9 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3502~)119 $02.00
Selenium in the Central Nervous System of Rats Exposed to 75-Se L-Selenomethionine and Sodium Selenite HENNING GRONB/EK 1 AND O L E THORLACIUS-USSING *,2
~lnstitute of Neurobiology, University of Aarhus, DK-8000 Aarhus C, Denmark; and 2Department of Surgery, Panders Centralsygehus, DK-8900 Panders, Denmark Received December 7, 1991; Accepted February 4, 1992
ABSTRACT The aim of the present study is to investigate the accumulation and retention of organic and inorganic selenium in the central nervous system (CNS) of the rat. Selenium accumulation was investigated after oral treatment (3.0 mg Se/L drinking water) or ip injection (1.7 mg Se/kg body wt) of rats exposed to 75-Se L-selenomethionine (SeMeth) or sodium selenite (NaSe). Significant higher concentrations were observed after exposure to organic compared to inorganic selenium after oral as well as ip administration. Highest concentrations in both experiments were observed in cerebellum followed by the nearly identical levels in the cerebral hemisphere and spinal cord independent of the chemical form of selenium or the route of administration. The difference in concentrations observed between the different parts of the CNS investigated in each group were, however, not significant. Retention of selenium in the CNS was investigated after a single ip injection (1.7 mg Se/kg body wt) of 75-Se SeMeth or NaSe. In both groups, we observed an initial fast excretion phase followed by a slower excretion phase resembling a first-order reaction. Organic selenium disappeared much slower from all parts of the central nervous system compared to NaSe after a single injection. Index Entries: Selenium; sodium selenite; selenomethionine; central nervous system; rat. *Author to whom all correspondence and reprint requests should be addressed, Biological Trace Element Research
] ]9
Vol. 35, 1992
120
Gronbaek and Thorlacius-Ussing
INTRODUCTION In the 1930s, Moxon (1) described ataxia, motor incoordination, hyperreflexia, paralysis, depression, and lack of vitality in seleniumintoxicated animals. The CNS seems to be a very sensitive organ to the toxicity of selenium. There seems, however, to be outstanding differences between the toxicity of organic a n d inorganic selenium. After intracranial injection, Ammar and Couri (2) observed sodium selenite to be much more toxic than selenomethionine. In the literature, there is very little information concerning the accumulation of organic and inorganic selenium in the CNS of animals exposed to high amounts of selenium. In particular, no comparative studies between NaSe and SeMeth have been published so far. The aims of the present study were to investigate: (1) accumulation of 75-Se selenium in the CNS of rats orally exposed to either NaSe or SeMeth in the drinking water, and (2) accumulation and disappearance in the CNS after a single toxic dose of NaSe and SeMeth.
MATERIAL AND METHODS The present study consisted of two experiments using male Wistar rats with a mean weight of 150 g at the beginning of the study. All rats were kept in plastic boxes in a room with a mean temperature of 21 _+ 2~ humidity 55 -+ 5%, and a 12-h light cycle (0600-1800). If nothing else is depicted, the rats were maintained on stock pellet (Altromin 1324, Altromin Special-futterwerke, GMBH) with a declared and tested content of 0.2 ppm selenium and tap water ad libitum.
Experiment 1 Two groups of 16 rats were orally exposed to either 75-Se NaSe or SeMeth in the drinking water over a 4-wk period. The water contained 3.0 mg Se/L and 3.7 MBq 75-Se/L drinking water. Four rats from each group were sacrificed after 7, 14, 21, and 28 d of exposure, and the selenium concentration estimated in the cerebral hemisphere, cerebellum, and spinal cord. No difference in water consumption was observed.
