Archlvu of
Arch. Toxicol. 36, 63--69 (1976)
TOXICOLOGY by Springer-Verlag 1976
Differences in Distribution and Excretion of Selenium and Cadmium or Mercury after Their Simultaneous Administration Subcutaneously in Equimolar Doses* L. Magos** and M. Webb MRC Toxicology Unit, MRC Laboratories Woodmansterne Road, Carshalton, Surrey, United Kingdom
Abstract. Forty-eight hours after the simultaneous administration of either 1.8 ~moles Se (as Na2SeO3) to 200 g male rats with equivalent doses of Cd (as CdCI2) or 0.5 ~moles Se with equivalent doses of Hg (as HgC12) the retention of selenium at the subcutaneous injection site was affected only when it was given in a single injection with the heavy metal. The retention of cadmium was increased only if selenium was injected separately and the retention of mercury was increased both in single and separate injections. When there was an increase in retention at the site of injection, this made up less than 8% of the dose for selenium and mercury and 3% for cadmium and thus could not explain either changes in distribution or the reported protective effects by one metal against the other. The shift in the distribution pattern of one metal caused by the other metal was similar, and independent of whether they were injected in single or separate injections. However the shift in the distribution of interacting metals showed some differences indicating that interaction could not be mediated through the formation of a stable complex between selenium on the one hand and mercury or cadmium on the other.
Key words: Selenium - Cadmium - Mercury - Interaction between mettals.
Zusammenfassung. 200 g schwere m/innliche Ratten erhielten gleichzeitig entweder 1,8 ~mol Selen (als Na2SeO3) und eine equivalente Dosis Kadmium (als CdCI2) oder 0,5 gmol Selen und eine equivalente Dosis Quecksilber (als HgClz). Die Retention des Selens an der Stelle der subcutanen Injektion 48 Std nach der Applikation wurde nur dann beeinflul3t, wenn dieses in einer einzigen Injektion mit dem Schwermetall gegeben wurde. Die Retention des Kadmiums nahm nur zu, wenn das Selen getrennt injiziert wurde, und die Retention des Quecksilbers war sowohl bei gemeinsamer als auch bei getrennter Injektion erh6ht. Wenn eine Zunahme der Retention an der Injektionsstelle gefunden wurde, so machte diese * Supported partly by International Atomic Energy Agency, Grant Number 1530/RB ** To whom offprint requests should be sent
64
L. Magos and M. Webb weniger als 8% der Dosis f/ir Selen ud Quecksilber und 3% ffir K a d m i u m aus. Dieses reicht nicht aus, um entweder die Unterschiede in der Verteilung oder den protektiven Effekt, den die Metalle untereinander aus/iben sollen, zu erklfiren. Die Verfinderung des Verteilungsmusters eines Metalls, verursacht durch ein anderes Metall, war/ihnlich und unabhfingig davon, ob sie in einer einzigen oder in getrennten Injektionen gegeben wurden. Allerdings zeigt die .~nderung der Verteilung von Metallen, die in Wechselwirkung treten, einige Unterschiede, die darauf hinweisen, dab dieser Effekt nicht auf der Bildung eines stabilen Komplexes zwischen Selen auf der einen Seite und Quecksilber und Kadmium auf der anderen beruht.
Sehliisselwiirter: Selen - K a d m i u m - Quecksilber - Wechselwirkung zwischen Metallen.
Introduction The protective effect of selenium against cadmium and mercury toxicity (Kar et al., 1960; Parizek, 197 I; G u n n e t al., 1968; Eybl et al., 1969) or the reversal of selenium toxicity by the same heavy metals (Hill, 1974) presents some interesting problems. It has been observed that to produce the maximum effect on the distribution of mercury, equimolar doses of selenium (as selenite) are necessary (Moffitt and Clary, 1974). Similarly equimolar doses of Hg are required to give an optimum protection against selenium (Hill, 1975). McConnel and Carpenter (1971) reported that cadmium mixed with selenium, but not when injected separately, slowed down the absorption of selenium from the injection site. On the b~isis of these observations it has been proposed that the protective effect observed between the two heavy metals and selenium is based on the formation of a heavy metal-selenium complex (Hill, 1975). However in the experiments in which retention of selenium was followed (McConnel and Carpenter, 1971) the Se dose was 57 ~moles/kg and the cadmium dose ranged from 2 8 . 5 - 2 2 8 ~moles/kg. These doses are extremely high: according to our experience in rats 22 ~moles/kg Cd given subcutaneously is able to cause local tissue damage judged from the palpable hardening of the tissue surrounding the injection site and 127 vmoles/kg is the LD50 (unpublished observation).
Materials and Methods
Animals and Experimental Schedule Male rats of Porton Wistar strain of approximately 200 g body weight were used. In control animals the radioactively labelled test metal was injected subcutaneously in 0.2 ml volume into the right hind leg above the knee. In experimental animals the test metal and the unlabeUed interfering metal was injected either mixed together in 0.2 ml volume into the same site or the tested metal was injeced into the leg and the interfering metal under the skin of the neck in the same volume. Animals were kept in metabolic cages, two rats in one cage, and urine and faeces were collected separately. Forty-eight hours after injection animals were killed by decapitation, blood from the cervical blood vessels was collected in a beaker and was weighed. Liver and kidneys were removed and both hind legs were removed by cutting through the hip joint.
