Chronic exposure to cadmium did not impair vitamin D metabolism in monkeys

ENVIRONMENTALRESEARCH46, 48-58 (1988)

Chronic Exposure to Cadmium Did Not Impair Vitamin D Metabolism in Monkeys HIROYUKI KAWASHIMA, *'1 HIROKO NOMIYAMA,t AND KAZUO NOMIYAMAt

Departments of *Pharmacology and ~Environmental Health, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi-ken, 329-04, Japan Received August, 1987 Vitamin D metabolism in primates with chronic exposure to cadmium was studied in relation to Itai-Itai disease. In a series of experiments, crab-eating monkeys were fed cadmium-contaminated rice (1.33 p,g Cd/g) or a diet containing 3 v,g/g cadmium chloride for 6 years. These treatments had no effect on the 1,25-dihydroxyvitamin D (1,25(OH)eD), 24,25dihydroxyvitamin D (24,25(OH)2D), and 25-hydroxyvitamin D (25(OH)D) in the serum. This is consistent with unchanged production of 1,25(OH)2D and 24,25(OH)2D by renal mitochondria prepared from the same animals. No indication of renal dysfunction was observed. In another series of experiments, rhesus monkeys were fed a diet containing 3, 10, 30, or 100 p~g/g cadmium for 9 years. Serum vitamin D metabolites and renal production of 24,25(OH)2D also remained unchanged. In contrast, renal 25(OH)D-l-hydroxylase (lhydroxylase), which is responsible for the production of 1,25(OH)2D, seemed to be suppressed in the animals fed 30 or 100 p,g/kg cadmium-contaminated diet (no statistical significance). These animals had indications of mild renal dysfunction, and there was a strong negative correlation between 1-hydroxylase and urinary concentration of either protein or ~2-microglobulin. These data suggest a slight change in the total enzyme activity, possibly due to mild renal dysfunction. Since substrate (25(OH)D) concentration is much lower and thus rate-limiting in vivo as compared with that in vitro assay system used in this study, the slight change of enzyme activity would not have been sufficient to affect the serum level of 1,25(OH)2D. No skeletal abnormality was observed in any of these animals. In view of these data, the length of cadmium exposure and the life span of animals as well as epidemiological data published elsewhere, factors other than cadmium may also be involved in the development of Itai-Itai disease. © 1988AcademicPress,Inc.

INTRODUCTION B o n e c h a n g e s in I t a i - I t a i d i s e a s e h a v e b e e n r e c o g n i z e d as a c o m b i n a t i o n o f o s t e o m a l a c i a a n d o s t e o p o r o s i s ( N o m i y a m a , 1986). S e v e r e e x p o s u r e to c a d m i u m h a s b e e n s u g g e s t e d to c a u s e r e n a l p r o x i m a l t u b u l a r d y s f u n c t i o n , t h e r e b y r e s u l t i n g in r e d u c t i o n o f 1 , 2 5 - d i h y d r o x y v i t a m i n D (1,25(OH)2D) p r o d u c t i o n , w h i c h , in t u r n , l e a d s to t h e d e v e l o p m e n t o f t h e b o n e d i s e a s e ( F e l d m a n a n d C o u s i n s , 1973). H o w e v e r , n o d e f i n i t i v e e v i d e n c e h a s b e e n a v a i l a b l e to s u p p o r t this h y p o t h e s i s in e i t h e r c l i n i c a l o r a n i m a l s t u d i e s . I n an a t t e m p t to e l u c i d a t e t h e m e c h a n i s m s u n d e r l y i n g t h e b o n e c h a n g e s s e e n in I t a i - I t a i d i s e a s e , w e h a v e s t u d i e d c h r o n i c e f f e c t s o f c a d m i u m i n t o x i c a t i o n in p r i m a t e s ( N o m i y a m a a n d N o m i y a m a , i n p r e s s ; N o m i y a m a et al., 1981).

1 Present address: Central Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 1-8, Azusawa 1-chome, Itabashi-ku, Tokyo 174, Japan. 48 0013-9351/88 $3.00 Copyright © 1988 by Academic Press, Inc. All tights of reproduction in any form reserved.

