9 1987 by The Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/87/1300-0237502.40
Influence of Cadmium on the Distribution of the Essential Trace Elements Zinc and Copper in the Liver and Kidneys of Rats YASUMASA KUNIFUJI, 1'* T E R U M A S A NAKAMURA, I AND MASAYUKI TAKASUGI 1'2
'Division of Clinical Chemistry, School of Medical Technology, and 2Kidney Center, School of/Vledicine, University of Occupational and Environmental Health, Yahatanishiku, Kitakyushu 807, Japan Received D e c e m b e r 15, 1986; Accepted January 6, 1987
ABSTRACT Cd induced changes of Zn and Cd distribution in the liver and kidneys were studied in relation to Cd metallothionein (MT) synthesis. Wistar male rats were given CdCI2 by sc injection of .8, 1.5, and 3.0 mg Cd/kg three times a week for three weeks. Cd levels of liver and kidneys increased with the increment of Cd dosage and 80-90% of Cd was found in the cytosol. The MT fractions contained 80-89% cytosolic Cd in the liver and 55-75% Cd in the kidneys. Zn concentrations in the liver increased following Cd administration, but Zn in the kidneys showed only slight increase. There was a distinct decrease of Cu concentration in the liver of the 3.0 mg group. In contrast, Cu concentrations in the kidneys increased about three times in the .8 and 1.5 mg Cd groups, but Cu in the 3.0 mg group showed only 1.5 times increase. The changes of these metal concentrations were observed mainly in the cytosol. Non-MT-Cd in the kidneys was maximum in the 1.5 mg group, but the 3.0 mg group showed significant decrease. In parallel with this decrease of Cd, Cu and Zn in the kidneys showed similar decrease. When the kidneys are injured, Zn and Cu appear to leak from this organ. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
237
Vol. 14, 1987
Kunifuji, Nakamura, and Takasugi
238
Index Entries: Effects of Cd on Zn and Cu distribution, in liver and kidneys; Cd rnetallothionein, in liver and kidneys; critical concentration of Cd, in kidneys; renal non-metallothionein-Cd, in kidneys.
INTRODUCTION Cadmium accumulates in various organs, especially the liver and kidneys, and leads to toxic effects following oral and parenteral administration into experimental animals. Most of the Cd accumulated in the liver and kidneys exists in a low molecular weight protein, metallothionein (MT). It is well known that thionein binds Cd, Zn, Cu, Bi, Hg, Au, and Ag, and these metals induce the synthesis of the corresponding MT. Cadmium administration influences the distribution of Zn and Cu in the liver and kidneys. As Cd competes with essential trace elements for ligands in biological systems, Cd may have an effect on the disposition and homeostasis of these elements, and will manifest its toxicity. Stonard and Webb (1) found that the accumulation of Cd following a long-term dietary administration of the metal to rats was associated with an increased concentration of Cu in the kidneys and Zn in the liver. In a similar experiment, other investigators confirmed the increased liver concentration of Zn, but found that the Cu concentration in the liver was increased only during the first three weeks of Cd administration (2). When an acute dose of Cd was administered to rats, Ashby et al. (3) observed that the Zn concentration in the liver was greatly increased, whereas the Cu concentration in the liver was only slightly increased. Some researchers assumed that the Zn accumulation in the liver in the presence of Cd was a result of the induction and synthesis of MT (4,5). The present study was designed to elucidate Cd induced changes of Zn and Cu concentration in the liver and kidneys, and to consider the role of metallothionein.