Interaction of Selenium with Cadmium and Mercury
65
Injection Solutions Cold stock solutions of CdC12, HgCI2 or Na2SeOa were prepared in saline and were labelled with the corresponding isotope (llSmCd, 2~ or 75Se) supplied by the Radiochemieal Centre (Amersham, Bucks) Volumes of the injection solution were 0.2 ml. The metal was injected either alone or mixed in a ratio of 1 : 1 with the other metal. Only one of them was labelled. The precipitate of Se and Cd was dissolved by adding 0.1 N HCI drop by drop to the solution before diluting to the final volume. Radioactivities in 0.2 ml injection solutions were approximately: ll5mCd: 1.60 ~Ci, Hg: 0.15 pCi; 75Se: 0.05 ~Ci.
Radioactive Measurements Live animals placed in a 14 x 6 x 6.5 cm plexiglass box were counted between two NaI crystal pairs (No. N656, EKCO Electronics Ltd., Southend on-Sea, Essex) facing each other. Faeces and urine samples, blood, liver and legs were counted in beakers in the same system with the following efficiencies: HSmCd:0.6%, 2~ 7.7%, 75Se: 28.0%. Kidneys were counted in a well shaped NaI crystal with efficiencies of 2.5%, 31.0% and 80.0% for llsmCd2+, 2~ and 755e4+ respectively. Injection standards in I ml acid permanganate solution were counted and concentrations in organs and in whole animals were estimated after corrections for geometrical differences by relating their radioactivities to standards. Radioactivity retained at the injection site was determined from the difference between the injected and the control leg. Radioactivity in the total blood was calculated on the assumption that blood made up 7.0% of the body weight.
Statistical Calculations Statistical differences were estimated by the Student t-test, in the case of radioactivity retained at the site of injection by one tailed test and in every other case by two tailed test.
Results C a d m i u m increased the retention o f selenium when the two metals were administered in a single subcutaneous injection, but had no effect on retention when they were injected separately (Table 1). Body burden of selenium at the end o f the 48 h period was higher when c a d m i u m was injected mixed with selenium than when c a d m i u m was injected separately. This effect was mainly due to a more pronounced decrease in the urinary excretion of selenium. Liver and kidney contents o f selenium were increased in both groups, but due to a smaller standard error the increase was more significant in the group which was given the two metals separately. M e r c u r y had a more profound effect on the distribution of selenium: body burden, liver, kidney and blood contents o f selenium increased and excretion decreased (Table 2). Retention at the injection site increased only when the two metals were injected simultaneously. The increase in the retention o f selenium at the injection site caused by heavy metals -- though statistically significant - amounted only to 6.7% o f the dose in the case o f mercury and to even less with cadmium. C o m p a r i s o n o f the control values showed some variation from one experiment to another and distribution in the experimental animals are c o m p a r e d only with their own controls.
66
L. Magos and M. Webb
Table 1. Effects of cadmium on the tissue contents and excretion of 75Se given as SeO 2 48 h after administration. 1.8 ~moles Se was injected either alone (controls) or mixed in a single injection with 1.8 ~moles Cd or the two metals were given separately 75Se contents in % of the dose Single injection
Injection site Body burden Liver Kidneys Blood Faeces Urine
Separate injection
Control N:-6
Experimental N=6
Control N=6
Experimental N=6
0.4 26.9 7.6 1.2 5.8 12.5 47.3
4.0 45.5 9.1 2.1 6.1 12.5 29.9
1.0 25.1 6.3 0.8 7.1 10.7 42.7
1.4 37.3 8.2 1.2 5.4 10.3 30.7
+ 0.15 _+ 1.10 + 0.37 + 0.12 _+ 0.32 _+ 0.74 + 2.24
• + + • • + +
0.71 a 2.36 a 0.89 0.62 0.37 1.01 1.21
+ 0.18 _+ 0.57 + 0.23 • 0.08 • 0.22 • 0.24 • 2.71
+ 0.17 + 2.30 a • 1.48a • 0.08 a _+ 0.82 • 1.21 • 2.23
a Significantly different from the control (P < 0.05) by the Student t-test. N - the number of animals, every faeces or urine sample was collected from two rats.
3"able 2. Effects of mercury on the tissue contents and excretion of 75Se given as SeO 2 48 h after administration. 0.5 p~moles Se was injected either alone (controls) or mixed in a single injection with 0.5 ~moles Hg or the two metals were injected separately 7SSe contents in % of the dose
Single injection
Injection site Body burden Liver Kidneys Blood Faeces (48 h) Urine (48 h)
Separate injection
Control N: 6
Experimental N=6
Control N=6
Experimental N=6
2.6 49.0 9.5 1.9 7.4 9.1 29.9
9.3 76.3 14.4 2.4 12.1 6.3 13.7
0.3 37.6 9.1 1.6 4.5 18.5 40.9
0.3 71.1 13.6 2.1 11.5 7.2 16.3
_+ 1.20 + 4.06 _+ 1.20 _+ 0.05 + 0.62 • 0.75 + 1.43
+ 1.73a +_ 1.68a + 0.91 a _+ 0.09 a _+ 1.09a + 0.89 +_ 0.92 a
+ 0.12 _+ 1.44 + 0.28 + 0.04 _+ 0.13 + 5.42 _+ 1.03
_+ 0.15 _+ 2.03 + 1.92a + 0.07 a _+ 0.52 _+ 0.17 • 1.38a
a Significally different from the control (P < 0.05) by the Student t-test. N the number of animals, every faeces or urine sample was collected from two rats.