CADMIUM EXPOSURE AND VITAMIN D METABOLISM

49

In this report, we will present data on renal vitamin D metabolism as well as serum vitamin D metabolites in these animals and will discuss our results in relation to Itai-Itai disease. MATERIALS AND METHODS

Animals. Nine male crab-eating monkeys, Macaca fascicularis, were divided into three groups. A group of four animals was fed a diet containing rice contaminated with 1.33 Ixg/g of cadmium, a group of three animals fed a diet containing 3.0 Ixg/g of cadmium chloride, and another group of two monkeys served as a control. The animals were kept on each diet for 6 years except for two which were killed after eating rice contaminated with cadmium for 2 years. Nine male rhesus monkeys, Macaca mulatta, were divided into five groups. Each group of two animals was fed a diet containing 0, 3, 10, or 30 Ixg/g cadmium, and one animal was fed a diet containing 100 ~g/g cadmium. The animals were kept on each diet for 9 years. The dietary content of calcium, phosphate, and vitamin D was 1.2%, 1.0%, and 2.4 Ix/g, respectively. Preparation of blood and tissue samples. After the treatment, the animals were anesthetized for histological, cytochemical, and biochemical analyses. Kidneys were removed and mitochondria were prepared by the method of Veith and Fraser (1979). The a c t i v i t i e s o f 2 5 - h y d r o x y v i t a m i n D - l - h y d r o x y l a s e (25( O H ) D - l - h y d r o x y l a s e , 1 - h y d r o x y l a s e ) a n d 25(OH)D-24-hydroxylase (24hydroxylase), the enzymes responsible for the production of 1,25(OH)zD 3 and 24,25-dihydroxyvitamin D (24,25(OH)2D3), respectively, were measured by the methods described below. The sera were stored at -20°C until use. Extraction of serum vitamin D metabolites. Serum samples (0.5 ml) were added to 12 × 75-mm borosilicate glass tubes and brought up to 1 ml with saline. Eight h u n d r e d c o u n t s p e r m i n u t e of 1,25(OH)2126,27-methyl-3H]D3, 24,25(OH)2126,27-methyl-3H]D3 or 25(OH)[26,27-methyl-3H]D3 (Amersham, Arlington Heights, IL) in 20 Ixl of ethanol was added to each serum sample and to a counting vial for monitoring recovery. The samples were then vortexed and allowed to stand for 10 min. One volume of acetonitrile was added to each serum sample. The samples were vortexed vigorously and then centrifuged for 10 min at 1500g. After centrifugation, the supernatant was decanted into a tube containing 0.5 ml 0.4 M KH2PO 4, pH 10.6, and vortexed. This extract was directly applied to a prewashed Sep Pak C18 (Waters Associates, Inc., Milford, MA). Excess salt was removed by washing the cartridge with 5 ml distilled water, and polar lipids were removed by washing the cartridge with 3 ml methanol/water (70/30, v/v). The vitamin D metabolites were then eluted with 3 ml acetonitrile, and this fraction was dried under a stream of nitrogen. The residue was dissolved in nhexane/isopropanol/methanol(94/5/l, v/v/v) and applied to a Zorbax CN (0.39 × 30 cm) column which was attached to a HPLC system (CCP&8000 series, Toyo Soda Manufacturing Co. Ltd., Tokyo, Japan) and eluted with the same solvent at the rate of 1.5 ml/min. The fractions corresponding to 1,25(OH)eD , 24,25(OH)2D, and 25(OH)D were collected separately, dried under nitrogen streams, and subjected to the binding assays. Production of l,25(OH)zD and 24,25(01-1)21) by the kidney. Mitochondria were

