Brain Research, 557 (1991) 280-294 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/9l/$03.50 ,4 DONIS f~h689939116935C
280
BRES 16935
Calcium deficiency enhances cadmium accumulation in the central nervous system Vincent A. Murphy, Everett C. Embrey*, Jack M. Rosenberg, Quentin R. Smith and Stanley I. Rapoport Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) (Accepted 16 April 1991) Key words: Cadmium; Calcium deficient; Brain; Serum; Liver; Kidney; Femur
Weanling male rats were administered 1 of 4 diets for 40 days: control (CONT), low Ca (LOCA), control plus Cd (CONT+Cd) or low Ca plus Cd (LOCA+Cd). After 40 days, Cd was analyzed in 7 brain regions, spinal cord, serum, liver, kidney, muscle and femur by atomic absorption spectrophotometry with Zeeman background correction. No significant difference in Cd between CONT and LOCA was found except in femur, where it was increased. In CONT+Cd rats, peripheral tissues showed an increase in Cd of 30-71 fold above CONT rats. Brain regions exhibited a more modest 7-10 fold change, and serum Cd was 8.5 times above control. LOCA + Cd rats showed a 25-fold increase of Cd above LOCA in serum, 25-100 fold in peripheral tissues, and a 14-20 times increase in brain. These findings show that brain Cd is increased during Ca deficiency, but that central nervous system Cd changes less than peripheral tissue Cd. This increase in brain Cd could alter brain function.
INTRODUCTION A n i m a l studies h a v e s h o w n that C a deficiency enhances C d toxicity and intestinal a b s o r p t i o n 7'1°A4"18"19. A l t h o u g h i n c r e a s e d q u a n t i t i e s o f Cd in p e r i p h e r a l tissue h a v e b e e n d o c u m e n t e d in animals on low C a diet, no i n f o r m a t i o n is a v a i l a b l e on brain Cd. C d can alter levels of n e u r o t r a n s m i t t e r s
in the brain 2, but b e c a u s e brain
u p t a k e of C d is low, the brain is m o r e resistant to C d toxicity than are p e r i p h e r a l tissues 1'8. M e a s u r e m e n t o f b o t h brain and s e r u m C d h a v e not b e e n d o n e in o t h e r studies, but clearly are necessary to d e t e r m i n e h o w the distribution of Cd in brain differs f r o m its distribution
in p e r i p h e r a l
tissues during C a
deficiency. T h e o b j e c t i v e s of this study w e r e to d e t e r m i n e Cd c h a n g e s in b r a i n d u r i n g Ca deficiency, to c o m p a r e t h e s e c h a n g e s with differing levels of Cd i n t a k e , and to e v a l u a t e d i f f e r e n c e s a m o n g tissues in C d distribution. This study analyzes brain, spinal cord, and o t h e r tissues for C d in rats fed e i t h e r low o r control C a diet, with or w i t h o u t Cd. MATERIALS AND METHODS Male Fischer-344 rats (Charles River, Wilmington, MA), 21 days
old and weighing 30-50 g, were fed one of 4 diets (Teklad, Madison, WI) for 40 days: (1) control Ca diet (CONT) - - 7 mg Ca/g; (2) control Ca diet with extra Cd (CONT+Cd) - - CONT diet as above with 20.1 gg/ml Cd(acetate) 2 dihydrate (Fluka Chemical Corp., Ronkonkoma, NY) in drinking water (equals 8.5 gg Cd/ml); (3) low Ca diet (LOCA) - - 0 . 1 mg Ca/g; and (4) low Ca diet with extra Cd (LOCA+Cd) - - low Ca diet as above with Cd acetate in drinking water (see group 2). To control for the effects of decreased food intake in rats fed a LOCA diet, 8 rats were fed 5.5 g of CONT food per day. Half of these rats received Cd in their drinking water (8.5 gg/ml). The animals were housed in a humidity and temperature controlled room with a 12 h light-dark cycle. The quantity of Cd was chosen to minimize toxicity, as Decker et al.6 found that Cd in drinking water up to 10 gg/ml did not alter growth. Cd content of the diets was 6.5 ng/g for CONT and 8.8 ng/g for LOCA animals. Diets were formulated to have equal amounts of chromium, copper, magnesium, manganese, phosphorus, and zinc. Sodium and potassium were elevated in the LOCA diet to replace missing Ca ~2. After 40 days on a diet, a rat was anesthetized with sodium pentobarbital, 30 #g/kg i.p. Blood was collected from the abdominal aorta, placed in polypropylene tubes to clot, and centrifuged at 2000 g to obtain serum. A 250 #1 aliquot of blood was mixed with 5/A Ca heparin (875 units/ml) for measurement of plasma ionized Ca ~a. Samples of liver, kidney, femur, and skeletal muscle were taken after the rat was exsanguinated by severing the aorta. The brain and cervical spinal cord were removed. The brain was dissected into 8 regions: olfactory bulb, cerebral cortex, caudate nucleus, hippocampus, thalamus, midbrain-coiliculi, cerebellum, and pons-medulla following the procedure of Chiueh et al. 4. Serum and tissue samples were frozen on dry ice and stored at -70 °C for later Cd determination. Cd was analyzed using a Zeeman 5100 PC atomic absorption
* Current address: Creighton University Medical School, California at 24th Street, Omaha, NE 68178, U.S.A. Correspondence: V.A. Murphy, Laboratory of Neurosciences, National Institute on Aging, Building 10, Room 6C103, National Institute of Health, Bethesda, MD 20892.
281 spectrophotometer with a H G A 600 graphite furnace and an AS-60 autosampler (Perkin-Elmer, Norwalk, CT). Pyrolytic-coated graphite tubes and L'vov platforms were used. The furnace program is given in Table I. Steps 1-6 were typical for CA, with step 7, air ashing, added to reduce accumulation of charred organic matrix on the platform. Two/A of an aqueous solution of NH4H2PO 4, 20 mg/ml (Aldrich Chemical Co., Milwaukee, WI), was placed onto the L'vov platform as the matrix modifier prior to addition of a 20 #1 aliquot of sample solution or of Cd standard. Standards containing 0.125, 0.25, 0.5, 1, 2, and 4 ng Cd/ml were prepared from a stock standard containing 1 mg Cd/ml (Fluka). Serum was diluted 3 fold with 0.2% (v/v) HNO 3 before furnace analysis. Tissues, 20 to 250 mg, were placed in polypropylene tubes, to which 0.5 mi of concentrated HNO 3 (Baker Ultrex II, Phillipsburg, N J) was added. One part blood was mixed with 2 parts concentrated HNO 3 in similar tubes. Blood and tissue samples were allowed to dissolve overnight at 60 °C, and then tissue samples were diluted to 1.0 ml with 0.2% HNO 3. Further dilution was done if necessary to keep the concentration below the highest standard. Kruskal-Wallis analysis and the Mann-Whitney U-test were used to determine significance between serum and tissue mean Cd among the different diet groups 16. Plasma ionized Ca was compared using one way analysis of variance (ANOVA) and Bonferroni t-statistics. The level of significance was set at P < 0.05.