C o n t r a r y t o t h e effect o f the t w o h e a v y m e t a l s o n t h e r e t e n t i o n o f selenium, t h e r e t e n t i o n o f c a d m i u m o r m e r c u r y b y s e l e n i u m w a s n o t h i g h e r if s e l e n i u m w a s a d m i n i s t e r e d w i t h t h e s e m e t a l s in a single as c o m p a r e d w i t h s e p a r a t e i n j e c t i o n s ( T a b l e s 3 a n d 4). In the c a s e o f c a d m i u m t h e r e w a s a slight, b u t s i g n i f i c a n t i n c r e a s e in t h e r e t e n t i o n o n l y if t h e m e t a l s w e r e i n j e c t e d s e p a r a t e l y . I n t h e c a s e o f m e r c u r y , selen i u m i n c r e a s e d t h e r e t e n t i o n b y 6 . 3 % in a single i n j e c t i o n a n d 7 . 2 % w h e n i n j e c t e d s e p a r a t e l y . T h e b o d y b u r d e n o f c a d m i u m w a s n o t c h a n g e d b y selenium, the liver
Interaction of Selenium with Cadmium and Mercury
67
Table 3. Effects of selenium on the distribution and excretion of us"Cd given as CdC12 48 h after administration. 1.8 ~tmoles Cd was injected either alone (controls) or mixed in a single injection with 1.8 ~moles Se or the two metals were injected separately
llSmCd contents in % of the dose Single injection
Injection site Body burden Liver Kidneys Blood Faeces (48 h) Urine (48 h)
Separate injection
Control N = 14
Experimental N = 14
Control N = 16
Experimental N = 16
15.5 86.9 48.0 2.7 1.0 6.3 0.7
16.1 89.0 37.4 2.8 3.0 5.8 0.9
15.8 85.4 49.8 2.7 0.9 5.6 0.8
18.1 85.5 34.0 2.6 3.1 7.1 1.4
• 0.79 • 1.45 • 0.86 • 0.12 • 0.15 • 0.72 _+ 0.09
• • • • • • •
0.56 1.82 1.27a 0.29 0.29 a 0.82 0.09
• 0.73 • 1.39 + 1.03 • 0.10 _+ 0.15 • 0.95 • 0.08
• 0.75 a • 1.63 • 0.62 a • 0.11 • 0.26 a • 1.47 _+ 0.34
a Significantly different from the control (P < 0.05) by the Student t-test. N the number of animals, every faeces or urine sample was collected from two rats.
Table 4. Effects of selenium on the tissue contents and excretion of 2~ given as HgCI 2 48 h after administration. 0.5 ~moles Hg was injected either alone (controls) or mixed in a single injection with 0.5 ~moles Se or the two metals were injected separately
2~
contents in % of the dose
Control N 8
Injection site Body burden Liver Kidneys Blood Faeces (48 h) Urine (48 h)
15.9 76.3 2.9 33.3 2.3 14.8 5.8
• • • • + • •
0.96 1.99 0.23 1.06 0.21 0.86 1.20
with Se Single injection N=8
Separate injection N=8
21.2 90.3 12.4 11.8 9.5 5.5 0.4
23.1 95.2 14.4 7.8 12.3 6.3 0.3
• • • • i • •
1.33a 1.72a 0.70 a 0.44 a 0.72 a 0.89 a 0.05 a
• • • + • • •
2.93 a 2.00 ~ 1.0P 0.98 a 0.77 a 1.50a 0.07 a
a Significantly different from the control (P < 0.05) by the Student t-test. N = the number of animals, every faeces or urine sample was collected from two rats.
content decreased and the blood content increased. Selenium had a more profound effect o n m e r c u r y w i t h i n c r e a s e s in b o d y b u r d e n a n d in the liver a n d b l o o d c o n t e n t o f m e r c u r y a n d w i t h d e c r e a s e s in t h e k i d n e y s , f a e c e s a n d urine. T h e shift in distribution w a s i n d e p e n d e n t o f w h e t h e r s e l e n i u m w a s i n j e c t e d s i m u l t a n e o u s l y w i t h the c a d m i u m or mercury, or was injected separately.