50

KAWASHIMA, NOMIYAMA, AND NOMIYAMA

prepared from the kidney, and incubations were performed as described previously (Kawashima, 1986). The kidneys were placed in an ice-cold homogenization medium (250 mM sucrose, 10 mM Hepes, 10 mM KC1, adjusted with NaOH to pH 7.42 at room temperature), and cortical tissues were obtained after removing capsules, papilla, ureter, and medulla. The tissues were homogenized in I0 vol (w/v) of ice-cold homogenization medium with a motor-driven Potter-Elvehjem homogenizer. The homogenate was centrifuged at 4000g and 4°C for 40 sec,and the supernatant was removed and then centrifuged at 9000g and 4°C for 20 min. The pellet was suspended in 10-15 vol of ice-cold incubation medium (125 mM KCI, 20 mM Hepes, 10 mM succinic acid, 2 mM M g S O 4, 1 mM dithiothreitol, 0.25 mM EDTA, adjusted to pH 7.2 at room temperature); the final concentration of mitochondrial protein was 2.5-5 mg/ml. Each incubation sample consisted of a 1.0-ml portion of mitochondrial suspension. Samples, in 25-ml Erlenmeyer flasks, were placed in a Dubnoff incubator at 25°C, shaking at 100 cycles/min. At 3 rain, 5 &g of 25(OH)D 3 (kindly provided by the Upjohn Co., Kalamazoo, MI) in 20 p,1 of ethanol was added. After substrate addition, the flask was gassed for 1 min with a direct flow of Oz/CO2 (95/5) at a rate of 0.5 liter/min. Immediately after gassing, the flask was sealed with a rubber stopper, incubated at 25°C for 15 min, and then the reaction was stopped by the addition of 3.75 ml of methanol/chloroform (2/1, v/v). Lipid extraction was performed by the method of Bligh and Dyer (1959). The extract was dried under an Nz gas stream and resolved in acetonitrile/water (I/I, v/v). The solution was decanted into a tube containing 0.5 ml 0.4 M KHzPO4, pH 10.6, and vortexed. The procedure used for the serum samples, as described above, was also applied for the analysis of this extract. Determination of 1,25(OH)zD. 1,25(OH)zD was determined as described previously (Kawashima, 1986) using calf thymic receptor for 1,25(OH)zD. Standard solutions of 1,25(OH)2D3 (generously given to us by Dr. Uskokovic of HoffmanLaRoche, Nuttley, NJ) (1-64 and 800 pg for the nonspecific binding determination) and samples (in 30 Ixl of ethanol) were added to 12 × 75-mm glass tubes on ice. Freeze-dried receptor was solubilized and diluted in a buffer containing 50 mM Tris-HC1, 500 mM KCI, 5 mM dithiothreitol, 10 mM N a z M o O 4 and 1.5 mM EDTA, pH 7.5; and then 450 ~l of receptor solution (approximately 0.7 mg of protein/tube) was added to the standards and samples on ice, followed by vortexmixing. The samples and standards were incubated in a 25°C water bath for 45 min with gentle shaking. At the end of 45 min, the tubes were transferred to an ice bath and allowed to cool for 5 min, and then each tube received 5000 cpm of 1,25(OH)zD[26,27-methyl-3H]D3 (160 Ci/mmole) (Amersham) in 25 ~1 of ethanol. The tubes were vortex-mixed, and the incubation was continued for 15 min in a 25°C water bath with shaking. The tubes were allowed to cool for 5 min in an ice bath, and 200 ~1 of dextran-coated charcoal suspension was added to each tube, followed by vortex-mixing. The tube contents were vortex-mixed again after 10 min; and after 20 rain of charcoal treatment, bound and free hormones were separated by centrifugation at 200g for 10 rain. The supernatant containing bound hormones was decanted into a scintillation vial, and radioactivity was determined in a liquid-scintillation counter (Aloka 7000, Aloka Ltd., Tokyo, Japan) (counting

CADMIUM EXPOSURE AND VITAMIN D METABOLISM

51

efficiency was approximately 50%). The intraassay coefficient of variation for measurement of 1,25(OH)2D was 7.8%. Determination of 25(OH)D and 24,25(OH)2D. 25(OH)D and 24,25(OH)2D were measured by radioligand binding assays with rat plasma vitamin D-binding protein (DBP). The rat plasma was diluted 1/5000 (v/v) in 0.05 i phosphate buffer (pH 7.5) containing 0.01% gelatin and 0.01% merthiolate (PBG buffer). The assay mixtures were placed in 12 × 75-mm borosilicate glass tubes, each consisting of 0.5 ml of 1/5000 dilution of vitamin D-deficient rat plasma in PBG buffer, 5000 cpm of either 25(OH)[26,27-methyl-3H]D3 (160 Ci/mmole) (Amersham) in 20 wl of ethanol, or a standard or unknown sample in 25 wl of ethanol. Standard curves for both 25(OH)D3 and 24,25(OH)2D3 (generously given to us by Dr. Uskokovic of Hoffman-LaRoche) were constructed over the range 0.1-0.6 ng. After 2 hr incubation at 4°C, each tube received 200 wl of dextran-coated charcoal in PBG buffer to separate bound and free hormones. After 30 min at 4°C, the tubes were spun at 1500g for 10 min in a refrigerated centrifuge. The supernatant was decanted into a scintillation vial, and radioactivity was determined as described above. Intraassay coefficients of variation for measurements of 25(OH)D and 24,25(OH)2D were 8.4 and 8.9%, respectively. Protein determination. The mitochondrial protein concentration was measured by the method of Lowry et al., with bovine serum albumin as the standard (1951). Determination of ~2-microglobulin and total protein in urine. The methods used for measuring f32-microglobulin were latex immunoassay by Bernard et al. (1981) and Tsuchiya-Biuret method by Piscator (1962). Calculations. The values of 1,25(OH)2D (pg/tube), 24,25(OH)2D (ng/tube), and 25(OH)D (ng/tube) were calculated by using logit-log plots of the data. Serum concentration of the vitamin D metabolites and the level of their renal production were obtained after correcting for recovery (approximately 50%) and sample volume. Statistical analysis. Statistical analysis was performed by Student's t test.

