help with
aluminum deterrmnations and medical asl~cts of the project We
also wish to thank Mary Scott and Betty Simon for the typing of this
manuscript
42
G_/
tt
/
41
/
40
0
I
2
TRIAL
FIG 5 A hypothetical extensmn of the data obtained from the last
electroretmogram test before sacrifice, by analysis group. Data
points and solid lines are from Fig 2, whereas dashed hnes represent
hypothetical continuations between data points
REFERENCES
1 Alfrey, A C., G R LeGendre and W D Kaehny The dtalysls
encephalopathy syndrome----possible alurmnum intoxication
New Engl J Med 294: 184--185, 1976
2 Berlyne, G M., J BenAn, E. Knopf, R Yagel, G Wemberger
and G M. Danovltch Aluminum toxicity in rats Lancet 1:
564-567, 1972
3 Brown, J L Flicker and intermittent stimulation In Vtslon
and Visual Perception, edited by C H Graham New York
John Wiley and Sons, 1965
4 Cicerone, C M. Cones survive rods in the light-damaged eye of
the albino rat Sctence 194: 1183-1185, 1976
5 Cornsweet, T. N The stawcase method in psychophyslcs Am
J Psychol. 75: 485--491, 1962
6 Crupper, D R and A. J. Dalton. Aluminum reduced neurofibrdlary
de~r~raUon, brmn eleemcal ~-tixqty and alterations in
acquisition and retention. Physiol Behav 1O: 935-945, 1973
7 Clapper, D R and A J Dalton Alterations m short-term retention, conditioned avoidance response acqmsttton and motivation following aluminum reduced neurofibrillary degeneration Physiol Behav 10: 925-933, 1973
8. Crupper, D R , S S Knshnan and A. J Dalton Brmn aluminum distributed in Alzhelmer's disease and exponmental
neurofibnllary degeneration. Science 180: 511-513, 1973
9 Clapper, D. R , S S Krishnan and S Quittkat Aluminum,
neurofibnUary degeneration and Alzhelmer's disease Brain 99:
67--80, 1976
10 Crapper, D R and G. J. Tomko Neuronal correlates of an
encephalopathy associated with aluminum neurofibnllary degeneration Brain Res 97: 253-264, 1975
11 Day, H G. and E V McCollum Mineral metabohsm, growth,
and symptomoiogy of rats on a diet extremely deficient m phosphorus J btol Chem 130: 269--283, 1939
12 Dialysis dementia alurmnum again9 Lancet. 1: 349, 1976.
13 Falconer, M W , H. R. Patterson and E. A Gustafson. Current
Drug Handbook. Ptuladelpbaa- W B Sannders, 1976-1978
14 Kaehny, W M., A. P Hegg and A C. Alfr~y Gastrointestinal
absorption from aluminum-contaimng antacids New Engl J
Med 296: 1389-1390, 1977
15 Lezak, M D. NeuropsychotoglcalAssessment. New York Oxford Umversay Press, 1976.
16 Mayor, G. H , J A Kaiser, D Makdam and P K Ku Aluminum absorption and distribution, effect of parathyroid hormone
Science 197: 1187-1189, 1977
17 Overman, S R Behavioral effects of symptomaUc developmental plumblsm in rats Unpublished doctoral dissertation,
Miclugan State University, 1976
18. Price, E O and W. Huck. Open field behavior of wild and
domestic Norway rats Atom Learn Behav 4: 125--130, 1976
t9 Rettan, R M The relatmn of the tralimakang test to organic
brain damage. J consult Psycho/ I9: 393-394, 1955.
20 Wechsler, D. The Measurement o f Adult Intelligence Baltimore Williams and Wilkins, 1955.