68
L. Magos and M. Webb
Discussion The present experiments showed that the retention of selenium was influenced by the presence of mercury or cadmium, but the extent of this effect expressed in percent of the dose was too small to explain differences in distribution. Though retention was not influenced when the heavy metals were injected at a different site, the shifts in body burden, excretion or distribution were in the same direction. If retention was increased by the formation of an insoluble complex, it seems reasonable to suppose that the heavy-metal selenium complex did not reach the blood as such, because the shift in the distribution was the same and independent of whether the two metals were injected in single or separate sites. Alternatively it might be that selenium absorbed from one site and the heavy metal from another site can compex together in the blood. There are, however, two observations which contradict the formation or existence of such a complex outside the injection site. Firstly, both cadmium and mercury increased the kidney contents of selenium, but selenium did not affect the content of cadmium and actually decreased the content of mercury in this organ. Liver level of selenium was lowered by cadmium or mercury but the liver level of mercury was increased by selenium. Selenium in this respect had an opposite effect on the liver content of cadmium which was decreased by selenium by more than 10%. In the case of an association between selenium and the heavy metals the shift in the distribution pattern ought to be similar. Secondly, contrary to the expected results, selenium given separately slightly increased the retention of the heavy metals at the injection site compared with experiments when selenium and the heavy metal were given together at the same site. Since the retention of cadmium or mercury alone at the injection site is approximately one order higher than that of selenium alone (see Tables) it is difficult to observe a few percent difference in retention in relation to the dose. Even so results suggest that the retention of selenium in the presence of mercury or cadmium or the retention of mercury and cadmium in the presence of selenium does not follow the same pattern and consequently might not be the result of the same mechanism. Thus it might be that local irritation, inflammatory reaction and tissue damage is responsible for the increased retention of selenium when injected with mercury or cadmium, and the retention of the two heavy metals is increased only after the metabolic conversion of selenium which takes place outside the injection site. It has been reported that plasma proteins do not bind selenite in vitro unless erythrocytes are present (Sandholm, 1974) which tranform selenite to a form which is able to be bound to plasma proteins, first of all to ~-lipoprotein and to unidentified fractions located electrophoretically between the al and a2 globulins (Sandholm, 1975). In the presence of Cd there is a shift in the binding of this transformed selenium to other protein fractions which bind cadmium and selenium in an atomic ratio of 1 : 1 (Gasiewicz and Smith, 1976). This binding is transitory and not affected by the preinjection level of plasma selenium (Gasiewicz and Smith, 1976) indicating that interactions at protein binding sites operate only at the distributory phase of selenium. It has been suggested that Se 2- forms a RS-Se-SR type of binding with proteins (Painter, 1941) by which their carrier capacity to heavy metals should be altered. Conversely cadmium or mercury through their affinity to thiol groups might affect the binding, metabolism or transport of selenium.
Interaction of Selenium with Cadmium and Mercury
69
In summary these experiments give evidence that if selenium forms an insoluble complex with cadmium or mercury, this cannot explain the effect of interaction on their distribution and the extent of change in their retention is too small to affect significantly their toxicity. Moreover, as the effect of selenium on cadmium or mercury differs from the effect of these heavy metals on the retention of selenium at the subcutaneous injection site, this makes questionable whether an insoluble complex is formed at all. Acknowledgement. The authors wish to thank Mr. A. R. Hudson for technical assistance.
References Eybl, V., Sykora, J., Mertl, F.: Einflul3 yon Natriumselenit, Natriumtellurit und Natriumsulfit auf Retention und Verteilung von Quecksilber bei M~iusen, Arch. Toxikol. 25, 296--305 (1969) Gasiewicz, T. A., Smith, J. C.: Interactions of cadmium and selenium in rat plasma in vivo and in vitro. Biochim. biophys. Acta (Amst.) 428, 113--122 (1976) Gunn, S. A.. Gould, T. C., Anderson, W. A. D.: Selectivity of organ response to cadmium injury and various protective measures. J. Path. Bact. 96, 89--96 (1968) Hill, C. H.: Reversal of selenium toxicity in chicks by mercury, copper and cadmium. J. Nutr. 104, 593-598 (1974) Hill, C. H.: Inter-relationships of selenium with other trace elements. Fed. Proc. 34, 2096--2100 (1975) Kar, A. B., Das, R. R. P., Mukerji, B.: Prevention of cadmium induced changes in the gonads of rat by zinc and selenium - a study in antagonism between metals in the biological systm. Proc. Nat. Inst. Sci. India 26 (Suppl), 40 (1960) McConnel, K. P., Carpenter, D. M.: Inter-relationship between selenium and specific trace elements. Proc. Soc. exp. Biol. (N.Y.) 137, 996--1001 (1971) Moffitt, A. E. Jr., Clary, J. J.: Selenite-induced binding of inorganic mercury in blood and other tissues in the rat. Res. Comm. Chem. Path. Pharmacol. 7, 593-603 (1974) Painter, E. P.: The chemistry and toxicity of selenium compounds with special reference to the selenium problem. Chem. Rev. 28, 197-213 (1941) Parizek, J.: The detoxifying effects of selenium: inter-relations between compounds of selenium and certain metals. In: Newer trace elements in nutrition (W. Meltz, W. E. Cornatzer, Eds.), pp. 85--122. New York: Dekker 1971 Sandholm, M.: Selenium carrier proteins is mouse plasma. Acta Pharmacol. Toxicol. 35, 424--428 (1974) Sandholm, M.: Function of erythrocytes in attaching selenite-Se onto specific plasma proteins. Acta Pharmacol. Toxicol. 36, 321--327 (1975)
Received May 3, 1976
Arch. Toxicol. 36, 63--69 (1976)
TOXICOLOGY by Springer-Verlag 1976
Differences in Distribution and Excretion of Selenium and Cadmium or Mercury after Their Simultaneous Administration Subcutaneously in Equimolar Doses* L. Magos** and M. Webb MRC Toxicology Unit, MRC Laboratories Woodmansterne Road, Carshalton, Surrey, United Kingdom
Abstract. Forty-eight hours after the simultaneous administration of either 1.8 ~moles Se (as Na2SeO3) to 200 g male rats with equivalent doses of Cd (as CdCI2) or 0.5 ~moles Se with equivalent doses of Hg (as HgC12) the retention of selenium at the subcutaneous injection site was affected only when it was given in a single injection with the heavy metal. The retention of cadmium was increased only if selenium was injected separately and the retention of mercury was increased both in single and separate injections. When there was an increase in retention at the site of injection, this made up less than 8% of the dose for selenium and mercury and 3% for cadmium and thus could not explain either changes in distribution or the reported protective effects by one metal against the other. The shift in the distribution pattern of one metal caused by the other metal was similar, and independent of whether they were injected in single or separate injections. However the shift in the distribution of interacting metals showed some differences indicating that interaction could not be mediated through the formation of a stable complex between selenium on the one hand and mercury or cadmium on the other.
