~#Copyright 1985 by The Humana Press Inc. All rights of anv nature whatsoever reserved.
0163-4984, 85, 07()5-0169S02.20
The Effect of Sodium Selenite Toxicity on Tissue Distribution of Zinc, Iron, and Copper in Rats S. Y. CHEN,* P. J. COLLIPP, AND J. M. HSU Nassau County ,Medical Center, Department of Pediatrics, East ,Meadow, NY 1I554, USA; and Veterans Administration ,Medical Center, Bay Pines, FL 33504, USA
ABSTRACT Male Sprague-Dawley rats were used to determine the effects of subtoxic and toxic concentrations of selenite in the drinking water on tissue distribution of zinc (Zn), iron (Fe), and copper (Cu). Se (as sodium selenite) was provided in drinking water at concentrations of 0, 2, 4, and 8 ppm. At 19 d, half of the rats in 4 and 8 ppm Sesupplemented groups were kept on drinking water alone for additional 13 d. All rats w e r e sacrificed at the end of 32 d of experiment. Heart, liver, and kidney were analyzed for the concentrations of Fe, Zn, and Cu by atomic absorption spectrophotometry and of Se bv a fluorometric method. Results indicated that rats receiving 4 and 8 ppm Se in drinking water showed a marked reduction in food intake and a reduced growth rate. These adverse effects were quickly reversed when high Se intake was discontinued. Se toxicitv caused minimal change in zinc status, reduced tissue iron concentrations and caused a marked increase in copper contents in heart, liver, and kidnev. The latter findings were only partly reversed after removal of Se in drinking water. The accumulation of Cu in the tissues of Se-toxic rats provides the evidence of some interaction between Se and Cu. Index Entries: Sodium selenite; toxicity and Zn, Fe, and Cu tissue distribution of; toxicity, Zn, Fe, and Cu tissue distribution in Se; tissue distribution, of Zn, Fe, and Cu in Se toxicity; zinc, Se toxicity and tissue distribution of iron, Se toxicity and tissue distribution of; copper, Se toxicity, and tissue distribution of; selenite, toxicity and Zn, Fe, and Cu tissue distribution of. *Author to w h o m all correspondence and reprint requests should be addressed. Biological Trace Element Research
] 69
VoL 7, 1985
Chen, Collipp, and Hsu
170
INTRODUCTION Selenium (Se) is an essential dietary constituent for animals and also for h u m a n s , but in excess, it is toxic. Although most reports of selenium intoxication have resulted from chronic ingestion of organic selenium, sodium selenite has also been reported to be toxic. Because of the important implications of Se toxicology to animals and h u m a n s , we have carried out a n u m b e r of studies designed to elucidate the effects of Se toxicity on tissue distribution of zinc (Zn), iron (Fe), and copper (Cu) in rats. We f o u n d that copper concentrations in liver, kidney, a n d heart were markedly higher in Se toxic rats than in nontoxic controls.
MATERIALS AND METHODS T w e n t y - f o u r male Sprague-Dawley rats, weighing 68-95 g were h o u s e d individually in stainless-steel, screen-covered plastic cages. They w e r e divided r a n d o m l y into eight groups of three rats each and treated as described in Table 1. Sodium selenite was provided in the drinking water which was given ad libitum. The rats were fed on a p o w d e r e d commercial laboratory feed, Ralston Purina rat chow that contained 0.1 p p m of Se, 58.0 p p m of Zn, 18.0 p p m of Cu, 198 ppm of Fe, and 23.0% protein. After 32 d of feeding, all rats were sacrificed and samples of blood, liver, kidney, and heart were collected. Concentrations of Zn, Cu, and Fe were estimated by the m e t h o d of Hsu and Smith (1). Commercially available solutions standardized for atomic absorption (Fisher Scientific Co., Fairlawn, NJ) were used throughout. These standards were frequently checked against appropriate references that were prepared from metals of the highest purity obtainable; no differences in absorption could be deTABLE 1 Experimental Design Group A C E (E2) B F (F2)