METHODS Twenty-four male rats of the Wistar strain (250-310 g) were divided into four groups of six rats each, and were kept in stainless-steel cages with free access to tap water and rat standard pellets (C-2, Japan CLEA Co., Ltd.) and a 12-h-light, 12-h-dark condition at 23~ They were injected sc with .8, 1.5, and 3.0 mg Cd/kg body wt. as CdCI2 in saline (.8, 1.5, and 3.0 mg groups) or normal saline (control), every other day three times a week over a period of three weeks. Body weight was recorded just before injection. All the rats were sacrificed by exsanguination at 48 h after the last injection. Then, liver, kidneys, adrenals, and testes were removed and weighed. Liver and kidneys were stored at -80~ until processed. Biological Trace Element Research
Vol. 14, 1987
Influence of Cd on Renal and
HepaticZn and Cu
239
Each organ was homogenized in 4 vol. of 100 mM Tris-HCl buffer (pH 7.4 at 25~ containing 250 mM glucose. One portion (2 mL) of the homogenate was used to determine Cd, Zn, and Cu concentrations. The remaining homogenate was centrifuged in a refrigerated ultracentrifuge (4~ for 60 min at 170,000 x g. Then the precipitate was resuspended with 10 mL of distilled deionized water. Each portion (2 mL) of the homogenate, its supernatant, and precipitate were wet-digested (6), and the Cd and Zn concentrations of the digests were measured with a Perkin-Elmer model 603 atomic absorption spectrophotometer (AAS) (flame), and Cu concentration was measured with a Hitachi model 170-70 Zeeman-corrected AAS (flameless). A portion (100 ILL) of supernatant was applied to an HPLC gel filtration column (TSKgel G3000SW, Toyo Soda, 7.5 mm ID x 60 cm with a precolumn 7.5 mm ID x 10 cm) and eluted with 50 mM Tris-HC1 buffer (pH 8.6 at 25~ at a flow rate of .5 mL/min, and eluate was collected with a Pharmacia FRAC-100 fraction collector (fraction size: 1 mL, 10% peak threshold mode). Eluate was monitored at a wavelength of 250 nm absorbance, and the MT fraction was determined. Then Cd and Zn concentrations of the MT fractions were measured with a Hitachi model 170-70 Zeeman-corrected AAS (flameless). For statistical analysis, a t-test using Fisher's distribution was used.
RESULTS
Animal Experiments Growth curves of control and Cd groups during the administration period are shown in Fig. 1. Growth of the 1.5 mg group was markedly retarded in comparison with that of the control and .8 mg groups. The body weight in the 3.0 mg group showed initial rapid decrement in the first 2 d and thereafter a slight and slower decrease, resulting in an average 10% weight loss at the end of experiment. Average organ weights of the control and Cd groups are shown in Table 1. The average liver weight in Cd groups was not significantly different from that in the control group except for the .8 mg group in which the average liver weight was 20% heavier than that of the control group. The average weight of both kidneys in the control group was about 2.4 g, and there were no significant differences among experiment groups. Liver and kidneys of all groups showed no histological alteration on light microscopic examination (H and E stain). The averag e weights of both adrenals in the control, .8, and 1.5 mg groups were about .05 g, and that in the 3.0 mg group was .07 g, but these differences were not statistically significant. The average weights of both testes in the control and .8 mg groups were about 3.0 g, whereas average weights in the 1.5 and 3.0 mg groups were about 1.0 g and showed macroscopically marked atrophy Biological Trace Element Research
VoL 14, 1987
Kunifuji, Nakamura, and Takasugi
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100 O~ "-'
Z
8060-
.1-
40-
20ILl
0 >-20 E3 0
m
-40
I
,
I
i
0
5
10
15
DAYS OF Cd TREATMENT Fig. 1. Effect of Cd (subcutaneously administered CdC12 in saline) on body weight gain. &:Cd injection @--@ :control group 9169 :.8 mg group I - - m :1.5 mg group []--[3:3.0 mg group w i t h w h i t i s h discoloration a n d microscopic d e g e n e r a t i o n of s e m i n i f e r o u s tubules.
Concentration of H e a v y Metals in the Liver and Kidneys The a d m i n i s t e r e d Cd was distributed 50-60% in the liver a n d 5-6% in the k i d n e y s . Metal c o n c e n t r a t i o n s in the liver a n d k i d n e y s are s h o w n in Tables 2 a n d 3. Eighty to n i n e t y p e r c e n t of Cd in the liver a n d k i d n e y s was f o u n d to be in the s u p e r n a t a n t . Table 1 Organ Weight (Liver, Kidneys, Adrenals, and Testes) of the Control and Cd Treated Rats (mean _+SD) Organs, g Dose group
n
Control .8 mg 1.5 mg 3.0 mg
6 6 6 6
Liver 11.6 13.2 11.2 11.3
_+ 1.1 ___ 1.3" + 1.5 _+ .8
Kidneys
Adrenals
2.4 2.4 2.4 2.3
.05 .05 .05 .07
+_ .2 ___ .2 ___ .3 ___ .2
_+ .01 --- .01 +-- .01 ___ .02
Testes 3.1 3.0 1.0 1.0
_+_ .1 ___ .1 +__ .1"* _+ .1"*
Significantly different from the control:*p < .05; **p < .01 Biological Trace Element Research
Vol. 14, 1987
Influence of Cd on Renal and Hepatic Zn and Cu
241
Zinc concentration increased significantly in the liver of the Cd groups. Eight-two to eighty-five percent of Zn was found in the supernatant. In the kidneys of Cd groups, Zn concentrations increased slightly. Sixty-seven to seventy-six percent of Zn was in the supernatant. Copper concentrations in the liver in the .8 and 1.5 mg groups were not significantly different from that in the control group, but Cu in the 3.0 mg group was decreased to two-thirds of the control group. In the kidneys, Cu concentrations of the Cd groups increased markedly, however that in the 3.0 mg group showed significant decrease compared to the 1.5 mg group. The increase of Cu concentration in kidneys was mainly found in the supernatant.