TABLE I
Description of the graphitefurnace program used in the analysis of Cd Step
1 2 3 4 5 6 7
Temperature (°C)
Time,s Ramp
Hold
10 30 10 1 0 1 1
10 15 30 10 5 6 20
80 160 600 20 1600 2600 600
Glasflow (ml/min)
Gas type
300 300 300 300 0 300 300
argon argon argon argon argon air
lated by adding the intake of each day to the sum of the intakes of prior days. These values were then plotted against days of treatment
t o o b t a i n l i n e a r s l o p e s , so
cumulative intake could be compared
statistically. F o r
diet intake these values (cumulative g diet/g rat per day) w e r e 0.095 + 0.001 ( S . E . ) f o r C O N T , 0 . 0 7 2 + 0.001 f o r L O C A , 0.091 + 0.001 f o r C O N T + C d
RESULTS
for LOCA+Cd. compared
Mean weight gain for each of the 4 diet groups of rats
LOCA
to CONT
a n d 0 . 0 7 0 + 0.001
rats had reduced
rats,
with
extra
diet intake
Cd
having no
influence on diet intake. Water intake values (cumulative
is s h o w n in Fig. 1. T h e t w o g r o u p s f e d c o n t r o l levels o f
ml water/g rat per day) were 0.127 + 0.002 for CONT,
Ca grew to similar weights, whereas the two groups given
0.141 + 0 . 0 0 4 f o r L O C A ,
low Ca diets and the reduced fed animals experienced
a n d 0 . 1 1 6 + 0.003 f o r L O C A + C d .
s i m i l a r , b u t r e t a r d e d g r o w t h . A s l o w e r r a t e o f g r o w t h in
a s i g n i f i c a n t l y g r e a t e r w a t e r i n t a k e ( P < 0.01 b y A N O V A
C a - d e p r i v e d d e v e l o p i n g r a t s h a s b e e n n o t e d p r e v i o u s l y 12.
and Bonferroni
P r o v i d i n g a d d i t i o n a l C d in d r i n k i n g w a t e r d i d n o t c a u s e
Although LOCA
a s i g n i f i c a n t r e d u c t i o n in b o d y w e i g h t .
t-statistics) t h a n
g r o u p s , c u m u l a t i v e d i e t a n d w a t e r i n t a k e s w e r e calcu-
expected
to
alter
LOCA
animals had
the other
3 groups.
rats had about a 20% greater water
intake than LOCA+Cd
From data gathered on diet and water intake in the 4
0.121 + 0 . 0 0 2 f o r C O N T + C d
rats, this change would not be
serum
volume
enough
to
TABLE II
Cd content in ratsfed diets differing in Ca and Cdfor 40 days Values are means + S.E.M. for 5 animals. See Materials and Methods for diets.
Cd content (ng/g)
Serum Olfactory bulb Cerebral cortex Caudate nucleus Hippocampus Thalamus Midbrain-coilic. Cerebellum Pons-Medulla Spinal cord Liver Kidney Muscle Femur Blood
explain
d i f f e r e n c e s in s e r u m C d . T h e t o t a l d o s e o f C d d e t e r m i n e d
CONT
CONT+ Cd
LOCA
L OCA + Cd
0.20 _+0.03 3.1 + 0.6 1.9 _+0.6 1.8 + 0.5 1.4 _+0.3 1.4 _+0.4 1.1 _+0.4 1.4 + 0.6 1.6 + 0.3 1.1 + 0.2 52 + 14 166 + 23 0.41 + 0.10 4.0 + 1.3 0.73 + 0.20
1.7 _+0.2 a 17.4 _+ 1.2a 13.7 + 0.4 a 12.6 + 0.6 a 9.5 + 0.4 a 10.0 + 0.4 ~ 9.3 _+0.4 ~ 11.5 + 0.7 ~ 11.7 + 0.4 a 11.3 + 1.03760 + 450" 8800 + 410" 15.7 + 1.1" 119 + 15a 26.2 + 1.2"
0.40 + 0.09 2.8 _+0.5 1.4 + 0.5 1.2 _+0.4 1.0 _+0.3 1.1 _+0.4 1.0 + 0.3 1.1 + 0.3 1.4 + 0.3 1.3 + 0.5 166 + 60 313 + 50 1.2 + 0.5 10.5 + 1.6" 0.97 + 0.35
9.7 + 0.7 a'b 37.5 _+ 1.2a'b 25.9 _+ 1.5 a'b 26.4 __.2.0 a'b 20.4 _+ 1.4 "'b 21.1 + 0.7 a'b 18.0 + 1.2 a'b 22.2 + 1.4a'b 22.1 + 0.5 ~'b 22.8 + 1.3 ~'b 16700 + 1110" 'b 31240 + 2000- ,b 26.4 + 0.7 a'b 530 + 49 a'b 111 _.+4.9a'b
" Differs from Cd content of CONT o r bCd content of L O C A + C d differs from that of CONT+Cd by Mann-Whitney U-test, P < 0.05.
282 250
skeletal muscle and spinal cord. Different tissues had a CO~K
wide range of Cd content (ng/g), with kidney having the highest value (167 in C O N T and 31300 in L O C A + C d ) in
LOCA 200
•
CONT+Cd
•
LCX]A+C~
all diet treatments and muscle the lowest (0.41 in C O N T and 26 in L O C A + C d ) .