D E1 F1
Treatment
Final body wt in 32 d, g
0 ppm Se in drinking water (DW) 273 + 6.5 2 ppm Se in DW 208 -4- 7.9" 4 ppm Se in DW 147 _ 7.5',~ Pair-fed of E 240 -+ 8.9 8 ppm Se in DW 65 + 0.3 ~ Pair-fed of F 156 --+ 6.1 19 d on 4 ppm Se in DW and 13 d received no Se in DW 202 - 6.5' 19 d on 8 ppm Se in DW and 13 d received no Se in DW 185 -+ 10.1'
'Statistical difference from group A (p < 0.001). 'Statistical difference from respective pair-fed groups (p < 0.01). ~Statistical difference from respective groups E and F (p < 0.01). Biological Trace Element Research
VoL 7, 1985
Selenite Toxicity and Zinc, Iron, and Copper Distribution
171
tected. For the m e a s u r e m e n t of Se, s a m p l e s of blood a n d o t h e r soft tissues w e r e lyophilized and digested with a m i x t u r e of nitric a n d perchloric acids. After digestion, the s a m p l e s t r e a t e d with 2,3d i a m i n o n a p h t h a l e n e a n d Se was extracted by n-hexane. The a m o u n t s of Se in the n - h e x a n e extract w e r e d e t e r m i n e d by a f l u o r o m e t e r as d e s c r i b e d in o u r p r e v i o u s publication (2). The significance of the d i f f e r e n c e bet w e e n the m e a n s of two g r o u p s of values was d e t e r m i n e d by S t u d e n t ' s t-test or D u n c a n ' s N e w Multiple-Range Test w h e n applicable.
RESULTS T h e effects of various a m o u n t s of Se intake in d r i n k i n g w a t e r on g r o w t h are s h o w n in Table 1. Two p p m of Se s u p p l e m e n t a t i o n h a d slight but significant r e d u c t i o n in bodv w e i g h t gain. The rats d r i n k i n g 4 p p m of Se ( G r o u p E) h a d a mild g r o w t h retardation in the first 15 d a n d then s h o w e d a g r a d u a l i m p r o v e m e n t in d e v e l o p m e n L The rats s u p p l e m e n t e d with 8 p p m of Se h a d b o d y weight loss, coarse hair, a n d general w e a k hess d u r i n g the entire experiment. In addition, these Se-intoxicated rats exhibited a s e v e r e r e d u c t i o n in food a n d w a t e r intake. After eliminating Se in d r i n k i n g water, the rats (Group E~ a n d F~) exhibited an i m m e d i a t e a n d m a r k e d i m p r o v e m e n t in general condition a n d w e i g h t gain, indicating that S e toxicity was reversible w h e n the intake of excess Se was stopped. Table 2 indicates Se retention in blood, heart, liver a n d k i d n e y as related to the a m o u n t of Se intake in drinking water. There was a direct
TABLE 2 Se Concentration in Blood, Liver, Kidney, and Heart
Group A C E B' F D:
Se in drinking water Blood, ppm Ixg/mL 0 2 4 0 8 0
0.33 0.74 0.78 0.34 0.98 0.35
_ • -+ • •
Heart, Liver, Kidney, ~xg/g (wet p.g/g (wet wt) ~,g/g (wet wt) wt) 0.02" 0.11" 0.13' 0.03 0.15 ~' 0.03
'Mean • SD. ~ignificant difference between 'Significant difference between ~ difference between ~Significant difference between 'B, pair-fed to group E. -;D, pair-fed to group F. Biological Trace Element Research
0.80 3.21 5.81 0.68 12.41 0.89
C and E and F and F and
• 0.08 1.11 • 0.25 __+2.51" 4.48 -+ 1.50~' • 1.4(Y 8.24 +__ 1.90' • 0.22 0.78 -+ 0.15 • 2.11"' 17.93 • 2.50 '.~ z 0.18 1.33 • 0.25
0.25 0.70 0.61 0.28 0.93 0.28
+-- 0.06 • 0.18" _ 0.10 +_ 0.09 +-- 0.26": • 0.05
A (p ~ 0.01/. A, B, D (p ~ 0.01). A, B, D (p ~ 0.01) and F and C (p ~ 0.05). E (p ~ 0.01/.