Experiment 2 Two groups of 40 rats were lightly anesthetized with pentobarbital at t -- 5 min. At t = 0, they were injected ip with either 75-Se NaSe or SeMeth (1.7 mg Se and 740 kBq 75-Se/kg body wt). Within 10 rain, each rat was whole body counted for the first time. Two hours later, four rats from each of the two groups were recounted, sacrificed, and the organs (the cerebral hemisphere, cerebellum, and the spinal cord) removed. This was also done 24 and 48 h later, and further 3, 6, 9, 14, 21, 40, and 80 Biological Trace Element Research
Vol. 35, 1992
Se in the Central Nervous System
121
d after the injection. The selenium concentrations observed in the individual organs 2 h after the injection were used as the 100% reference value. The content of labeled selenium in the cerebral hemisphere, cerebellum, and the spinal cord was estimated. The procedures for acquiring the individual parts of the central nervous system were equal for all rats. Before the sacrifice, each rat was whole body counted for determining the whole body content of labeled selenium. Afterward, the rats were anesthetized by an overdose of pentobarbital, exsanguinated by v. porta puncture, and the organs removed. The wet weights of the organs were recorded before the selenium content was determined using a well counter. The organs were counted together with standards containing known concentrations of labeled selenium to correct for background irradiation and physical decay of the isotope. The standards were prepared from the initial drinking or injection solutions. Counting values < 10,000 were not accepted, which assures an SE below 1%. The slope of the elimination curve for the organs assembling a first-order equation was estimated by linear logarithmic regression and T1/2 determined. All statistics were performed using BMDP (3) and P-values estimated by a Mann-Whitney test.
RESULTS Experiment 1 Selenium accumulated in all parts of the CNS, and the contents in the individual parts are shown in Figs. 1A and B. Significantly more selenium accumulated in all parts of the CNS after exposure to SeMeth compared to NaSe after 1 wk (p < 0.001). This difference lasted for the rest of the period. After 14 d of exposure, the highest amount of selenium was observed in both groups in the cerebellum (1.6 • 0.5 [SD] lag/g vs 0.20 + 0.03 [SD] IJ,g/g wet wt) followed by the cerebral hemisphere (1.4 • 0.4 [SD]. ~g/g vs 0.18 • 0.07 SD lag/g) and the spinal cord (1.2 • 0.3 [SD] lag/g vs 0.15 _+ 0.03 [SD] tag/g).
Experiment 2 Distribution of Selenium in the CIXlS The selenium content in the individual parts of the CNS after an ip injection of SeMeth or NaSe is shown in Figs. 2A and B. In both groups, highest selenium concentrations were observed 2 h after the injection, and the group treated with SeMeth contained significantly more selenium compared to the NaSe group (p < 0.05). Two hours after the injection, the cerebellum contained 0.79 -- 0.12 (SD) lag/g vs 0.58 • 0.1 (SD) lag/g wet wt, the cerebral hemisphere 0.67 -- 0.08 lag/g vs 0.42 -40.07 ~g/g, and the spinal cord 0.75 • 0.07 lag/g vs 0.43 • 0.03 lag/g. Biological Trace Element Research
Vol. 35, 1992
Gronbaek and Thorlacius-Ussing
122
A pp;~
Se 9 V
2.5
days
[] s
days
[] 2 X
days
20
days
1,5
Q.5
~+++I Cepe}~{ I l u m
~ Cex~el~al
l~emispbe~e
E|
Spinal
iiiii co~,t
Fig. 1A. Selenium concentrations (l~g Se/g wet wt) in the CNS of rats orally exposed to 75-Se SeMeth (3.0 mg Se/L drinking water).
B ppt~
Se
2.5 9 ?
days
[] 14 d a y s [] 21 d a y s 28
days
1.5
0.5
ml]i~i~!i!i] Ce~e~ellu~
, Ce~e~ral
hemisphere
Spinal
cox,4
Fig. lB. Selenium concentrations (~g Se/g wet wt) in the CNS of rats orally exposed to 75-Se NaSe (3.0 mg Se/L drinking water). Biological Trace Element Research
Vol. 35, 1992
Se in the Central Nervous System
123
A Se
pp~
I-
0.8
9
Cer, e ~ e l
luM
B
Cere~x, al hel~i s p h e r e
~
Spinal
cord
i'i '0 2
24
48
3
6
9
14
R1
42
82
~ays
Fig. 2A. Selenium concentrations (~g Se/g wet wt) in the CNS after an ip injection of 75-Se SeMeth (1.7 mg Se/kg body wt).