Key words: Selenium - Cadmium - Mercury - Interaction between mettals.
Zusammenfassung. 200 g schwere m/innliche Ratten erhielten gleichzeitig entweder 1,8 ~mol Selen (als Na2SeO3) und eine equivalente Dosis Kadmium (als CdCI2) oder 0,5 gmol Selen und eine equivalente Dosis Quecksilber (als HgClz). Die Retention des Selens an der Stelle der subcutanen Injektion 48 Std nach der Applikation wurde nur dann beeinflul3t, wenn dieses in einer einzigen Injektion mit dem Schwermetall gegeben wurde. Die Retention des Kadmiums nahm nur zu, wenn das Selen getrennt injiziert wurde, und die Retention des Quecksilbers war sowohl bei gemeinsamer als auch bei getrennter Injektion erh6ht. Wenn eine Zunahme der Retention an der Injektionsstelle gefunden wurde, so machte diese * Supported partly by International Atomic Energy Agency, Grant Number 1530/RB ** To whom offprint requests should be sent
64
L. Magos and M. Webb weniger als 8% der Dosis f/ir Selen ud Quecksilber und 3% ffir K a d m i u m aus. Dieses reicht nicht aus, um entweder die Unterschiede in der Verteilung oder den protektiven Effekt, den die Metalle untereinander aus/iben sollen, zu erklfiren. Die Verfinderung des Verteilungsmusters eines Metalls, verursacht durch ein anderes Metall, war/ihnlich und unabhfingig davon, ob sie in einer einzigen oder in getrennten Injektionen gegeben wurden. Allerdings zeigt die .~nderung der Verteilung von Metallen, die in Wechselwirkung treten, einige Unterschiede, die darauf hinweisen, dab dieser Effekt nicht auf der Bildung eines stabilen Komplexes zwischen Selen auf der einen Seite und Quecksilber und Kadmium auf der anderen beruht.
Sehliisselwiirter: Selen - K a d m i u m - Quecksilber - Wechselwirkung zwischen Metallen.
Introduction The protective effect of selenium against cadmium and mercury toxicity (Kar et al., 1960; Parizek, 197 I; G u n n e t al., 1968; Eybl et al., 1969) or the reversal of selenium toxicity by the same heavy metals (Hill, 1974) presents some interesting problems. It has been observed that to produce the maximum effect on the distribution of mercury, equimolar doses of selenium (as selenite) are necessary (Moffitt and Clary, 1974). Similarly equimolar doses of Hg are required to give an optimum protection against selenium (Hill, 1975). McConnel and Carpenter (1971) reported that cadmium mixed with selenium, but not when injected separately, slowed down the absorption of selenium from the injection site. On the b~isis of these observations it has been proposed that the protective effect observed between the two heavy metals and selenium is based on the formation of a heavy metal-selenium complex (Hill, 1975). However in the experiments in which retention of selenium was followed (McConnel and Carpenter, 1971) the Se dose was 57 ~moles/kg and the cadmium dose ranged from 2 8 . 5 - 2 2 8 ~moles/kg. These doses are extremely high: according to our experience in rats 22 ~moles/kg Cd given subcutaneously is able to cause local tissue damage judged from the palpable hardening of the tissue surrounding the injection site and 127 vmoles/kg is the LD50 (unpublished observation).
Materials and Methods
Animals and Experimental Schedule Male rats of Porton Wistar strain of approximately 200 g body weight were used. In control animals the radioactively labelled test metal was injected subcutaneously in 0.2 ml volume into the right hind leg above the knee. In experimental animals the test metal and the unlabeUed interfering metal was injected either mixed together in 0.2 ml volume into the same site or the tested metal was injeced into the leg and the interfering metal under the skin of the neck in the same volume. Animals were kept in metabolic cages, two rats in one cage, and urine and faeces were collected separately. Forty-eight hours after injection animals were killed by decapitation, blood from the cervical blood vessels was collected in a beaker and was weighed. Liver and kidneys were removed and both hind legs were removed by cutting through the hip joint.