150 O)
Table III shows mean tissue/serum Cd ratios. As brain regions were similar, only cerebral cortex data are
100' n"
presented. The effect of extra Cd was to increase the 50'
01 0
tissue/serum ratio of all tissues except brain and spinal cord. Reduction of dietary Ca resulted in a decrease in 20
10
30
tissue/serum Cd ratios with the exception of liver in animals fed Cd. In Ca-deficient animals not fed addi-
40
D a y s on Diet
Fig. 1. Effect of altered Ca and Cd intake on rat body weight. Rats, 21 days old, were fed normal (7 mg Ca/g) or low Ca (0.1 mg Ca/g) diets with Cd, 8.5/~g/ml, (CONT+Cd or LOCA+Cd) or without Cd (CONT or LOCA) in the drinking water for 40 days. Each point represents the mean weight of 7-9 rats.
from the cumulative diet and water intake was (in/~g/g
tional Cd, only the brain/serum and spinal cord/serum ratios were decreased compared to C O N T rats. Serum and tissue Cd in rats given reduced amounts of C O N T diet with or without extra Cd along with tissue/ serum ratios are shown in Table IV. Values of Cd content in reduced-fed C O N T animals did not differ significantly from values in normal-fed C O N T animals (P > 0.05).
normal-fed animals. After 40 days on the diet, C O N T plasma ionized Ca
Serum and tissue Cd in reduced-fed animals given additional dietary Cd was similar to normal fed C O N T + C d rats with the exceptions of kidney Cd being 30% lower and femur Cd being 140% higher. Generally the tissue/serum ratios in rats after reduced feeding were
was 1.41 + 0.01/zmol/ml for 5 rats, compared to 0.77 _+ 0.04/~mol/ml for L O C A and 1.35 + 0.01 for reduced-fed CONT. The addition of Cd to the diet had no effect on any of these mean values. Blood p H of C O N T animals was 7.44 _ 0.01; p H in the other diet groups was similar.
similar to those in rats after normal feeding, but the liver/serum ratio in reduced-fed C O N T and the femur/ serum ratio in reduced-fed C O N T + C d were increased. All tissues from the reduced-fed C O N T + C d group had lower Cd content than those in the L O C A + C d group.
rat): 0.026 for L O C A , 0.025 for CONT, 41.0 for L O C A + C d , and 41.5 for C O N T + C d . Total dose of Cd in reduced-fed animals was essentially the same as in
Table II lists Cd content for serum, blood, 8 brain regions, spinal cord and 4 peripheral tissues. No significant difference in Cd content was found between L O C A and C O N T in serum, blood or tissue, except in the femur. The groups fed extra Cd had greater serum, blood and tissue Cd than either the L O C A or C O N T groups. Mean Cd content in L O C A + C d rats was 6 times larger than C O N T + C d animals in serum; 4 times larger in liver, kidney, blood and femur; and 2 times larger in brain,
DISCUSSION O u r results show that Cd increases within the nervous system in Cd-supplemented animals and that the level of Cd is critically sensitive to the level of dietary Ca. As compared to rats given a control diet, brain Cd was 5-10 times higher in rats given additional dietary Cd and 10-20 times in Ca-deficient rats given Cd. These results suggest
TABLE III Tissue~serum Cd ratios in rats fed diets differing in Ca and Cd for 40 days
Values are means + S.E.M. for 5 animals. Ratio
Cerebral cortex Spinal cord Liver Skeletal muscle Kidney Femur
CONT
CONT+ Cd
L 0 CA
9.2 + 1.7 5.8 + 0.8 251 + 40 2.0 + 0.2 867 + 75 19.4 + 3.6
8.7 + 1.1 7.1 + 0.9 2350 + 397a 10.0 + 1.5a 5550 + 704a 72.3 + 6.0~
3.1 + 0.6a 3.0 + 0.6a 365 + 86 1.7 + 0.4 880 + 126 23.5 + 3.9
LOCA + Cd
2.7 -t- 0.2a,b 2.4 + 0.2a'b 1744 + 140" 2.8 + 0.1~ 3330 + 438a'~ 55.3 + 5.9a'b
a Differs from ratio of CONT or bratio of LOCA+Cd differs from ratio of CONT+Cd Mann-Whitney U-test, P < 0.05.
283 TABLE IV Cd content and tissue~serum ratios in rats fed reduced amounts o f CO N T diet for 40 days
Values are means + S.E.M. for 4 animals. Tissue~serum ratio
Cd content (ng/g)
Serum Cerebral cortex Spinal cord Liver Skeletal muscle Kidney Femur
CONT
CONT+ Cd
0.23 + 0.01 1.8 + 0.4 0.84 + 0.20 103 + 6.5 0.64 + 0.11 254 + 20 4.2 + 0.4
2.1 + 0.6a'¢ 17.1 + 1.8a'c 9.0 + 1.4ax 5407 + 758a'c 14.7 + 2.8~'c 6400 + 337~-¢ 300 + 43a-c
CONT
8.2 + 2.5 3.8 + 1.1 455 + 37 3.0 + 0.7 1122 + 116 18.2 + 0.2
CO NT+ Cd
9.5 + 1.8c 4.8 + 0.8 2905 + 442~ 7.6 + 0.9a 3719 + 854~ 161 + 26a-¢
a Differs from Cd content or ratio of reduced-fed CONT, bCd content or ratio of reduced-fed CONT+Cd differs from Cd content or ratio of normal-fed CONT+Cd (Tables II and III) and ¢Cd content or ratio of reduced-fed CONT+Cd differs from Cd content or ratio of LOCA+Cd (Tables II and III) by Mann-Whitney U-test, P < 0.05.