B ppM
Se
9
Ce~e~ellu~
hemisphere
Cerebral
0.8-
Spinal
coma
0.6-
0,4-
0.2-
2
24
48
4
10
20
3~
40
60
80
d~ys
Fig. 2B. Selenium concentrations (p~g Se/g wet wt) in the CNS after an ip injection of 75-Se NaSe (1.7 mg Se/kg body wt). Biological Trace Element Research
Vol. 35, 1992
124
Grenbaek and Thorlacius-Ussing
The initial insignificant difference in selenium concentration between the different parts of the CNS lasted for the first 3 wk of the experiment. Thereafter, the selenium concentrations were nearly equal in all parts of the CNS.
Retention of Selenium Retention of selenium in the CNS of rats injected with SeMeth is shown in a semilogarithmic plot in Fig. 3A. Within 48 h after the injection, 20-30% of selenium disappeared from the organs investigated. The spinal cord had an initial faster excretion compared to cerebellum and the cerebral hemisphere, though at the end of the study, the organs retained almost the same amount of selenium (6%). Generally, the excretion of selenium was characterized by an initial fast disappearance followed by a slow excretion phase. The slow excretion phase started at day 6, and T1/2 was almost the same for the three parts of the CNS investigated: Cerebellum (T1/2 = 25.5 + 4.3 d), the cerebral hemisphere (TI/2 = 25.3 -+ 4.4 d), and the spinal cord (T1/2 = 29.2 _+ 4.3 d). The retention of selenium in the group injected with NaSe is shown in a semilogarithmic plot in Fig. 3B. A very fast disappearance of selenium was observed from both cerebellum and the cerebral hemisphere in that approx 70% disappeared within 48 h. Practically no labeled selenium was retained after 60 d (1-2%). Both organs showed a biphasic excretion pattern with a slow phase starting at day 6. Tin was estimated to 12.9 + 1.9 d for cerebellum and 13.6 _+ 1.9 d for the cerebral hemisphere. Selenium disappeared more slowly from the spinal cord during the first period after the injection. Tl/2 after 6 d was, however, practically identical to the others (Tin -- 12.9 + 0.8 d), and nearly all selenium had disappeared after 60 d.
DISCUSSION Our results show that selenium accumulates in the CNS of the rat independent of the chemical form or the route of administration. In both experiments, the cerebellum contained the highest level followed by nearly identical levels in the cerebral hemisphere and the spinal cord. This distribution in the CNS seems to be a general phenomenon, since the present results are in accordance with those of others (4,5), although they used only trace amounts of NaSe. The distribution of selenium in the CNS is generally fairly homogeneous, and the difference observed between different parts of the CNS seems to reflect the content of gray matter compared to white matter as observed in the rat brain (5) and human brain (6). In our study, the CNS generally contained substantially smaller amounts of exogenous selenium compared to whole-body as well as fullblood content (7,8). This might indicate a regulatory mechanism that is Biological Trace Element Research
Vol. 35, 1992
Se in the Central Nervous System
125
A 1s
~"~
"
.
--I
10
i ZO
I
I
i
I
30
4s
5s
6~
I 7~
Cer, e~ratl
he~i sP}lePe
i
80
!, 90
~ays
Fig. 3A. Retention of selenium in the CNS after an ip injection of 75-Se SeMeth.
