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.