that Ca deficiency may increase the risk of Cd neurotoxicity in animals exposed to Cd. Central nervous tissue may be protected from Cd toxicity. After administration of Cd, increases in brain concentrations are small compared to those in kidney and liver 3'8'9'14. Studies using l°9Cd show that entry of Cd into brain is very slow 1'13. However, brain Cd does increase with elevation of the metal in the diet (Table II and ref. 5). Rats given Cd (0.5-1/~g/g per day for 20-60 days) similar to the quantity in our study (1/~g/g per day for 40 days) had altered brain neurotransmitters 2. Abnormal neurological symptoms in workers exposed to Cd and impaired intelligence in children with elevated hair Cd also have been reported 2. In rats fed 100/~g/g for 67 days, Clark et al.5 found the highest Cd content in the olfactory bulb and hippocampus (314 and 275 ng/g) and the lowest in the tegmentum (130 ng/g). Using Z e e m a n background correction, we found in C O N T + C d rats that midbrain-colliculi Cd (9.3 ng/g) was lowest, that olfactory bulb Cd (17 ng/g) was the highest, but that the hippocampus (9.5 ng/g) was among the regions with the lowest Cd content. In L O C A + C d animals, brain regional distribution of Cd was unchanged, even though Cd content was increased 2 fold from C O N T + C d rats. Along with the elevation in central nervous tissue Cd, we found even greater increases in peripheral tissue Cd (Table II). Ca deficiency induced increases found in liver, kidney, and bone Cd agree with previous investigations 1°' 14,18,19. The kidney Cd content in L O C A + C d rats (32 /zg/g) was less than the critical value of Cd (160-400/~g/g) that is reported to produce proteinuria in humans 16. Ca deficiency did not alter tissue Cd when compared to ad libitum or reduced-fed controls, with the exception of femur. In brain, spinal cord, serum and muscle, Cd was near detection limits in all groups not fed Cd leading
to increased error and inability to obtain statistical differences for these tissues. In tissues with significant Cd content (liver, kidney, and femur), the Ca-deficient animals have higher metal contents but this was not significant in liver and kidney. The coefficients of variation of these tissues were decreased at least 50% in reduced-fed animals and in animals fed Cd, suggesting that variations in eating behavior and absorption at trace levels led to increased inconsistency. The ability of brain and muscle to better regulate tissue Ca could explain why increases in brain and muscle Cd were only half those in liver when C O N T + C d and L O C A + C d animals were compared. During Ca deficiency, tissue Ca, corrected for interstitial and blood Ca, is maintained better in brain and skeletal muscle (8593% of control, ref. 11 and unpublished data) than in liver (70% of control, unpublished data). If Cd and Ca compete for tissue binding sites 2, then tissues which retain Ca better during Ca deficiency would have less sites available for Cd, and thus less ability to accumulate Cd. Metallothionein synthesis could explain differences in tissue Cd accumulation. Metallothionein, a major Cd binding protein, is a determinant of tissue Cd TM. It is induced by protein-calorie malnutrition, Ca deficiency or Cd 14'15. A combination of factors ( L O C A + C d ) can induce more metallothionein than any one alone ( C O N T + C d ) . Because dietary Cd induces metallothionein in liver and kidney 14'15, the capacity of these tissues to accumulate Cd is increased. However, metallothionein is not elevated in brain 15, which may explain why the brain/serum ratio remained unchanged, whereas the ratios for the other tissues increased (Table IV). A 3-fold decrease seen in the brain/serum [Cd] ratio, but not in the other tissue/serum ratios between the C O N T and L O C A groups could also be explained by this.
284 A n increase in serum Cd might increase the fraction of Cd available for distribution to tissues, which also would elevate the tissue/serum ratio. A change in the serumfree fraction of Cd also could explain the decrease in
suggests that Cd distribution is n o n s a t u r a b l e from serum [Cd] of 8-30 ng/ml. No data are available from 1 to 10 ng/ml. To summarize,
brain minimizes Cd
accumulation
tissue/serum ratios in L O C A + C d group compared to C O N T + C d group. If Cd and Ca compete for binding
relative to peripheral tissues. Despite this ability, brain Cd does increase with elevated dietary Cd, and neuro-
sites on serum proteins 2, then during low plasma Ca, the free fraction of Cd would be expected to decrease. A
toxicity would be possible with e n v i r o n m e n t a l exposure.
decrease in tissue/serum ratios could also result from a
Ca deficiency further increases body Cd and changes tissue Cd distribution. Differences in Cd distribution
dependence of serum to tissue distribution on serum Cd. Kotsonis and Klaassen 9 found similar linear relations
could be explained by altered tissue [Ca], metallothionein, and Cd tissue and serum binding.
between Cd dose and both serum and tissue Cd, which REFERENCES 1 Arvidson, B. and Tj~ilve, H., Distribution of 1°9Cd in the nervous system of rats after intravenous injection, Acta Neuropathol., 69 (1986) 111-116. 2 Babitch, J.A., Cadmium neurotoxicity. In S.C. Bondy and K.N. Prasad (Eds.), Metal Neurotoxicity, CRC Press, Boca Raton, FL, 1988, pp. 141-166. 3 Cahill, A.L., Nyberg, D. and Ehret, C.E, Tissue distribution of cadmium and metallothionein as a function of time of day and dosage, Environ. Res., 31 (1983) 54-65. 4 Chiueh, C.C., Sun, C.L., Kopin, I.J., Fredericks, W.R. and Rapoport, S.I., Entry of [3H]norepinephrine, [125I]albuminand Evans blue from blood to brain following unilateral osmotic opening of the blood-brain barrier, Brain Research, 145 (1978) 291-301. 5 Clark, D.E., Nation, J.R., Bourgeois, A.J., Hare, M.E, Baker, D.M. and Hinderberger, E.J., The regional distribution of cadmium in the brains of orally exposed adult rats, Neurotoxicology, 6 (1985) 109-114. 6 Decker, L.E., Byerrum, R.U., Decker, C.E, Hoppert, C.A. and Langham, R.E, Chronic toxicity studies I. Cadmium administered in drinking water to rats, AMA Arch. Ind. Health, 18 (1958) 228-231. 7 Hamilton, D.L. and Smith, M.W., Inhibition of intestinal calcium uptake by cadmium and the effect of a low calcium diet on cadmium retention, Environ. Res., 15 (1978) 175-184. 8 Horner, D.B. and Smith, J.C., The distribution of tracer doses of cadmium in the normal rat, Arch. Environ. Contain. Toxicol., 3 (1975) 307-318. 9 Kotsonis, F.N. and Klaassen, C.D., Toxicity and distribution of cadmium administered to rats at sublethal doses, Toxicol. AppL PharmacoL, 41 (1977) 667-680.