Vol. 7. 1985
Chen, Collipp, and Hsu
172
correlation between the intake of Se and Se concentrations in the liver and kidney. C o m p a r e d to control rats (Group A) and pair-fed animals (Groups B and D), rats that received 2, 4, and 8 p p m of Se in drinking water s h o w e d that Se content in kidney was about 4, 8, and 13-fold increased, respectively. Similar results (Table 2) were obtained in the liver. The a m o u n t of Se in the heart and blood were considerably less than those in the kidney and liver. However, significant increases were also observed in Se-intoxicated rats compared to control animals. The comparisons of Se concentration in various tissues between the rats receiving Se treatment for 32 d, and the rats receiving plain drinking water for 13 d after 19 d of Se treatment are graphically illustrated in Figs. 1 to 4. In every instance, results clearlv indicate that the excess Se in the blood and other soft tissues can be completely eliminated by r e m o v i n g Se from drinking water. Table 3 discloses the effects of Se intake on Zn distribution in various tissues. Se toxicity appeared to have no effect on cardiac Zn content. H o w e v e r , drinking water containing 2 ppm of Se (Group C) reduced Zn contents in both liver and kidney as compared to control rats (Group A) (p < 0.01). The pair-fed rats (Group B) also had r e d u c e d Zn concentration (p < 0.01) in liver and kidney, possibly related to their r e d u c e d food intake. In contrast, the rats drinking plain water for 13 d after 19 d on 4 p p m of Se treatment (Group E~) had an increase of hepatic Zn content. A Se Content in Blood
! ].2Z I ~ 1.1-!
~c
0.9~
=-
0.7 -~
O
'St+' in DW, {2 day>,
Se in DX", 19 day~ L_.'] N() Se, 1{ days
0.6s
"J
05-
v~
0.40.30.2 J
0.1-
~
e ()f)pm
4ppm
8ppm
Fig. 1. Blood selenium concentration in rats receMng sodium selenite in drinking water. Biological Trace Element Research
Vol. 7, 1985
Selenite Toxicity and Zinc, Iron, and Copper Distribution
173
Se C o n t e n t in Heart 13f ~ U Se in DW,?>2 days I 2 I--1 Se in DW, 19 days ].lNo Se , 1~> days 10
E a~
0.9-
-~
0.8-
0163-4984, 85, 07()5-0169S02.20
The Effect of Sodium Selenite Toxicity on Tissue Distribution of Zinc, Iron, and Copper in Rats S. Y. CHEN,* P. J. COLLIPP, AND J. M. HSU Nassau County ,Medical Center, Department of Pediatrics, East ,Meadow, NY 1I554, USA; and Veterans Administration ,Medical Center, Bay Pines, FL 33504, USA
ABSTRACT Male Sprague-Dawley rats were used to determine the effects of subtoxic and toxic concentrations of selenite in the drinking water on tissue distribution of zinc (Zn), iron (Fe), and copper (Cu). Se (as sodium selenite) was provided in drinking water at concentrations of 0, 2, 4, and 8 ppm. At 19 d, half of the rats in 4 and 8 ppm Sesupplemented groups were kept on drinking water alone for additional 13 d. All rats w e r e sacrificed at the end of 32 d of experiment. Heart, liver, and kidney were analyzed for the concentrations of Fe, Zn, and Cu by atomic absorption spectrophotometry and of Se bv a fluorometric method. Results indicated that rats receiving 4 and 8 ppm Se in drinking water showed a marked reduction in food intake and a reduced growth rate. These adverse effects were quickly reversed when high Se intake was discontinued. Se toxicitv caused minimal change in zinc status, reduced tissue iron concentrations and caused a marked increase in copper contents in heart, liver, and kidnev. The latter findings were only partly reversed after removal of Se in drinking water. The accumulation of Cu in the tissues of Se-toxic rats provides the evidence of some interaction between Se and Cu. Index Entries: Sodium selenite; toxicity and Zn, Fe, and Cu tissue distribution of; toxicity, Zn, Fe, and Cu tissue distribution in Se; tissue distribution, of Zn, Fe, and Cu in Se toxicity; zinc, Se toxicity and tissue distribution of iron, Se toxicity and tissue distribution of; copper, Se toxicity, and tissue distribution of; selenite, toxicity and Zn, Fe, and Cu tissue distribution of. *Author to w h o m all correspondence and reprint requests should be addressed. Biological Trace Element Research
] 69
VoL 7, 1985
Chen, Collipp, and Hsu
170
INTRODUCTION Selenium (Se) is an essential dietary constituent for animals and also for h u m a n s , but in excess, it is toxic. Although most reports of selenium intoxication have resulted from chronic ingestion of organic selenium, sodium selenite has also been reported to be toxic. Because of the important implications of Se toxicology to animals and h u m a n s , we have carried out a n u m b e r of studies designed to elucidate the effects of Se toxicity on tissue distribution of zinc (Zn), iron (Fe), and copper (Cu) in rats. We f o u n d that copper concentrations in liver, kidney, a n d heart were markedly higher in Se toxic rats than in nontoxic controls.