RESULTS As shown in Fig. 1, exposure of crab-eating monkeys to cadmium for 2 to 6 years had no effects on their serum 1,25(OH)ED, 24,25(OH)ED and 25(OH)D. This is consistent with the data that neither 1-hydroxylase nor 24-hydroxylase activity in the cadmium-treated animals was different from those of the control animals (Fig. 2). It is well known that normal values for serum 1,25(OH)2D vary considerably among species and with age. Serum levels of 1,25(OH)ED in monkeys appear to be higher than those in humans, as is also the case for rats. In animals who were exposed to cadmium for 9 years, serum concentrations of 25(OH)D did not change appreciably (Fig. 3). Serum 24,25(OH)ED seems to decrease in a dosedependent manner (Fig. 3), although statistical significance was not obtained due to the small number of animals used. It is known, however, in several species including humans that the concentration of this metabolite as well as others shows considerable variation among different animals depending on the animal's nutritional and physiological condition. Thus it is unlikely that the seemingly different

52

KAWASHIMA, 15C

AND NOMIYAMA

25(OH)D o

l OO

NOMIYAMA,

o

o

o oo

o

o

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0

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I

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200

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1,25(OH)2D 0 o o

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24,25(OH)2D

o 10

0

o

0

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o

I 0

I I00

I 200 Cadmium,

I 300

I 400

ug/g

FIG. 1. Effect of 2 to 6 years exposure to cadmium on serum vitamin D metabolites in crab-eating m o n k e y s . Each point represents a mean of duplicate assays.

levels of 24,25(OH)2D in Fig. 3 are biologically significant. This explanation is consistent with the data indicating that renal 24-hydroxylase activity was not affected in these animals as shown in Fig. 4. Similarly, serum concentrations of 1,25(OH)2D in these animals appear to be unaffected (Fig. 3). By contrast, renal 1-hydroxylase activity in animals exposed to either 30 or 100 txg/kg cadmium, seems to be suppressed (Fig. 4), although they did not reach statistical difference because of the small number of animals used. It may be that these differences are not significant for the same reason that the differences in 24,25(OH)2D are not significant as described above. However, there is a significant negative linear correlation between 1-hydroxylase activity and the logarithm of urinary protein or [32-microglobulin as shown in Figs. 5 and 6, suggesting that cadmium exposure may indeed impair 1-hydroxylase in these animals. Urinary protein excretion in the group exposed to the highest dose of cadmium

CADMIUM EXPOSURE AND VITAMIN D METABOLISM

25(OH)D-l-hydroxyllase

40O

o

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53

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%

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Cadmium

FIG. 2. Effect of 2 to 6 years exposure to cadmium on the renal 1- and 24-hydroxylase activities in the crab-eating monkeys. Each point represents a mean of duplicate assays.

(100 Ixg/g, 100 ppm group) began to rise at 48 weeks on the diet, and half of the animals in this group had 100 mg/100 ml/day or higher urinary protein excretion. However, urine protein was maintained at less than 500 mg/day except for one animal which had some occasional severe proteinuria (more than 600 mg/day). Proteinuria was not observed in any other groups. Urine [3z-microglobulin in the 100 ppm group began to rise at 138 weeks of exposure and some of them reached a level higher than 10,000 txg/liter after 171 weeks. After 195 weeks other animals of this group also had increased excretion of 132-microglobulin which remained less than 10,000 p~g/liter. In the 30 ppm group, urine 132-microglobulin was also elevated after 311 weeks and remained less than 5000 p~g/liter with occasional transient rises over 10,000 ~g/liter. These changes in urinary protein and 13e-microglobulin excretion were milder than those seen in subjects with Itai-Itai disease.