B
~,
9 CePeb~al hemisphere
+ Ce~Pe~,ellum )K ~ p i n a l
0
10
20
30
4~
50
60
70
80
cord
90
days
Fig. 3B. Retention of selenium in the CNS after an ip injection of 75-Se NaSe. Biological Trace Element Research
Vol. 35, 1992
126
Gr~nbaek and Thorlacius-Ussing
able to protect the CNS from selenium intoxication. Indeed, this mechanism seems to be more effective against selenite intoxication compared to organic selenium, since earlier results (7,8) demonstrated a greater gradient from blood to the CNS in animals exposed to NaSe compared to SeMeth. Ammar and Couri (2) have earlier demonstrated NaSe to be much more toxic after intracranial injection compared to iv injection, and a protection from selenite may therefore be important. They also demonstrated that the total amount of selenium in the CNS was rather unimportant, whereas the chemical form was important, since the minimal lethal dose of NaSe was 200 times that for SeMeth after intracranial administration. Indeed, the selenium content in the CNS seems to be strictly regulated, in that the selenium concentration in the CNS remained relatively high during depletion studies (9). In experiment 1, the selenium concentration in the CNS of NaSe-treated animals reached a maximal concentration within 2 wk and stayed nearly constant thereafter. It is indeed remarkable that the concentration reached a level only two times higher than the normal CNS level estimated by Prohaska and Ganther (5). In the SeMeth-treated animals, maximal concentrations also appeared after 2 wk, but this level was seven times the estimated normal level. Our results also demonstrate for the first time a significantly higher content of selenium in the CNS after exposure to organic selenium compared to inorganic selenium in rats exposed to high amounts of selenium. This difference was especially remarkable after long-term exposure at which the cerebellum content in the SeMetb-exposed animals exceeded the content in the NaSe-treated animals by a factor of 8 compared to a factor of 1.4 2 h after the ip injection. This result may indicate that either SeMeth is incorporated in more stable complexes than selenite or the transport over the blood-brain barrier may be more favorable for SeMeth. Both mechanisms seem to operate in the case of SeMeth, since the CNS contained higher amounts of selenium just after the injection of a single dose. On the other hand, the disappearance of selenium from the CNS of SeMeth-treated animals was much slower than in the NaSetreated rats. The concentration of the selenoenzyme glutathionperoxidase (GSHpx) in the CNS is generally low (10), although Prohaska and Ganther (5) observed a higher concentration compared to that observed by DeMarchena et al. (10). In other organs of the rat, SeMeth has been demonstrated (11,12) to be as potent as NaSe to increase the amount of GSHpx. During supplementation with SeMeth, however, there was observed a larger proportion of selenium in other proteins. Thus, the high selenium concentration in the CNS after exposure to SeMeth could not solely be explained by an increase in GSHpx, but more likely by a general incorporation of SeMeth into specific and unspecific selenium-containing proteins (for review, see 13). After administration of SeMeth, selenium Biological Trace Element Research
VoL 35, 1992
Se in the Central Nervous System
127
seems to be incorporated in specific selenium-containing proteins w h e n available in smaller doses, but incorporated in the general protein pool w h e n available in excess (14). An earlier study has demonstrated four selenium-containing proteins in the CNS (5) and a recent study at least seven selenium-containing proteins (9) after administration of selenite. In conclusion, our study demonstrates that selenium accumulates in the CNS of rats exposed to high levels of either organic or inorganic selenium. The level was, however, lower in the CNS compared to most other organs, which may reflect a protective mechanism against the extreme neurotoxicity of selenium.
ACKNOWLEDGMENTS The authors gratefully acknowledge the skillful technical assistance rendered by Else Maria S. Hansen, Karin Wiedemann, and Thorkild Nielsen. We further want to express our thanks to Prof. C. Frederickson, Dallas, for valuable comments on the manuscript. This study was supported by grants from NOVO's Foundation, Fonden at 1870; Konsul Johannes Fogh-Nielsen og hustru Ella Fogh-Nielsens Fond, and Jemo Pharm. Ltd., Hillerod, Denmark.
REFERENCES 1. A. L. Moxon, Saulh. Dak. Agric. Expt. Sta. Bull. 311, 1-91 (1937). 2. E. M. Ammar and D. Couri, Neurotoxicology 2, 383--386 (1981). 3. W. J. Dixon, BMDP Statistical Software. University of California Press, Berkeley, 1983. 4. G. A. Trapp and J. Millam, ]. Neurochem. 24, 593-595 (1975). 5. J. R. Prohaska and H. E. Ganther, ]. Neurochem. 27, 1379-1387 (1976). 6. A. H6ch, V. Demmel, H. Schicha, K. Kasparek, and L. E. Feinendegen, Brain 98, 49-64 (1975). 7. O. Thorlacius-Ussing and F. T. Jensen, Biol. Trace Element Res. 15, 277-287 (1988). 8. H. Gronb~ek and O. Thorlacius-Ussing, in Selenium in Biology and Medicine, A. Wendel, ed., Springer Verlag, Heidelberg, 1989, pp. 130-132. 9. D. Behne, H. Hilmert, S. Scheid, H. Gessner, and W. Elger, Biochem. Biophys. Acta 966, 12-21 (1988). 10. O. DeMarchena, M. Guarnieri, and G. McKhann, J. Neurochem. 22, 773-776 (1974). 11. J. T. Deagen, J. A. Butler, M. A. Beilstein, and P. D. Whanger, J. Nutr. 117, 91-98 (1987). 12. M. A. Beilstein and P. D. Whanger, J. Inorg. Biochem. 33, 31-46 (1988). 13. R. A. Sunde, Annu. Rev. Nutr. 10, 451-474 (1990). 14. C. Ip, J. Nat. Cancer Inst. 80, 258-262 (1988).