Interaction of Selenium with Cadmium and Mercury
65
Injection Solutions Cold stock solutions of CdC12, HgCI2 or Na2SeOa were prepared in saline and were labelled with the corresponding isotope (llSmCd, 2~ or 75Se) supplied by the Radiochemieal Centre (Amersham, Bucks) Volumes of the injection solution were 0.2 ml. The metal was injected either alone or mixed in a ratio of 1 : 1 with the other metal. Only one of them was labelled. The precipitate of Se and Cd was dissolved by adding 0.1 N HCI drop by drop to the solution before diluting to the final volume. Radioactivities in 0.2 ml injection solutions were approximately: ll5mCd: 1.60 ~Ci, Hg: 0.15 pCi; 75Se: 0.05 ~Ci.
Radioactive Measurements Live animals placed in a 14 x 6 x 6.5 cm plexiglass box were counted between two NaI crystal pairs (No. N656, EKCO Electronics Ltd., Southend on-Sea, Essex) facing each other. Faeces and urine samples, blood, liver and legs were counted in beakers in the same system with the following efficiencies: HSmCd:0.6%, 2~ 7.7%, 75Se: 28.0%. Kidneys were counted in a well shaped NaI crystal with efficiencies of 2.5%, 31.0% and 80.0% for llsmCd2+, 2~ and 755e4+ respectively. Injection standards in I ml acid permanganate solution were counted and concentrations in organs and in whole animals were estimated after corrections for geometrical differences by relating their radioactivities to standards. Radioactivity retained at the injection site was determined from the difference between the injected and the control leg. Radioactivity in the total blood was calculated on the assumption that blood made up 7.0% of the body weight.
Statistical Calculations Statistical differences were estimated by the Student t-test, in the case of radioactivity retained at the site of injection by one tailed test and in every other case by two tailed test.
Results C a d m i u m increased the retention o f selenium when the two metals were administered in a single subcutaneous injection, but had no effect on retention when they were injected separately (Table 1). Body burden of selenium at the end o f the 48 h period was higher when c a d m i u m was injected mixed with selenium than when c a d m i u m was injected separately. This effect was mainly due to a more pronounced decrease in the urinary excretion of selenium. Liver and kidney contents o f selenium were increased in both groups, but due to a smaller standard error the increase was more significant in the group which was given the two metals separately. M e r c u r y had a more profound effect on the distribution of selenium: body burden, liver, kidney and blood contents o f selenium increased and excretion decreased (Table 2). Retention at the injection site increased only when the two metals were injected simultaneously. The increase in the retention o f selenium at the injection site caused by heavy metals -- though statistically significant - amounted only to 6.7% o f the dose in the case o f mercury and to even less with cadmium. C o m p a r i s o n o f the control values showed some variation from one experiment to another and distribution in the experimental animals are c o m p a r e d only with their own controls.
66
L. Magos and M. Webb
Table 1. Effects of cadmium on the tissue contents and excretion of 75Se given as SeO 2 48 h after administration. 1.8 ~moles Se was injected either alone (controls) or mixed in a single injection with 1.8 ~moles Cd or the two metals were given separately 75Se contents in % of the dose Single injection
Injection site Body burden Liver Kidneys Blood Faeces Urine
Separate injection
Control N:-6
Experimental N=6
Control N=6
Experimental N=6
0.4 26.9 7.6 1.2 5.8 12.5 47.3
4.0 45.5 9.1 2.1 6.1 12.5 29.9
1.0 25.1 6.3 0.8 7.1 10.7 42.7
1.4 37.3 8.2 1.2 5.4 10.3 30.7
+ 0.15 _+ 1.10 + 0.37 + 0.12 _+ 0.32 _+ 0.74 + 2.24
• + + • • + +
0.71 a 2.36 a 0.89 0.62 0.37 1.01 1.21
+ 0.18 _+ 0.57 + 0.23 • 0.08 • 0.22 • 0.24 • 2.71
+ 0.17 + 2.30 a • 1.48a • 0.08 a _+ 0.82 • 1.21 • 2.23
a Significantly different from the control (P < 0.05) by the Student t-test. N - the number of animals, every faeces or urine sample was collected from two rats.
3"able 2. Effects of mercury on the tissue contents and excretion of 75Se given as SeO 2 48 h after administration. 0.5 p~moles Se was injected either alone (controls) or mixed in a single injection with 0.5 ~moles Hg or the two metals were injected separately 7SSe contents in % of the dose
Single injection
Injection site Body burden Liver Kidneys Blood Faeces (48 h) Urine (48 h)
Separate injection
Control N: 6
Experimental N=6
Control N=6
Experimental N=6
2.6 49.0 9.5 1.9 7.4 9.1 29.9
9.3 76.3 14.4 2.4 12.1 6.3 13.7
0.3 37.6 9.1 1.6 4.5 18.5 40.9
0.3 71.1 13.6 2.1 11.5 7.2 16.3
_+ 1.20 + 4.06 _+ 1.20 _+ 0.05 + 0.62 • 0.75 + 1.43
+ 1.73a +_ 1.68a + 0.91 a _+ 0.09 a _+ 1.09a + 0.89 +_ 0.92 a
+ 0.12 _+ 1.44 + 0.28 + 0.04 _+ 0.13 + 5.42 _+ 1.03
_+ 0.15 _+ 2.03 + 1.92a + 0.07 a _+ 0.52 _+ 0.17 • 1.38a
a Significally different from the control (P < 0.05) by the Student t-test. N the number of animals, every faeces or urine sample was collected from two rats.