MATERIALS AND METHODS T w e n t y - f o u r male Sprague-Dawley rats, weighing 68-95 g were h o u s e d individually in stainless-steel, screen-covered plastic cages. They w e r e divided r a n d o m l y into eight groups of three rats each and treated as described in Table 1. Sodium selenite was provided in the drinking water which was given ad libitum. The rats were fed on a p o w d e r e d commercial laboratory feed, Ralston Purina rat chow that contained 0.1 p p m of Se, 58.0 p p m of Zn, 18.0 p p m of Cu, 198 ppm of Fe, and 23.0% protein. After 32 d of feeding, all rats were sacrificed and samples of blood, liver, kidney, and heart were collected. Concentrations of Zn, Cu, and Fe were estimated by the m e t h o d of Hsu and Smith (1). Commercially available solutions standardized for atomic absorption (Fisher Scientific Co., Fairlawn, NJ) were used throughout. These standards were frequently checked against appropriate references that were prepared from metals of the highest purity obtainable; no differences in absorption could be deTABLE 1 Experimental Design Group A C E (E2) B F (F2)
D E1 F1
Treatment
Final body wt in 32 d, g
0 ppm Se in drinking water (DW) 273 + 6.5 2 ppm Se in DW 208 -4- 7.9" 4 ppm Se in DW 147 _ 7.5',~ Pair-fed of E 240 -+ 8.9 8 ppm Se in DW 65 + 0.3 ~ Pair-fed of F 156 --+ 6.1 19 d on 4 ppm Se in DW and 13 d received no Se in DW 202 - 6.5' 19 d on 8 ppm Se in DW and 13 d received no Se in DW 185 -+ 10.1'
'Statistical difference from group A (p < 0.001). 'Statistical difference from respective pair-fed groups (p < 0.01). ~Statistical difference from respective groups E and F (p < 0.01). Biological Trace Element Research
VoL 7, 1985
Selenite Toxicity and Zinc, Iron, and Copper Distribution
171
tected. For the m e a s u r e m e n t of Se, s a m p l e s of blood a n d o t h e r soft tissues w e r e lyophilized and digested with a m i x t u r e of nitric a n d perchloric acids. After digestion, the s a m p l e s t r e a t e d with 2,3d i a m i n o n a p h t h a l e n e a n d Se was extracted by n-hexane. The a m o u n t s of Se in the n - h e x a n e extract w e r e d e t e r m i n e d by a f l u o r o m e t e r as d e s c r i b e d in o u r p r e v i o u s publication (2). The significance of the d i f f e r e n c e bet w e e n the m e a n s of two g r o u p s of values was d e t e r m i n e d by S t u d e n t ' s t-test or D u n c a n ' s N e w Multiple-Range Test w h e n applicable.