10 Larsson, S.-K. and Piscator, M., Effect of cadmium on skeletal tissue in normal and calcium-deficient rats, Israel J. Med. Sci., 7 (1971) 495-498. 11 Murphy, V.A. and Rapoport, S.I., Increased transfer of 45Ca into brain and cerebrospinat fluid from plasma during chronic hypocalcemia in rats, Brain Research, 454 (1988) 315-320. 12 Murphy, V.A., Smith, Q.R. and Rapoport, S.I., Homeostasis of brain and cerebrospinal fluid calcium concentrations during chronic hypo- and hypercalcemia, J. Neurochem., 47 (1986) 1735-1741. 13 Murphy, V.A., Smith, Q.R. and Rapoport, S.I., Rates of tracer cadmium uptake into various tissues, The Pharmacologist, 31 (1989) 137. 14 Nath, R., Prasad, R., Palinal, V.K. and Chopra, R.K., Molecular basis of cadmium toxicity, Prog. Food Nutr. Sci., 8 (1984) 109-163. 15 Onosaka, S., Tanaka, K. and Cherian, M.G., Effects of cadmium and zinc on tissue levels of metallothionein, Environ. Health Perspect. 54 (1984) 76-72. 16 Sachs, L., Applied Statistics: A Handbook of Techniques, 5th edn,, Springer, New York, 1982. 17 Shaikh, Z.A. and Smith, L.M., Biological indicators of cadmium exposure and toxicity. In H. Mislin and O. Ravera (Eds.), Cadmium In The Environment, Birkh~iuser, Boston, pp. 124130. 18 Takashima, M., Nishino, K. and Itokawa, Y., Effect of cadmium administration on growth, excretion, and tissue accumulation of cadmium and histological alterations in calcium-sufficient and -deficient rats: an equalized feeding study, Toxicol. Appl. Pharmacol., 45 (1978) 591-598. 19 Washko, P.W. and Cousins, R.J., Metabolism of ~°9Cdin rats fed normal and low calcium diets, J. Toxicol. Environ. Health, 1 (1976) 1055-1066.
280
BRES 16935
Calcium deficiency enhances cadmium accumulation in the central nervous system Vincent A. Murphy, Everett C. Embrey*, Jack M. Rosenberg, Quentin R. Smith and Stanley I. Rapoport Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) (Accepted 16 April 1991) Key words: Cadmium; Calcium deficient; Brain; Serum; Liver; Kidney; Femur
Weanling male rats were administered 1 of 4 diets for 40 days: control (CONT), low Ca (LOCA), control plus Cd (CONT+Cd) or low Ca plus Cd (LOCA+Cd). After 40 days, Cd was analyzed in 7 brain regions, spinal cord, serum, liver, kidney, muscle and femur by atomic absorption spectrophotometry with Zeeman background correction. No significant difference in Cd between CONT and LOCA was found except in femur, where it was increased. In CONT+Cd rats, peripheral tissues showed an increase in Cd of 30-71 fold above CONT rats. Brain regions exhibited a more modest 7-10 fold change, and serum Cd was 8.5 times above control. LOCA + Cd rats showed a 25-fold increase of Cd above LOCA in serum, 25-100 fold in peripheral tissues, and a 14-20 times increase in brain. These findings show that brain Cd is increased during Ca deficiency, but that central nervous system Cd changes less than peripheral tissue Cd. This increase in brain Cd could alter brain function.
INTRODUCTION A n i m a l studies h a v e s h o w n that C a deficiency enhances C d toxicity and intestinal a b s o r p t i o n 7'1°A4"18"19. A l t h o u g h i n c r e a s e d q u a n t i t i e s o f Cd in p e r i p h e r a l tissue h a v e b e e n d o c u m e n t e d in animals on low C a diet, no i n f o r m a t i o n is a v a i l a b l e on brain Cd. C d can alter levels of n e u r o t r a n s m i t t e r s
in the brain 2, but b e c a u s e brain
u p t a k e of C d is low, the brain is m o r e resistant to C d toxicity than are p e r i p h e r a l tissues 1'8. M e a s u r e m e n t o f b o t h brain and s e r u m C d h a v e not b e e n d o n e in o t h e r studies, but clearly are necessary to d e t e r m i n e h o w the distribution of Cd in brain differs f r o m its distribution
in p e r i p h e r a l
tissues during C a
deficiency. T h e o b j e c t i v e s of this study w e r e to d e t e r m i n e Cd c h a n g e s in b r a i n d u r i n g Ca deficiency, to c o m p a r e t h e s e c h a n g e s with differing levels of Cd i n t a k e , and to e v a l u a t e d i f f e r e n c e s a m o n g tissues in C d distribution. This study analyzes brain, spinal cord, and o t h e r tissues for C d in rats fed e i t h e r low o r control C a diet, with or w i t h o u t Cd. MATERIALS AND METHODS Male Fischer-344 rats (Charles River, Wilmington, MA), 21 days
old and weighing 30-50 g, were fed one of 4 diets (Teklad, Madison, WI) for 40 days: (1) control Ca diet (CONT) - - 7 mg Ca/g; (2) control Ca diet with extra Cd (CONT+Cd) - - CONT diet as above with 20.1 gg/ml Cd(acetate) 2 dihydrate (Fluka Chemical Corp., Ronkonkoma, NY) in drinking water (equals 8.5 gg Cd/ml); (3) low Ca diet (LOCA) - - 0 . 1 mg Ca/g; and (4) low Ca diet with extra Cd (LOCA+Cd) - - low Ca diet as above with Cd acetate in drinking water (see group 2). To control for the effects of decreased food intake in rats fed a LOCA diet, 8 rats were fed 5.5 g of CONT food per day. Half of these rats received Cd in their drinking water (8.5 gg/ml). The animals were housed in a humidity and temperature controlled room with a 12 h light-dark cycle. The quantity of Cd was chosen to minimize toxicity, as Decker et al.6 found that Cd in drinking water up to 10 gg/ml did not alter growth. Cd content of the diets was 6.5 ng/g for CONT and 8.8 ng/g for LOCA animals. Diets were formulated to have equal amounts of chromium, copper, magnesium, manganese, phosphorus, and zinc. Sodium and potassium were elevated in the LOCA diet to replace missing Ca ~2. After 40 days on a diet, a rat was anesthetized with sodium pentobarbital, 30 #g/kg i.p. Blood was collected from the abdominal aorta, placed in polypropylene tubes to clot, and centrifuged at 2000 g to obtain serum. A 250 #1 aliquot of blood was mixed with 5/A Ca heparin (875 units/ml) for measurement of plasma ionized Ca ~a. Samples of liver, kidney, femur, and skeletal muscle were taken after the rat was exsanguinated by severing the aorta. The brain and cervical spinal cord were removed. The brain was dissected into 8 regions: olfactory bulb, cerebral cortex, caudate nucleus, hippocampus, thalamus, midbrain-coiliculi, cerebellum, and pons-medulla following the procedure of Chiueh et al. 4. Serum and tissue samples were frozen on dry ice and stored at -70 °C for later Cd determination. Cd was analyzed using a Zeeman 5100 PC atomic absorption
* Current address: Creighton University Medical School, California at 24th Street, Omaha, NE 68178, U.S.A. Correspondence: V.A. Murphy, Laboratory of Neurosciences, National Institute on Aging, Building 10, Room 6C103, National Institute of Health, Bethesda, MD 20892.