Cd and Zn Concentrations in lVlT of Liver and Kidneys HPLC gel filtration profiles of liver supernatant obtained from the control and 3.0 mg groups are shown in Fig. 2. The elution position of MT was confirmed by continuous 250 nm absorbance monitoring and AAS analyses of Cd and Zn. MT was eluted in 30.6-36.5 min and was separated into two peaks, MT II and MT l, respectively (7). Cadmium recovery after HPLC separation was 103% in the 3.0 mg group. Zinc recovery was 95% and 82% in the 3.0 mg and the control group, respectively. Cadmium and Zn contents of MT fractions are summarized in Table 4. The Cd concentration of MT fractions in the liver and kidneys was increased directly proportional to the dosage of Cd. In comparison to the control group, the Zn concentration of liver MT fractions was markedly increased by Cd administration. The supernatant of Cd groups contained 40-50% of Zn in MT fractions. In the kidneys, the Zn concentration of MT fractions in the Cd groups did not differ from that in the control group.
DISCUSSION This study was conducted to investigate the effects of Cd or Zn and Cu levels in the liver and kidneys of rats after sc Cd injection. The results of Zn and Cu analyses (Table 2 and 3) indicate that Cd administration affects markedly the metabolism of essential trace elements, Zn and Cu. The Cd-induced increase of liver Zn concentration agrees with previous investigations. Ashby et al. (3) obtained a Cd-induced increase of liver Zn concentration after a sc injection of CdC12 into rats, and this increase in liver Zn is correlated with an increase of Cd-induced liver MT. By oral administration, Stonard and Webb (1), Wesenberg et al. (8), and Weigel et al. (9) reported a similar increase in liver Zn concentration. The Cu concentration of the liver in the 3.0 mg group was decreased to twothirds of the control. Other researchers did not observe the decrease of Cu concentration (1,3,9). In the kidneys, the increase of Cu concentration Biological Trace Element Research
Vol. 14, 1987
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Table 2 Cadmium, Zinc, and Copper Concentration (p,g/g Wet Wt) in Liver of Control and Cd Treated Rats (mean _+ SD) Cd dose group Control .8 mg 1.5 mg 3.0 mg
Cd n
Total
Sup."
Ppt?
6
ND
ND
ND
6 6 6
100.6 + 9.5 185.4 N _+ 15.5 307.8,~, +_ 41.7
88.4 -+ 8.3 159.8 ~ _+ 13.4 270.7,~, +- 53.3
14.7 _+ 2.0 25.6 ~ _+ 2.3 36.4,~, _+ 6.5
Zn Total Control .8 mg 1.5 mg 3.0 mg
34.3 75.6** 88.811 85.4**
Sup.
+ 2.5 _+ 4.8 _+ 4.6 _+ 10.9
Ppt.
22.9 _+ 1.7 62.2** _+ 3.9 73.2~ + 4.5 72.2** _+ 17.2
11.1 15.1"* 17.2"~ 14.5",*
+ .5 _+ .9 _+ .7 _+ 1.8
Cu Total Control .8 mg 1.5 mg 3.0 mg
2.6 2.8 2.6 1.7~
_+_ _+ +_ _+
Sup. .1 .4 .3 .3""
2.1 2.1 1.7 1.2~
_+ _+ _+ +
Ppt. .1 .4 .3 .2``
.8 .9 .9 .6~
+ + + _+
.1 .2 .6 .1``
~ ~'Precipitate. N~Not detectable. Significantly different from the control: *p < .05; **p < .01. Significantly different from the Cd .8 mg group: ep < .05; ~ p < .01. Significantly different from the Cd 1.5 rng group: Ap < .05; ``p < .01.