DISCUSSION In the present study, we showed that neither a 9-year exposure of rhesus monkeys to cadmium nor 2- or 6-year exposures of crab-eating monkeys to cadmium affected serum levels of vitamin D metabolites, 1,25(OH)2D, 24,25(OH)zD and 25(OH)D. This is in accord with the data indicating that exposure of crab-eating monkeys to cadmium for 2 to 6 years had no effect on any of the renal 1- and 24-hydroxylase activities, which are responsible for the production of 1,25(OH)2D and 24,25(OH)2D. This is consistent with the data that these animals show no signs of renal dysfunction judging from the lack of detectable urinary protein and

54

KAWASHIMA, NOMIYAMA, AND NOMIYAMA

1201

25(OH)Doo

o° o

%

80

o

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o

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30(

20(

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30

100

24,25(OH)2D oo

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0 0

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Cadmium,

lO

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lO0

ug/g

FIG. 3. Effect of 9 years exposure to c a d m i u m on serum vitamin D metabolites in the rhesus m o n k e y s . Each point represents a m e a n of duplicate assays.

no changes in urinary ~32-microglobulin and in creatinine clearance (Nomiyama and Nomiyama, in press, Nomiyama et al., 1981). In contrast, 1-hydroxylase in rhesus monkeys exposed to cadmium for 9 years seems to be reduced, although no statistical significance was observed because of the small number of animals used. However, when 1-hydroxylase was plotted against the logarithm of urinary protein or [32-microglobulin, a strong negative linear correlation became evident. The latter data suggest that renal 1-hydroxylase might have been suppressed to some extent by cadmium due to mild renal dysfunction. The unaffected serum levels of 1,25(OH)zD despite the reduction in the 1hydroxylase activity may be explained by suppressed catabolism of the metabolite. To examine this possibility, renal homogenate was incubated with 1,25(OH)z[26,27-methyl-3H]D at 37°C for 15 rain. No difference was observed between the cadmium-treated group and the control group (data not shown). Since the kidney is a major site for the catabolism of 1,25(OHhD, it is reasonable to assume that degradation of 1,25(OH)zD may not be enhanced by the exposure to

CADMIUM EXPOSURE AND VITAMIN D METABOLISM

55

c

g

c~

25(OH)D-l-hydroxylase

g 200C

0 0

~2

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E m

I 30

1~0

25(OH)D-24-hydroxylase 0

% 0 2 0

u

O0

0

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c~

b Cadmium,

pg/g

FIG. 4. Effect of 9 years exposure to cadmium on the renal 1- and 24-hydroxylase activities in the rhesus monkeys. Each point represents a mean of duplicate assays.

cadmium. Another possibility is that the change in 1-hydroxylase activity is too small to affect the serum level of this metabolite. To fully activate the enzyme so that a subtle difference in the enzyme activity is detectable, the substrate concentration in the assay system of 1-hydroxylase was elevated to a level approximately 10,000 times higher than that found in vivo. In the normal in vivo situation, however, the substrate concentration is much lower and this may be a ratelimiting factor. Thus, it is not surprising that the slight changes in renal function O =

150

-

O Y = 207.8

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66.7

X

loo

c~ 50

O o °o

,2 0

log(urinary

protein)

FIG. 5. Correlation between renal 1-hydroxylase and urinary protein excretion in the cadmiumintoxicated rhesus monkeys.

56

KAWASHIMA, NOMIYAMA, AND NOMIYAMA = 150

O

~ 0

. 100

o/ 0

~

Y = 250.7 - 97.8 X r =

0.7966

\

58

0 1

o 2

log(g2-microgl

obul in)

FIG. 6. Correlation between renal 1-hydroxylase and urinary [32-microglobulinin the cadmiumintoxicated rhesus monkeys.