281 spectrophotometer with a H G A 600 graphite furnace and an AS-60 autosampler (Perkin-Elmer, Norwalk, CT). Pyrolytic-coated graphite tubes and L'vov platforms were used. The furnace program is given in Table I. Steps 1-6 were typical for CA, with step 7, air ashing, added to reduce accumulation of charred organic matrix on the platform. Two/A of an aqueous solution of NH4H2PO 4, 20 mg/ml (Aldrich Chemical Co., Milwaukee, WI), was placed onto the L'vov platform as the matrix modifier prior to addition of a 20 #1 aliquot of sample solution or of Cd standard. Standards containing 0.125, 0.25, 0.5, 1, 2, and 4 ng Cd/ml were prepared from a stock standard containing 1 mg Cd/ml (Fluka). Serum was diluted 3 fold with 0.2% (v/v) HNO 3 before furnace analysis. Tissues, 20 to 250 mg, were placed in polypropylene tubes, to which 0.5 mi of concentrated HNO 3 (Baker Ultrex II, Phillipsburg, N J) was added. One part blood was mixed with 2 parts concentrated HNO 3 in similar tubes. Blood and tissue samples were allowed to dissolve overnight at 60 °C, and then tissue samples were diluted to 1.0 ml with 0.2% HNO 3. Further dilution was done if necessary to keep the concentration below the highest standard. Kruskal-Wallis analysis and the Mann-Whitney U-test were used to determine significance between serum and tissue mean Cd among the different diet groups 16. Plasma ionized Ca was compared using one way analysis of variance (ANOVA) and Bonferroni t-statistics. The level of significance was set at P < 0.05.
TABLE I
Description of the graphitefurnace program used in the analysis of Cd Step
1 2 3 4 5 6 7
Temperature (°C)
Time,s Ramp
Hold
10 30 10 1 0 1 1
10 15 30 10 5 6 20
80 160 600 20 1600 2600 600
Glasflow (ml/min)
Gas type
300 300 300 300 0 300 300
argon argon argon argon argon air
lated by adding the intake of each day to the sum of the intakes of prior days. These values were then plotted against days of treatment
t o o b t a i n l i n e a r s l o p e s , so
cumulative intake could be compared
statistically. F o r
diet intake these values (cumulative g diet/g rat per day) w e r e 0.095 + 0.001 ( S . E . ) f o r C O N T , 0 . 0 7 2 + 0.001 f o r L O C A , 0.091 + 0.001 f o r C O N T + C d
RESULTS
for LOCA+Cd. compared
Mean weight gain for each of the 4 diet groups of rats
LOCA
to CONT
a n d 0 . 0 7 0 + 0.001
rats had reduced
rats,
with
extra
diet intake
Cd
having no
influence on diet intake. Water intake values (cumulative
is s h o w n in Fig. 1. T h e t w o g r o u p s f e d c o n t r o l levels o f
ml water/g rat per day) were 0.127 + 0.002 for CONT,
Ca grew to similar weights, whereas the two groups given
0.141 + 0 . 0 0 4 f o r L O C A ,
low Ca diets and the reduced fed animals experienced
a n d 0 . 1 1 6 + 0.003 f o r L O C A + C d .
s i m i l a r , b u t r e t a r d e d g r o w t h . A s l o w e r r a t e o f g r o w t h in
a s i g n i f i c a n t l y g r e a t e r w a t e r i n t a k e ( P < 0.01 b y A N O V A
C a - d e p r i v e d d e v e l o p i n g r a t s h a s b e e n n o t e d p r e v i o u s l y 12.
and Bonferroni
P r o v i d i n g a d d i t i o n a l C d in d r i n k i n g w a t e r d i d n o t c a u s e
Although LOCA
a s i g n i f i c a n t r e d u c t i o n in b o d y w e i g h t .
t-statistics) t h a n
g r o u p s , c u m u l a t i v e d i e t a n d w a t e r i n t a k e s w e r e calcu-
expected
to
alter
LOCA
animals had
the other
3 groups.
rats had about a 20% greater water
intake than LOCA+Cd
From data gathered on diet and water intake in the 4
0.121 + 0 . 0 0 2 f o r C O N T + C d
rats, this change would not be
serum
volume
enough
to
TABLE II
Cd content in ratsfed diets differing in Ca and Cdfor 40 days Values are means + S.E.M. for 5 animals. See Materials and Methods for diets.
Cd content (ng/g)
Serum Olfactory bulb Cerebral cortex Caudate nucleus Hippocampus Thalamus Midbrain-coilic. Cerebellum Pons-Medulla Spinal cord Liver Kidney Muscle Femur Blood
explain
d i f f e r e n c e s in s e r u m C d . T h e t o t a l d o s e o f C d d e t e r m i n e d
CONT
CONT+ Cd
LOCA
L OCA + Cd
0.20 _+0.03 3.1 + 0.6 1.9 _+0.6 1.8 + 0.5 1.4 _+0.3 1.4 _+0.4 1.1 _+0.4 1.4 + 0.6 1.6 + 0.3 1.1 + 0.2 52 + 14 166 + 23 0.41 + 0.10 4.0 + 1.3 0.73 + 0.20
1.7 _+0.2 a 17.4 _+ 1.2a 13.7 + 0.4 a 12.6 + 0.6 a 9.5 + 0.4 a 10.0 + 0.4 ~ 9.3 _+0.4 ~ 11.5 + 0.7 ~ 11.7 + 0.4 a 11.3 + 1.03760 + 450" 8800 + 410" 15.7 + 1.1" 119 + 15a 26.2 + 1.2"
0.40 + 0.09 2.8 _+0.5 1.4 + 0.5 1.2 _+0.4 1.0 _+0.3 1.1 _+0.4 1.0 + 0.3 1.1 + 0.3 1.4 + 0.3 1.3 + 0.5 166 + 60 313 + 50 1.2 + 0.5 10.5 + 1.6" 0.97 + 0.35
9.7 + 0.7 a'b 37.5 _+ 1.2a'b 25.9 _+ 1.5 a'b 26.4 __.2.0 a'b 20.4 _+ 1.4 "'b 21.1 + 0.7 a'b 18.0 + 1.2 a'b 22.2 + 1.4a'b 22.1 + 0.5 ~'b 22.8 + 1.3 ~'b 16700 + 1110" 'b 31240 + 2000- ,b 26.4 + 0.7 a'b 530 + 49 a'b 111 _.+4.9a'b
" Differs from Cd content of CONT o r bCd content of L O C A + C d differs from that of CONT+Cd by Mann-Whitney U-test, P < 0.05.