in Cd g r o u p s in the p r e s e n t s t u d y was in a c c o r d a n c e with the observation by S t o n a r d a n d W e b b (1). H o w e v e r , Weigel et al. (9) did not observe C d - i n d u c e d c h a n g e s of Cu c o n c e n t r a t i o n in the kidneys. T h e r e is s o m e d i s a g r e e m e n t in the literature about the effects of Cd on Z n a n d Cu levels of liver a n d kidneys. These d i s a g r e e m e n t s m a y result from the difference of Cd dosage, as well as route a n d period of administration. The source of i n c r e a s e d Z n a n d Cu in the liver a n d k i d n e y s m a y be either s e c o n d a r y to an e n h a n c e d Z n a n d Cu absorption from the small intestine or a d e c r e a s e of biliary a n d / o r intestinal excretion. Starcher et al. (10) s h o w e d Z n absorption was directly proportional to intestinal MT levels a n d i m p l y a significant role for MT in Z n absorption. S u g a w a r a (11) failed to s h o w an increase of Z n u p t a k e from the jejun u m of Cd e x p o s e d rats. Evans et al. (12) d e m o n s t r a t e d that Z n h o m e o stasis in rats was m a i n t a i n e d by Z n secretion from the intestine. Suzuki
Biological Trace Element Research
Vol. 14, 1987
Influence of Cd on Renal and Hepatic Zn and Cu
243
Table 3 Cadmium, Zinc, and Copper Concentration (l~g/g Wet Wt) in Kidneys of Control and Cd Treated Rats (mean (range)) Cd dose group
Total
Cd Sup?
Control .8 mg 1.5 mg 3.0 mg
ND
ND
ND
64.3 (62.3 - 66.3)" 119.7 ~ (119.2 - 120.1) 145.2",(143.5 - 146.9)
52.6 (51.0 - 54.1) 99.8" (99.0 - 100.6) 124.55 (123.4 - 125.5)
14.3 (14.3 - 14.3) 24.1" (23.9 - 24.3) 27.6" (27.0 - 28.1)
Ppt. "
Zn Total Control .8 nag 1.5 rng 3.0 mg
23.9 (22.5 - 25.3) 31.2 (30.9 ~ 31.4) 26.2" (26.2 - 26.2) 25.6~ (25.6 - 25.6)
Sup. 13.8 21.0 19.5 19.5
(13.5 (19.1 (18.6 (18.4
-
Ppt. 13.9) 22.8) 20.4) 20.4)
8.7 (8.1 9.8 (9.7 7.8o (7.7 6.8" (6.6 -
9.3) 9.8) 7.8) 7.0)
Cu Total Control .8 mg 1.5 mg 3.0 mg
2.6 (2.5 - 2.7) 7.2 (5.8 ~ 8.5) 7.4** (7.3 ~ 7.5) 3.9"" (3.8 ~ 4.0)
Sup. 1.1 (0.9 ~ 1.2) 3.6 (2.4 ~ 4.7) 5.0* (4.8 ~ 5.2) 2.2"" (2.1 ~ 2.3)
Ppt. 1.3 2.0 1.9 1.2
(1.2 (1.8 (1.8 (1.1
~ ~ ~ ~
1.3) 2.1) 2.0) 1.2)
~ value in parenthesis stands for the maximum and minimum values obtained from the pooled sample of three rats. ~Supernatant. 'Precipitate. Significantly different from the control: *p < .05; **p < 0.01. Significantly different from the Cd .8 mg group: ~ < .05; Np < .01. Significantly different from the Cd .5 mg group: Ap < .05; ~ p < .01.
(12) o b s e r v e d no significant c h a n g e of p l a s m a Z n levels in Cd e x p o s e d rats. A c c u m u l a t e d Z n in the liver of Cd e x p o s e d rats m a y be c a u s e d by inhibition of biliary a n d intestinal Z n excretion. A s h b y et al. (3) s h o w e d biliary excretion of Cu was m a r k e d l y inhibited after Cd a d m i n i s t r a t i o n . These a u t h o r s a n d Suzuki (13) o b s e r v e d an increase of p l a s m a Cu a n d c e r u l o p l a s m i n levels following Cd administration. In the plasma, 90% of total Cu was in h i g h m o l e c u l a r w e i g h t protein fraction (ceruloplasmin) a n d 8% in MT fraction. Metal c o m p o s i t i o n of MT in the p l a s m a was different from that of liver a n d k i d n e y s , a n d p l a s m a MT c o n t a i n e d Cd a n d Cu, but not Z n (13). I n t r a v e n o u s l y injected Cd-MT a c c u m u l a t e d m a i n l y in the k i d n e y s (14,15,16). Plasma MT m a y be filtrated t h r o u g h renal glomeruli a n d reabsorbed into proximal tubules. Cu rich p l a s m a MT m a y also contribute to the Cu increase in the kidneys.