C o n t r a r y t o t h e effect o f the t w o h e a v y m e t a l s o n t h e r e t e n t i o n o f selenium, t h e r e t e n t i o n o f c a d m i u m o r m e r c u r y b y s e l e n i u m w a s n o t h i g h e r if s e l e n i u m w a s a d m i n i s t e r e d w i t h t h e s e m e t a l s in a single as c o m p a r e d w i t h s e p a r a t e i n j e c t i o n s ( T a b l e s 3 a n d 4). In the c a s e o f c a d m i u m t h e r e w a s a slight, b u t s i g n i f i c a n t i n c r e a s e in t h e r e t e n t i o n o n l y if t h e m e t a l s w e r e i n j e c t e d s e p a r a t e l y . I n t h e c a s e o f m e r c u r y , selen i u m i n c r e a s e d t h e r e t e n t i o n b y 6 . 3 % in a single i n j e c t i o n a n d 7 . 2 % w h e n i n j e c t e d s e p a r a t e l y . T h e b o d y b u r d e n o f c a d m i u m w a s n o t c h a n g e d b y selenium, the liver
Interaction of Selenium with Cadmium and Mercury
67
Table 3. Effects of selenium on the distribution and excretion of us"Cd given as CdC12 48 h after administration. 1.8 ~tmoles Cd was injected either alone (controls) or mixed in a single injection with 1.8 ~moles Se or the two metals were injected separately
llSmCd contents in % of the dose Single injection
Injection site Body burden Liver Kidneys Blood Faeces (48 h) Urine (48 h)
Separate injection
Control N = 14
Experimental N = 14
Control N = 16
Experimental N = 16
15.5 86.9 48.0 2.7 1.0 6.3 0.7
16.1 89.0 37.4 2.8 3.0 5.8 0.9
15.8 85.4 49.8 2.7 0.9 5.6 0.8
18.1 85.5 34.0 2.6 3.1 7.1 1.4
• 0.79 • 1.45 • 0.86 • 0.12 • 0.15 • 0.72 _+ 0.09
• • • • • • •
0.56 1.82 1.27a 0.29 0.29 a 0.82 0.09
• 0.73 • 1.39 + 1.03 • 0.10 _+ 0.15 • 0.95 • 0.08
• 0.75 a • 1.63 • 0.62 a • 0.11 • 0.26 a • 1.47 _+ 0.34
a Significantly different from the control (P < 0.05) by the Student t-test. N the number of animals, every faeces or urine sample was collected from two rats.
Table 4. Effects of selenium on the tissue contents and excretion of 2~ given as HgCI 2 48 h after administration. 0.5 ~moles Hg was injected either alone (controls) or mixed in a single injection with 0.5 ~moles Se or the two metals were injected separately
2~
contents in % of the dose
Control N 8
Injection site Body burden Liver Kidneys Blood Faeces (48 h) Urine (48 h)
15.9 76.3 2.9 33.3 2.3 14.8 5.8
• • • • + • •
0.96 1.99 0.23 1.06 0.21 0.86 1.20
with Se Single injection N=8
Separate injection N=8
21.2 90.3 12.4 11.8 9.5 5.5 0.4
23.1 95.2 14.4 7.8 12.3 6.3 0.3
• • • • i • •
1.33a 1.72a 0.70 a 0.44 a 0.72 a 0.89 a 0.05 a
• • • + • • •
2.93 a 2.00 ~ 1.0P 0.98 a 0.77 a 1.50a 0.07 a
a Significantly different from the control (P < 0.05) by the Student t-test. N = the number of animals, every faeces or urine sample was collected from two rats.
content decreased and the blood content increased. Selenium had a more profound effect o n m e r c u r y w i t h i n c r e a s e s in b o d y b u r d e n a n d in the liver a n d b l o o d c o n t e n t o f m e r c u r y a n d w i t h d e c r e a s e s in t h e k i d n e y s , f a e c e s a n d urine. T h e shift in distribution w a s i n d e p e n d e n t o f w h e t h e r s e l e n i u m w a s i n j e c t e d s i m u l t a n e o u s l y w i t h the c a d m i u m or mercury, or was injected separately.