282 250
skeletal muscle and spinal cord. Different tissues had a CO~K
wide range of Cd content (ng/g), with kidney having the highest value (167 in C O N T and 31300 in L O C A + C d ) in
LOCA 200
•
CONT+Cd
•
LCX]A+C~
all diet treatments and muscle the lowest (0.41 in C O N T and 26 in L O C A + C d ) .
150 O)
Table III shows mean tissue/serum Cd ratios. As brain regions were similar, only cerebral cortex data are
100' n"
presented. The effect of extra Cd was to increase the 50'
01 0
tissue/serum ratio of all tissues except brain and spinal cord. Reduction of dietary Ca resulted in a decrease in 20
10
30
tissue/serum Cd ratios with the exception of liver in animals fed Cd. In Ca-deficient animals not fed addi-
40
D a y s on Diet
Fig. 1. Effect of altered Ca and Cd intake on rat body weight. Rats, 21 days old, were fed normal (7 mg Ca/g) or low Ca (0.1 mg Ca/g) diets with Cd, 8.5/~g/ml, (CONT+Cd or LOCA+Cd) or without Cd (CONT or LOCA) in the drinking water for 40 days. Each point represents the mean weight of 7-9 rats.
from the cumulative diet and water intake was (in/~g/g
tional Cd, only the brain/serum and spinal cord/serum ratios were decreased compared to C O N T rats. Serum and tissue Cd in rats given reduced amounts of C O N T diet with or without extra Cd along with tissue/ serum ratios are shown in Table IV. Values of Cd content in reduced-fed C O N T animals did not differ significantly from values in normal-fed C O N T animals (P > 0.05).
normal-fed animals. After 40 days on the diet, C O N T plasma ionized Ca
Serum and tissue Cd in reduced-fed animals given additional dietary Cd was similar to normal fed C O N T + C d rats with the exceptions of kidney Cd being 30% lower and femur Cd being 140% higher. Generally the tissue/serum ratios in rats after reduced feeding were
was 1.41 + 0.01/zmol/ml for 5 rats, compared to 0.77 _+ 0.04/~mol/ml for L O C A and 1.35 + 0.01 for reduced-fed CONT. The addition of Cd to the diet had no effect on any of these mean values. Blood p H of C O N T animals was 7.44 _ 0.01; p H in the other diet groups was similar.
similar to those in rats after normal feeding, but the liver/serum ratio in reduced-fed C O N T and the femur/ serum ratio in reduced-fed C O N T + C d were increased. All tissues from the reduced-fed C O N T + C d group had lower Cd content than those in the L O C A + C d group.
rat): 0.026 for L O C A , 0.025 for CONT, 41.0 for L O C A + C d , and 41.5 for C O N T + C d . Total dose of Cd in reduced-fed animals was essentially the same as in
Table II lists Cd content for serum, blood, 8 brain regions, spinal cord and 4 peripheral tissues. No significant difference in Cd content was found between L O C A and C O N T in serum, blood or tissue, except in the femur. The groups fed extra Cd had greater serum, blood and tissue Cd than either the L O C A or C O N T groups. Mean Cd content in L O C A + C d rats was 6 times larger than C O N T + C d animals in serum; 4 times larger in liver, kidney, blood and femur; and 2 times larger in brain,
DISCUSSION O u r results show that Cd increases within the nervous system in Cd-supplemented animals and that the level of Cd is critically sensitive to the level of dietary Ca. As compared to rats given a control diet, brain Cd was 5-10 times higher in rats given additional dietary Cd and 10-20 times in Ca-deficient rats given Cd. These results suggest
TABLE III Tissue~serum Cd ratios in rats fed diets differing in Ca and Cd for 40 days
Values are means + S.E.M. for 5 animals. Ratio
Cerebral cortex Spinal cord Liver Skeletal muscle Kidney Femur
CONT
CONT+ Cd
L 0 CA
9.2 + 1.7 5.8 + 0.8 251 + 40 2.0 + 0.2 867 + 75 19.4 + 3.6
8.7 + 1.1 7.1 + 0.9 2350 + 397a 10.0 + 1.5a 5550 + 704a 72.3 + 6.0~
3.1 + 0.6a 3.0 + 0.6a 365 + 86 1.7 + 0.4 880 + 126 23.5 + 3.9
LOCA + Cd
2.7 -t- 0.2a,b 2.4 + 0.2a'b 1744 + 140" 2.8 + 0.1~ 3330 + 438a'~ 55.3 + 5.9a'b
a Differs from ratio of CONT or bratio of LOCA+Cd differs from ratio of CONT+Cd Mann-Whitney U-test, P < 0.05.
283 TABLE IV Cd content and tissue~serum ratios in rats fed reduced amounts o f CO N T diet for 40 days
Values are means + S.E.M. for 4 animals. Tissue~serum ratio
Cd content (ng/g)
Serum Cerebral cortex Spinal cord Liver Skeletal muscle Kidney Femur
CONT
CONT+ Cd
0.23 + 0.01 1.8 + 0.4 0.84 + 0.20 103 + 6.5 0.64 + 0.11 254 + 20 4.2 + 0.4
2.1 + 0.6a'¢ 17.1 + 1.8a'c 9.0 + 1.4ax 5407 + 758a'c 14.7 + 2.8~'c 6400 + 337~-¢ 300 + 43a-c
CONT
8.2 + 2.5 3.8 + 1.1 455 + 37 3.0 + 0.7 1122 + 116 18.2 + 0.2
CO NT+ Cd
9.5 + 1.8c 4.8 + 0.8 2905 + 442~ 7.6 + 0.9a 3719 + 854~ 161 + 26a-¢
a Differs from Cd content or ratio of reduced-fed CONT, bCd content or ratio of reduced-fed CONT+Cd differs from Cd content or ratio of normal-fed CONT+Cd (Tables II and III) and ¢Cd content or ratio of reduced-fed CONT+Cd differs from Cd content or ratio of LOCA+Cd (Tables II and III) by Mann-Whitney U-test, P < 0.05.