Biological Trace Elernent Research
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E r 04
1.0 ~-
Control
3.0rag Group
0.8-
0
bJ c.1
MT
0.6-
Z
,
Influence of Cadmium on the Distribution of the Essential Trace Elements Zinc and Copper in the Liver and Kidneys of Rats YASUMASA KUNIFUJI, 1'* T E R U M A S A NAKAMURA, I AND MASAYUKI TAKASUGI 1'2
'Division of Clinical Chemistry, School of Medical Technology, and 2Kidney Center, School of/Vledicine, University of Occupational and Environmental Health, Yahatanishiku, Kitakyushu 807, Japan Received D e c e m b e r 15, 1986; Accepted January 6, 1987
ABSTRACT Cd induced changes of Zn and Cd distribution in the liver and kidneys were studied in relation to Cd metallothionein (MT) synthesis. Wistar male rats were given CdCI2 by sc injection of .8, 1.5, and 3.0 mg Cd/kg three times a week for three weeks. Cd levels of liver and kidneys increased with the increment of Cd dosage and 80-90% of Cd was found in the cytosol. The MT fractions contained 80-89% cytosolic Cd in the liver and 55-75% Cd in the kidneys. Zn concentrations in the liver increased following Cd administration, but Zn in the kidneys showed only slight increase. There was a distinct decrease of Cu concentration in the liver of the 3.0 mg group. In contrast, Cu concentrations in the kidneys increased about three times in the .8 and 1.5 mg Cd groups, but Cu in the 3.0 mg group showed only 1.5 times increase. The changes of these metal concentrations were observed mainly in the cytosol. Non-MT-Cd in the kidneys was maximum in the 1.5 mg group, but the 3.0 mg group showed significant decrease. In parallel with this decrease of Cd, Cu and Zn in the kidneys showed similar decrease. When the kidneys are injured, Zn and Cu appear to leak from this organ. *Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research
237
Vol. 14, 1987
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238
Index Entries: Effects of Cd on Zn and Cu distribution, in liver and kidneys; Cd rnetallothionein, in liver and kidneys; critical concentration of Cd, in kidneys; renal non-metallothionein-Cd, in kidneys.
INTRODUCTION Cadmium accumulates in various organs, especially the liver and kidneys, and leads to toxic effects following oral and parenteral administration into experimental animals. Most of the Cd accumulated in the liver and kidneys exists in a low molecular weight protein, metallothionein (MT). It is well known that thionein binds Cd, Zn, Cu, Bi, Hg, Au, and Ag, and these metals induce the synthesis of the corresponding MT. Cadmium administration influences the distribution of Zn and Cu in the liver and kidneys. As Cd competes with essential trace elements for ligands in biological systems, Cd may have an effect on the disposition and homeostasis of these elements, and will manifest its toxicity. Stonard and Webb (1) found that the accumulation of Cd following a long-term dietary administration of the metal to rats was associated with an increased concentration of Cu in the kidneys and Zn in the liver. In a similar experiment, other investigators confirmed the increased liver concentration of Zn, but found that the Cu concentration in the liver was increased only during the first three weeks of Cd administration (2). When an acute dose of Cd was administered to rats, Ashby et al. (3) observed that the Zn concentration in the liver was greatly increased, whereas the Cu concentration in the liver was only slightly increased. Some researchers assumed that the Zn accumulation in the liver in the presence of Cd was a result of the induction and synthesis of MT (4,5). The present study was designed to elucidate Cd induced changes of Zn and Cu concentration in the liver and kidneys, and to consider the role of metallothionein.
METHODS Twenty-four male rats of the Wistar strain (250-310 g) were divided into four groups of six rats each, and were kept in stainless-steel cages with free access to tap water and rat standard pellets (C-2, Japan CLEA Co., Ltd.) and a 12-h-light, 12-h-dark condition at 23~ They were injected sc with .8, 1.5, and 3.0 mg Cd/kg body wt. as CdCI2 in saline (.8, 1.5, and 3.0 mg groups) or normal saline (control), every other day three times a week over a period of three weeks. Body weight was recorded just before injection. All the rats were sacrificed by exsanguination at 48 h after the last injection. Then, liver, kidneys, adrenals, and testes were removed and weighed. Liver and kidneys were stored at -80~ until processed. Biological Trace Element Research
Vol. 14, 1987
Influence of Cd on Renal and
HepaticZn and Cu
239
Each organ was homogenized in 4 vol. of 100 mM Tris-HCl buffer (pH 7.4 at 25~ containing 250 mM glucose. One portion (2 mL) of the homogenate was used to determine Cd, Zn, and Cu concentrations. The remaining homogenate was centrifuged in a refrigerated ultracentrifuge (4~ for 60 min at 170,000 x g. Then the precipitate was resuspended with 10 mL of distilled deionized water. Each portion (2 mL) of the homogenate, its supernatant, and precipitate were wet-digested (6), and the Cd and Zn concentrations of the digests were measured with a Perkin-Elmer model 603 atomic absorption spectrophotometer (AAS) (flame), and Cu concentration was measured with a Hitachi model 170-70 Zeeman-corrected AAS (flameless). A portion (100 ILL) of supernatant was applied to an HPLC gel filtration column (TSKgel G3000SW, Toyo Soda, 7.5 mm ID x 60 cm with a precolumn 7.5 mm ID x 10 cm) and eluted with 50 mM Tris-HC1 buffer (pH 8.6 at 25~ at a flow rate of .5 mL/min, and eluate was collected with a Pharmacia FRAC-100 fraction collector (fraction size: 1 mL, 10% peak threshold mode). Eluate was monitored at a wavelength of 250 nm absorbance, and the MT fraction was determined. Then Cd and Zn concentrations of the MT fractions were measured with a Hitachi model 170-70 Zeeman-corrected AAS (flameless). For statistical analysis, a t-test using Fisher's distribution was used.