seen in the present study failed to change the serum concentration of 1,25(OH)2D appreciably. It has been recently reported that among the residents in a cadmium-polluted area in Japan, approximately 20% reduction in plasma 1,25(OH)zD level was observed, with a marked elevation of [32-microglobulin in the urine as well as in the serum (Nogawa et al., 1987). The authors suggested that impaired vitamin D metabolism due to defects in the proximal tubules may be a mechanism leading to the bone disease caused by cadmium intoxication (Nogawa et al., 1987). It is known, however, that renal defects caused by severe cadmium intoxication can be seen not only in the proximal tubule cells, but also in the glomeruli (Lauwerys et al., 1979). It is also known that reduction in plasma 1,25(OH)2D observed in patients with nephrotic syndrome is mainly due to a secondary effect of proteinuria (glomerular proteinuria), i.e., the majority of circulating metabolites of vitamin D including 1,25(OH)2 D and its precursor, 25(OH)D, are bound to the serum vitamin D binding protein (DBP) and are lost in the urine with the protein (DiDomenico et al., in press). Thus the depressed plasma 1,25(OH)2D among subjects in the report by Nogawa et al. (1987) may have been due to either glomerular proteinuria or defects in the proximal tubules where 1-hydroxylase is exclusively localized (Kawashima and Kurokawa, 1986) or caused by a combination of the both. As described above, cadmium exposure caused less toxic effects in our animals as compared with those seen in patients with Itai-Itai disease. Since urinary cadmium excretion and renal cadmium accumulation in the animals in our experiments are higher than those o b s e r v e d in humans ( N o m i y a m a and Nomiyama, in press; Nomiyama et al., 1981; Ellis et al., 1984; Roels et al., 1981), the rhesus monkeys in the treated groups could have been exposed to higher amounts of cadmium as compared to humans with Itai-Itai disease or to humans with lower plasma 1,25(OH)zD (Nogawa et al., 1987). In addition, the length of cadmium exposure in monkeys (9 years) seems to be at least no less than that in humans with Itai-Itai disease (30 years) on the basis of the life spans of both species (20 and 80 years, respectively). Taken together, one may assume that monkeys are more resistant to cadmium exposure.

CADMIUM EXPOSURE AND VITAMIN D METABOLISM

57

Cadmium may directly perturb bone cell function(s), as suggested by Kawamura et al. using rats with exposure to cadmium (1978). In their animal model, they were able to show that lower calcium intake (low calcium diet) caused more prominent accumulation of cadmium in bone as well as more severe bone lesion as compared with normal calcium intake. Since these animals did not show hyperparathyroidism, osteomalacia observed in the animals could not have been due to uremic renal osteodystrophy (Kawamura et al., 1978). On the basis of both histological studies on bone, kidney, and parathyroid gland and renal clearance studies, it is postulated that cadmium acts directly on the bone causing an osteomalacia, and in addition, tubular dysfunction caused by cadmium together with disturbances in calcium reabsorption may play a role in the calcium loss in those animals (Kawamura et al., 1978). In our monkeys even with 100 ppm cadmium (twice higher than that in the study of Kawamura et al., 1978), no bone lesions were observed. It should be noted that in our experiment calcium content in the diet was 1.2%, which is more than twice higher than that in the study of Kawamura et al. (0.5%). This may be important in view of relatively lower intake of calcium as well as protein together with severe exposure to cadmium in patients with Itai-Itai disease. Exposure of primates to higher amounts of cadmium with lower calcium intake might cause bone changes similar to those in Itai-Itai disease, provided it produces a similar renal dysfunction, possibly including impaired vitamin D metabolism. Although we cannot rule out such a possibility, the present data suggest that factors other than cadmium may also be involved in the development of the bone disease observed in subjects in the area heavily contaminated with cadmium. Such factors may include aging, history of pregnancy and delivery, and sex hormones as well as other environmental parameters. Dietary calcium may also play a role in the development of the disease. These remain to be studied in the future.

ACKNOWLEDGMENT We are grateful to Drs. F. Akahori, T. Masaoka, N. Arai, and K. Kobayashi for generously providing the kidney tissues and blood samples for our experiments, and we thank Miss Y. Nakajima for her technical assistance. This study is supported in part by a Grant-in-Aid for Scientific Research from the Japan Environment Agency, the Japan Ministry of Health and Welfare, the Japan Ministry of Education, Science and Culture (61215029), and by a Grant-in-Aid from the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

REFERENCES Bernard, A. M., Vysko~il, A., and Lauwerys, R. R. (198l). Determination of ~2-microglobulin in human urine and serum by latex immunoassay. Clin. Chem. 27, 832-837. Bfigh, E. G., and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canad. J. Biochem. Physiol. 37, 911-917. DiDomeuico, N., Kawashima, H., and Kurokawa, K. Nephrotic syndrome and calcium metabolism. In "Renal Osteodystrophy" (G. R. D. Catto and J. W. Coburn, Eds.), Martinus, Hague/ Boston/London, in press. Ellis, K. J., Yuen, K., Yasumura, S., and Cohn, S. H. (1984). Dose-response analysis of cadmium in man: Body burden vs kidney dysfunction. Environ. Res. 33, 216-226. Feldman, S. L., and Cousins, R. J. (1973). Influence of cadmium on the metabolism of 25hydroxycholecalciferol in chicks. Nutr. Rep. Int. 8, 251-259.

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