that Ca deficiency may increase the risk of Cd neurotoxicity in animals exposed to Cd. Central nervous tissue may be protected from Cd toxicity. After administration of Cd, increases in brain concentrations are small compared to those in kidney and liver 3'8'9'14. Studies using l°9Cd show that entry of Cd into brain is very slow 1'13. However, brain Cd does increase with elevation of the metal in the diet (Table II and ref. 5). Rats given Cd (0.5-1/~g/g per day for 20-60 days) similar to the quantity in our study (1/~g/g per day for 40 days) had altered brain neurotransmitters 2. Abnormal neurological symptoms in workers exposed to Cd and impaired intelligence in children with elevated hair Cd also have been reported 2. In rats fed 100/~g/g for 67 days, Clark et al.5 found the highest Cd content in the olfactory bulb and hippocampus (314 and 275 ng/g) and the lowest in the tegmentum (130 ng/g). Using Z e e m a n background correction, we found in C O N T + C d rats that midbrain-colliculi Cd (9.3 ng/g) was lowest, that olfactory bulb Cd (17 ng/g) was the highest, but that the hippocampus (9.5 ng/g) was among the regions with the lowest Cd content. In L O C A + C d animals, brain regional distribution of Cd was unchanged, even though Cd content was increased 2 fold from C O N T + C d rats. Along with the elevation in central nervous tissue Cd, we found even greater increases in peripheral tissue Cd (Table II). Ca deficiency induced increases found in liver, kidney, and bone Cd agree with previous investigations 1°' 14,18,19. The kidney Cd content in L O C A + C d rats (32 /zg/g) was less than the critical value of Cd (160-400/~g/g) that is reported to produce proteinuria in humans 16. Ca deficiency did not alter tissue Cd when compared to ad libitum or reduced-fed controls, with the exception of femur. In brain, spinal cord, serum and muscle, Cd was near detection limits in all groups not fed Cd leading
to increased error and inability to obtain statistical differences for these tissues. In tissues with significant Cd content (liver, kidney, and femur), the Ca-deficient animals have higher metal contents but this was not significant in liver and kidney. The coefficients of variation of these tissues were decreased at least 50% in reduced-fed animals and in animals fed Cd, suggesting that variations in eating behavior and absorption at trace levels led to increased inconsistency. The ability of brain and muscle to better regulate tissue Ca could explain why increases in brain and muscle Cd were only half those in liver when C O N T + C d and L O C A + C d animals were compared. During Ca deficiency, tissue Ca, corrected for interstitial and blood Ca, is maintained better in brain and skeletal muscle (8593% of control, ref. 11 and unpublished data) than in liver (70% of control, unpublished data). If Cd and Ca compete for tissue binding sites 2, then tissues which retain Ca better during Ca deficiency would have less sites available for Cd, and thus less ability to accumulate Cd. Metallothionein synthesis could explain differences in tissue Cd accumulation. Metallothionein, a major Cd binding protein, is a determinant of tissue Cd TM. It is induced by protein-calorie malnutrition, Ca deficiency or Cd 14'15. A combination of factors ( L O C A + C d ) can induce more metallothionein than any one alone ( C O N T + C d ) . Because dietary Cd induces metallothionein in liver and kidney 14'15, the capacity of these tissues to accumulate Cd is increased. However, metallothionein is not elevated in brain 15, which may explain why the brain/serum ratio remained unchanged, whereas the ratios for the other tissues increased (Table IV). A 3-fold decrease seen in the brain/serum [Cd] ratio, but not in the other tissue/serum ratios between the C O N T and L O C A groups could also be explained by this.
284 A n increase in serum Cd might increase the fraction of Cd available for distribution to tissues, which also would elevate the tissue/serum ratio. A change in the serumfree fraction of Cd also could explain the decrease in
suggests that Cd distribution is n o n s a t u r a b l e from serum [Cd] of 8-30 ng/ml. No data are available from 1 to 10 ng/ml. To summarize,
brain minimizes Cd
accumulation
tissue/serum ratios in L O C A + C d group compared to C O N T + C d group. If Cd and Ca compete for binding
relative to peripheral tissues. Despite this ability, brain Cd does increase with elevated dietary Cd, and neuro-
sites on serum proteins 2, then during low plasma Ca, the free fraction of Cd would be expected to decrease. A
toxicity would be possible with e n v i r o n m e n t a l exposure.
decrease in tissue/serum ratios could also result from a
Ca deficiency further increases body Cd and changes tissue Cd distribution. Differences in Cd distribution
dependence of serum to tissue distribution on serum Cd. Kotsonis and Klaassen 9 found similar linear relations
could be explained by altered tissue [Ca], metallothionein, and Cd tissue and serum binding.
between Cd dose and both serum and tissue Cd, which REFERENCES 1 Arvidson, B. and Tj~ilve, H., Distribution of 1°9Cd in the nervous system of rats after intravenous injection, Acta Neuropathol., 69 (1986) 111-116. 2 Babitch, J.A., Cadmium neurotoxicity. In S.C. Bondy and K.N. Prasad (Eds.), Metal Neurotoxicity, CRC Press, Boca Raton, FL, 1988, pp. 141-166. 3 Cahill, A.L., Nyberg, D. and Ehret, C.E, Tissue distribution of cadmium and metallothionein as a function of time of day and dosage, Environ. Res., 31 (1983) 54-65. 4 Chiueh, C.C., Sun, C.L., Kopin, I.J., Fredericks, W.R. and Rapoport, S.I., Entry of [3H]norepinephrine, [125I]albuminand Evans blue from blood to brain following unilateral osmotic opening of the blood-brain barrier, Brain Research, 145 (1978) 291-301. 5 Clark, D.E., Nation, J.R., Bourgeois, A.J., Hare, M.E, Baker, D.M. and Hinderberger, E.J., The regional distribution of cadmium in the brains of orally exposed adult rats, Neurotoxicology, 6 (1985) 109-114. 6 Decker, L.E., Byerrum, R.U., Decker, C.E, Hoppert, C.A. and Langham, R.E, Chronic toxicity studies I. Cadmium administered in drinking water to rats, AMA Arch. Ind. Health, 18 (1958) 228-231. 7 Hamilton, D.L. and Smith, M.W., Inhibition of intestinal calcium uptake by cadmium and the effect of a low calcium diet on cadmium retention, Environ. Res., 15 (1978) 175-184. 8 Horner, D.B. and Smith, J.C., The distribution of tracer doses of cadmium in the normal rat, Arch. Environ. Contain. Toxicol., 3 (1975) 307-318. 9 Kotsonis, F.N. and Klaassen, C.D., Toxicity and distribution of cadmium administered to rats at sublethal doses, Toxicol. AppL PharmacoL, 41 (1977) 667-680.
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