RESULTS
Animal Experiments Growth curves of control and Cd groups during the administration period are shown in Fig. 1. Growth of the 1.5 mg group was markedly retarded in comparison with that of the control and .8 mg groups. The body weight in the 3.0 mg group showed initial rapid decrement in the first 2 d and thereafter a slight and slower decrease, resulting in an average 10% weight loss at the end of experiment. Average organ weights of the control and Cd groups are shown in Table 1. The average liver weight in Cd groups was not significantly different from that in the control group except for the .8 mg group in which the average liver weight was 20% heavier than that of the control group. The average weight of both kidneys in the control group was about 2.4 g, and there were no significant differences among experiment groups. Liver and kidneys of all groups showed no histological alteration on light microscopic examination (H and E stain). The averag e weights of both adrenals in the control, .8, and 1.5 mg groups were about .05 g, and that in the 3.0 mg group was .07 g, but these differences were not statistically significant. The average weights of both testes in the control and .8 mg groups were about 3.0 g, whereas average weights in the 1.5 and 3.0 mg groups were about 1.0 g and showed macroscopically marked atrophy Biological Trace Element Research
VoL 14, 1987
Kunifuji, Nakamura, and Takasugi
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100 O~ "-'
Z
8060-
.1-
40-
20ILl
0 >-20 E3 0
m
-40
I
,
I
i
0
5
10
15
DAYS OF Cd TREATMENT Fig. 1. Effect of Cd (subcutaneously administered CdC12 in saline) on body weight gain. &:Cd injection @--@ :control group 9169 :.8 mg group I - - m :1.5 mg group []--[3:3.0 mg group w i t h w h i t i s h discoloration a n d microscopic d e g e n e r a t i o n of s e m i n i f e r o u s tubules.
Concentration of H e a v y Metals in the Liver and Kidneys The a d m i n i s t e r e d Cd was distributed 50-60% in the liver a n d 5-6% in the k i d n e y s . Metal c o n c e n t r a t i o n s in the liver a n d k i d n e y s are s h o w n in Tables 2 a n d 3. Eighty to n i n e t y p e r c e n t of Cd in the liver a n d k i d n e y s was f o u n d to be in the s u p e r n a t a n t . Table 1 Organ Weight (Liver, Kidneys, Adrenals, and Testes) of the Control and Cd Treated Rats (mean _+SD) Organs, g Dose group
n
Control .8 mg 1.5 mg 3.0 mg
6 6 6 6
Liver 11.6 13.2 11.2 11.3
_+ 1.1 ___ 1.3" + 1.5 _+ .8
Kidneys
Adrenals
2.4 2.4 2.4 2.3
.05 .05 .05 .07
+_ .2 ___ .2 ___ .3 ___ .2
_+ .01 --- .01 +-- .01 ___ .02
Testes 3.1 3.0 1.0 1.0
_+_ .1 ___ .1 +__ .1"* _+ .1"*
Significantly different from the control:*p < .05; **p < .01 Biological Trace Element Research
Vol. 14, 1987
Influence of Cd on Renal and Hepatic Zn and Cu
241
Zinc concentration increased significantly in the liver of the Cd groups. Eight-two to eighty-five percent of Zn was found in the supernatant. In the kidneys of Cd groups, Zn concentrations increased slightly. Sixty-seven to seventy-six percent of Zn was in the supernatant. Copper concentrations in the liver in the .8 and 1.5 mg groups were not significantly different from that in the control group, but Cu in the 3.0 mg group was decreased to two-thirds of the control group. In the kidneys, Cu concentrations of the Cd groups increased markedly, however that in the 3.0 mg group showed significant decrease compared to the 1.5 mg group. The increase of Cu concentration in kidneys was mainly found in the supernatant.