68
L. Magos and M. Webb
Discussion The present experiments showed that the retention of selenium was influenced by the presence of mercury or cadmium, but the extent of this effect expressed in percent of the dose was too small to explain differences in distribution. Though retention was not influenced when the heavy metals were injected at a different site, the shifts in body burden, excretion or distribution were in the same direction. If retention was increased by the formation of an insoluble complex, it seems reasonable to suppose that the heavy-metal selenium complex did not reach the blood as such, because the shift in the distribution was the same and independent of whether the two metals were injected in single or separate sites. Alternatively it might be that selenium absorbed from one site and the heavy metal from another site can compex together in the blood. There are, however, two observations which contradict the formation or existence of such a complex outside the injection site. Firstly, both cadmium and mercury increased the kidney contents of selenium, but selenium did not affect the content of cadmium and actually decreased the content of mercury in this organ. Liver level of selenium was lowered by cadmium or mercury but the liver level of mercury was increased by selenium. Selenium in this respect had an opposite effect on the liver content of cadmium which was decreased by selenium by more than 10%. In the case of an association between selenium and the heavy metals the shift in the distribution pattern ought to be similar. Secondly, contrary to the expected results, selenium given separately slightly increased the retention of the heavy metals at the injection site compared with experiments when selenium and the heavy metal were given together at the same site. Since the retention of cadmium or mercury alone at the injection site is approximately one order higher than that of selenium alone (see Tables) it is difficult to observe a few percent difference in retention in relation to the dose. Even so results suggest that the retention of selenium in the presence of mercury or cadmium or the retention of mercury and cadmium in the presence of selenium does not follow the same pattern and consequently might not be the result of the same mechanism. Thus it might be that local irritation, inflammatory reaction and tissue damage is responsible for the increased retention of selenium when injected with mercury or cadmium, and the retention of the two heavy metals is increased only after the metabolic conversion of selenium which takes place outside the injection site. It has been reported that plasma proteins do not bind selenite in vitro unless erythrocytes are present (Sandholm, 1974) which tranform selenite to a form which is able to be bound to plasma proteins, first of all to ~-lipoprotein and to unidentified fractions located electrophoretically between the al and a2 globulins (Sandholm, 1975). In the presence of Cd there is a shift in the binding of this transformed selenium to other protein fractions which bind cadmium and selenium in an atomic ratio of 1 : 1 (Gasiewicz and Smith, 1976). This binding is transitory and not affected by the preinjection level of plasma selenium (Gasiewicz and Smith, 1976) indicating that interactions at protein binding sites operate only at the distributory phase of selenium. It has been suggested that Se 2- forms a RS-Se-SR type of binding with proteins (Painter, 1941) by which their carrier capacity to heavy metals should be altered. Conversely cadmium or mercury through their affinity to thiol groups might affect the binding, metabolism or transport of selenium.
Interaction of Selenium with Cadmium and Mercury
69
In summary these experiments give evidence that if selenium forms an insoluble complex with cadmium or mercury, this cannot explain the effect of interaction on their distribution and the extent of change in their retention is too small to affect significantly their toxicity. Moreover, as the effect of selenium on cadmium or mercury differs from the effect of these heavy metals on the retention of selenium at the subcutaneous injection site, this makes questionable whether an insoluble complex is formed at all. Acknowledgement. The authors wish to thank Mr. A. R. Hudson for technical assistance.
References Eybl, V., Sykora, J., Mertl, F.: Einflul3 yon Natriumselenit, Natriumtellurit und Natriumsulfit auf Retention und Verteilung von Quecksilber bei M~iusen, Arch. Toxikol. 25, 296--305 (1969) Gasiewicz, T. A., Smith, J. C.: Interactions of cadmium and selenium in rat plasma in vivo and in vitro. Biochim. biophys. Acta (Amst.) 428, 113--122 (1976) Gunn, S. A.. Gould, T. C., Anderson, W. A. D.: Selectivity of organ response to cadmium injury and various protective measures. J. Path. Bact. 96, 89--96 (1968) Hill, C. H.: Reversal of selenium toxicity in chicks by mercury, copper and cadmium. J. Nutr. 104, 593-598 (1974) Hill, C. H.: Inter-relationships of selenium with other trace elements. Fed. Proc. 34, 2096--2100 (1975) Kar, A. B., Das, R. R. P., Mukerji, B.: Prevention of cadmium induced changes in the gonads of rat by zinc and selenium - a study in antagonism between metals in the biological systm. Proc. Nat. Inst. Sci. India 26 (Suppl), 40 (1960) McConnel, K. P., Carpenter, D. M.: Inter-relationship between selenium and specific trace elements. Proc. Soc. exp. Biol. (N.Y.) 137, 996--1001 (1971) Moffitt, A. E. Jr., Clary, J. J.: Selenite-induced binding of inorganic mercury in blood and other tissues in the rat. Res. Comm. Chem. Path. Pharmacol. 7, 593-603 (1974) Painter, E. P.: The chemistry and toxicity of selenium compounds with special reference to the selenium problem. Chem. Rev. 28, 197-213 (1941) Parizek, J.: The detoxifying effects of selenium: inter-relations between compounds of selenium and certain metals. In: Newer trace elements in nutrition (W. Meltz, W. E. Cornatzer, Eds.), pp. 85--122. New York: Dekker 1971 Sandholm, M.: Selenium carrier proteins is mouse plasma. Acta Pharmacol. Toxicol. 35, 424--428 (1974) Sandholm, M.: Function of erythrocytes in attaching selenite-Se onto specific plasma proteins. Acta Pharmacol. Toxicol. 36, 321--327 (1975)
Received May 3, 1976