RESULTS T h e effects of various a m o u n t s of Se intake in d r i n k i n g w a t e r on g r o w t h are s h o w n in Table 1. Two p p m of Se s u p p l e m e n t a t i o n h a d slight but significant r e d u c t i o n in bodv w e i g h t gain. The rats d r i n k i n g 4 p p m of Se ( G r o u p E) h a d a mild g r o w t h retardation in the first 15 d a n d then s h o w e d a g r a d u a l i m p r o v e m e n t in d e v e l o p m e n L The rats s u p p l e m e n t e d with 8 p p m of Se h a d b o d y weight loss, coarse hair, a n d general w e a k hess d u r i n g the entire experiment. In addition, these Se-intoxicated rats exhibited a s e v e r e r e d u c t i o n in food a n d w a t e r intake. After eliminating Se in d r i n k i n g water, the rats (Group E~ a n d F~) exhibited an i m m e d i a t e a n d m a r k e d i m p r o v e m e n t in general condition a n d w e i g h t gain, indicating that S e toxicity was reversible w h e n the intake of excess Se was stopped. Table 2 indicates Se retention in blood, heart, liver a n d k i d n e y as related to the a m o u n t of Se intake in drinking water. There was a direct
TABLE 2 Se Concentration in Blood, Liver, Kidney, and Heart
Group A C E B' F D:
Se in drinking water Blood, ppm Ixg/mL 0 2 4 0 8 0
0.33 0.74 0.78 0.34 0.98 0.35
_ • -+ • •
Heart, Liver, Kidney, ~xg/g (wet p.g/g (wet wt) ~,g/g (wet wt) wt) 0.02" 0.11" 0.13' 0.03 0.15 ~' 0.03
'Mean • SD. ~ignificant difference between 'Significant difference between ~ difference between ~Significant difference between 'B, pair-fed to group E. -;D, pair-fed to group F. Biological Trace Element Research
0.80 3.21 5.81 0.68 12.41 0.89
C and E and F and F and
• 0.08 1.11 • 0.25 __+2.51" 4.48 -+ 1.50~' • 1.4(Y 8.24 +__ 1.90' • 0.22 0.78 -+ 0.15 • 2.11"' 17.93 • 2.50 '.~ z 0.18 1.33 • 0.25
0.25 0.70 0.61 0.28 0.93 0.28
+-- 0.06 • 0.18" _ 0.10 +_ 0.09 +-- 0.26": • 0.05
A (p ~ 0.01/. A, B, D (p ~ 0.01). A, B, D (p ~ 0.01) and F and C (p ~ 0.05). E (p ~ 0.01/.
Vol. 7. 1985
Chen, Collipp, and Hsu
172
correlation between the intake of Se and Se concentrations in the liver and kidney. C o m p a r e d to control rats (Group A) and pair-fed animals (Groups B and D), rats that received 2, 4, and 8 p p m of Se in drinking water s h o w e d that Se content in kidney was about 4, 8, and 13-fold increased, respectively. Similar results (Table 2) were obtained in the liver. The a m o u n t of Se in the heart and blood were considerably less than those in the kidney and liver. However, significant increases were also observed in Se-intoxicated rats compared to control animals. The comparisons of Se concentration in various tissues between the rats receiving Se treatment for 32 d, and the rats receiving plain drinking water for 13 d after 19 d of Se treatment are graphically illustrated in Figs. 1 to 4. In every instance, results clearlv indicate that the excess Se in the blood and other soft tissues can be completely eliminated by r e m o v i n g Se from drinking water. Table 3 discloses the effects of Se intake on Zn distribution in various tissues. Se toxicity appeared to have no effect on cardiac Zn content. H o w e v e r , drinking water containing 2 ppm of Se (Group C) reduced Zn contents in both liver and kidney as compared to control rats (Group A) (p < 0.01). The pair-fed rats (Group B) also had r e d u c e d Zn concentration (p < 0.01) in liver and kidney, possibly related to their r e d u c e d food intake. In contrast, the rats drinking plain water for 13 d after 19 d on 4 p p m of Se treatment (Group E~) had an increase of hepatic Zn content. A Se Content in Blood
! ].2Z I ~ 1.1-!
~c
0.9~
=-
0.7 -~
O
'St+' in DW, {2 day>,
Se in DX", 19 day~ L_.'] N() Se, 1{ days
0.6s
"J
05-
v~
0.40.30.2 J
0.1-
~
e ()f)pm
4ppm
8ppm
Fig. 1. Blood selenium concentration in rats receMng sodium selenite in drinking water. Biological Trace Element Research
Vol. 7, 1985
Selenite Toxicity and Zinc, Iron, and Copper Distribution
173
Se C o n t e n t in Heart 13f ~ U Se in DW,?>2 days I 2 I--1 Se in DW, 19 days ].lNo Se , 1~> days 10
E a~
0.9-
-~
0.8-