Cd and Zn Concentrations in lVlT of Liver and Kidneys HPLC gel filtration profiles of liver supernatant obtained from the control and 3.0 mg groups are shown in Fig. 2. The elution position of MT was confirmed by continuous 250 nm absorbance monitoring and AAS analyses of Cd and Zn. MT was eluted in 30.6-36.5 min and was separated into two peaks, MT II and MT l, respectively (7). Cadmium recovery after HPLC separation was 103% in the 3.0 mg group. Zinc recovery was 95% and 82% in the 3.0 mg and the control group, respectively. Cadmium and Zn contents of MT fractions are summarized in Table 4. The Cd concentration of MT fractions in the liver and kidneys was increased directly proportional to the dosage of Cd. In comparison to the control group, the Zn concentration of liver MT fractions was markedly increased by Cd administration. The supernatant of Cd groups contained 40-50% of Zn in MT fractions. In the kidneys, the Zn concentration of MT fractions in the Cd groups did not differ from that in the control group.
DISCUSSION This study was conducted to investigate the effects of Cd or Zn and Cu levels in the liver and kidneys of rats after sc Cd injection. The results of Zn and Cu analyses (Table 2 and 3) indicate that Cd administration affects markedly the metabolism of essential trace elements, Zn and Cu. The Cd-induced increase of liver Zn concentration agrees with previous investigations. Ashby et al. (3) obtained a Cd-induced increase of liver Zn concentration after a sc injection of CdC12 into rats, and this increase in liver Zn is correlated with an increase of Cd-induced liver MT. By oral administration, Stonard and Webb (1), Wesenberg et al. (8), and Weigel et al. (9) reported a similar increase in liver Zn concentration. The Cu concentration of the liver in the 3.0 mg group was decreased to twothirds of the control. Other researchers did not observe the decrease of Cu concentration (1,3,9). In the kidneys, the increase of Cu concentration Biological Trace Element Research
Vol. 14, 1987
Kunifuji, Nakamura, and Takasugi
242
Table 2 Cadmium, Zinc, and Copper Concentration (p,g/g Wet Wt) in Liver of Control and Cd Treated Rats (mean _+ SD) Cd dose group Control .8 mg 1.5 mg 3.0 mg
Cd n
Total
Sup."
Ppt?
6
ND
ND
ND
6 6 6
100.6 + 9.5 185.4 N _+ 15.5 307.8,~, +_ 41.7
88.4 -+ 8.3 159.8 ~ _+ 13.4 270.7,~, +- 53.3
14.7 _+ 2.0 25.6 ~ _+ 2.3 36.4,~, _+ 6.5
Zn Total Control .8 mg 1.5 mg 3.0 mg
34.3 75.6** 88.811 85.4**
Sup.
+ 2.5 _+ 4.8 _+ 4.6 _+ 10.9
Ppt.
22.9 _+ 1.7 62.2** _+ 3.9 73.2~ + 4.5 72.2** _+ 17.2
11.1 15.1"* 17.2"~ 14.5",*
+ .5 _+ .9 _+ .7 _+ 1.8
Cu Total Control .8 mg 1.5 mg 3.0 mg
2.6 2.8 2.6 1.7~
_+_ _+ +_ _+
Sup. .1 .4 .3 .3""
2.1 2.1 1.7 1.2~
_+ _+ _+ +
Ppt. .1 .4 .3 .2``
.8 .9 .9 .6~
+ + + _+
.1 .2 .6 .1``
~ ~'Precipitate. N~Not detectable. Significantly different from the control: *p < .05; **p < .01. Significantly different from the Cd .8 mg group: ep < .05; ~ p < .01. Significantly different from the Cd 1.5 rng group: Ap < .05; ``p < .01.
in Cd g r o u p s in the p r e s e n t s t u d y was in a c c o r d a n c e with the observation by S t o n a r d a n d W e b b (1). H o w e v e r , Weigel et al. (9) did not observe C d - i n d u c e d c h a n g e s of Cu c o n c e n t r a t i o n in the kidneys. T h e r e is s o m e d i s a g r e e m e n t in the literature about the effects of Cd on Z n a n d Cu levels of liver a n d kidneys. These d i s a g r e e m e n t s m a y result from the difference of Cd dosage, as well as route a n d period of administration. The source of i n c r e a s e d Z n a n d Cu in the liver a n d k i d n e y s m a y be either s e c o n d a r y to an e n h a n c e d Z n a n d Cu absorption from the small intestine or a d e c r e a s e of biliary a n d / o r intestinal excretion. Starcher et al. (10) s h o w e d Z n absorption was directly proportional to intestinal MT levels a n d i m p l y a significant role for MT in Z n absorption. S u g a w a r a (11) failed to s h o w an increase of Z n u p t a k e from the jejun u m of Cd e x p o s e d rats. Evans et al. (12) d e m o n s t r a t e d that Z n h o m e o stasis in rats was m a i n t a i n e d by Z n secretion from the intestine. Suzuki
Biological Trace Element Research
Vol. 14, 1987
Influence of Cd on Renal and Hepatic Zn and Cu
243
Table 3 Cadmium, Zinc, and Copper Concentration (l~g/g Wet Wt) in Kidneys of Control and Cd Treated Rats (mean (range)) Cd dose group
Total
Cd Sup?
Control .8 mg 1.5 mg 3.0 mg
ND
ND
ND
64.3 (62.3 - 66.3)" 119.7 ~ (119.2 - 120.1) 145.2",(143.5 - 146.9)
52.6 (51.0 - 54.1) 99.8" (99.0 - 100.6) 124.55 (123.4 - 125.5)
14.3 (14.3 - 14.3) 24.1" (23.9 - 24.3) 27.6" (27.0 - 28.1)
Ppt. "
Zn Total Control .8 nag 1.5 rng 3.0 mg
23.9 (22.5 - 25.3) 31.2 (30.9 ~ 31.4) 26.2" (26.2 - 26.2) 25.6~ (25.6 - 25.6)
Sup. 13.8 21.0 19.5 19.5
(13.5 (19.1 (18.6 (18.4
-
Ppt. 13.9) 22.8) 20.4) 20.4)
8.7 (8.1 9.8 (9.7 7.8o (7.7 6.8" (6.6 -
9.3) 9.8) 7.8) 7.0)
Cu Total Control .8 mg 1.5 mg 3.0 mg
2.6 (2.5 - 2.7) 7.2 (5.8 ~ 8.5) 7.4** (7.3 ~ 7.5) 3.9"" (3.8 ~ 4.0)
Sup. 1.1 (0.9 ~ 1.2) 3.6 (2.4 ~ 4.7) 5.0* (4.8 ~ 5.2) 2.2"" (2.1 ~ 2.3)
Ppt. 1.3 2.0 1.9 1.2
(1.2 (1.8 (1.8 (1.1
~ ~ ~ ~
1.3) 2.1) 2.0) 1.2)
~ value in parenthesis stands for the maximum and minimum values obtained from the pooled sample of three rats. ~Supernatant. 'Precipitate. Significantly different from the control: *p < .05; **p < 0.01. Significantly different from the Cd .8 mg group: ~ < .05; Np < .01. Significantly different from the Cd .5 mg group: Ap < .05; ~ p < .01.
(12) o b s e r v e d no significant c h a n g e of p l a s m a Z n levels in Cd e x p o s e d rats. A c c u m u l a t e d Z n in the liver of Cd e x p o s e d rats m a y be c a u s e d by inhibition of biliary a n d intestinal Z n excretion. A s h b y et al. (3) s h o w e d biliary excretion of Cu was m a r k e d l y inhibited after Cd a d m i n i s t r a t i o n . These a u t h o r s a n d Suzuki (13) o b s e r v e d an increase of p l a s m a Cu a n d c e r u l o p l a s m i n levels following Cd administration. In the plasma, 90% of total Cu was in h i g h m o l e c u l a r w e i g h t protein fraction (ceruloplasmin) a n d 8% in MT fraction. Metal c o m p o s i t i o n of MT in the p l a s m a was different from that of liver a n d k i d n e y s , a n d p l a s m a MT c o n t a i n e d Cd a n d Cu, but not Z n (13). I n t r a v e n o u s l y injected Cd-MT a c c u m u l a t e d m a i n l y in the k i d n e y s (14,15,16). Plasma MT m a y be filtrated t h r o u g h renal glomeruli a n d reabsorbed into proximal tubules. Cu rich p l a s m a MT m a y also contribute to the Cu increase in the kidneys.
Biological Trace Elernent Research
Vol. 14, 1987
Kunifuji, Nakamura, and Takasugi
244
E r 04
1.0 ~-
Control
3.0rag Group
0.8-
0
bJ c.1
MT
0.6-
Z
,