Biological Trace Element Research
Vol. 35, 1992
Selenium in the Central Nervous System of Rats Exposed to 75-Se L-Selenomethionine and Sodium Selenite HENNING GRONB/EK 1 AND O L E THORLACIUS-USSING *,2
~lnstitute of Neurobiology, University of Aarhus, DK-8000 Aarhus C, Denmark; and 2Department of Surgery, Panders Centralsygehus, DK-8900 Panders, Denmark Received December 7, 1991; Accepted February 4, 1992
ABSTRACT The aim of the present study is to investigate the accumulation and retention of organic and inorganic selenium in the central nervous system (CNS) of the rat. Selenium accumulation was investigated after oral treatment (3.0 mg Se/L drinking water) or ip injection (1.7 mg Se/kg body wt) of rats exposed to 75-Se L-selenomethionine (SeMeth) or sodium selenite (NaSe). Significant higher concentrations were observed after exposure to organic compared to inorganic selenium after oral as well as ip administration. Highest concentrations in both experiments were observed in cerebellum followed by the nearly identical levels in the cerebral hemisphere and spinal cord independent of the chemical form of selenium or the route of administration. The difference in concentrations observed between the different parts of the CNS investigated in each group were, however, not significant. Retention of selenium in the CNS was investigated after a single ip injection (1.7 mg Se/kg body wt) of 75-Se SeMeth or NaSe. In both groups, we observed an initial fast excretion phase followed by a slower excretion phase resembling a first-order reaction. Organic selenium disappeared much slower from all parts of the central nervous system compared to NaSe after a single injection. Index Entries: Selenium; sodium selenite; selenomethionine; central nervous system; rat. *Author to whom all correspondence and reprint requests should be addressed, Biological Trace Element Research
] ]9
Vol. 35, 1992
120
Gronbaek and Thorlacius-Ussing
INTRODUCTION In the 1930s, Moxon (1) described ataxia, motor incoordination, hyperreflexia, paralysis, depression, and lack of vitality in seleniumintoxicated animals. The CNS seems to be a very sensitive organ to the toxicity of selenium. There seems, however, to be outstanding differences between the toxicity of organic a n d inorganic selenium. After intracranial injection, Ammar and Couri (2) observed sodium selenite to be much more toxic than selenomethionine. In the literature, there is very little information concerning the accumulation of organic and inorganic selenium in the CNS of animals exposed to high amounts of selenium. In particular, no comparative studies between NaSe and SeMeth have been published so far. The aims of the present study were to investigate: (1) accumulation of 75-Se selenium in the CNS of rats orally exposed to either NaSe or SeMeth in the drinking water, and (2) accumulation and disappearance in the CNS after a single toxic dose of NaSe and SeMeth.
MATERIAL AND METHODS The present study consisted of two experiments using male Wistar rats with a mean weight of 150 g at the beginning of the study. All rats were kept in plastic boxes in a room with a mean temperature of 21 _+ 2~ humidity 55 -+ 5%, and a 12-h light cycle (0600-1800). If nothing else is depicted, the rats were maintained on stock pellet (Altromin 1324, Altromin Special-futterwerke, GMBH) with a declared and tested content of 0.2 ppm selenium and tap water ad libitum.
Experiment 1 Two groups of 16 rats were orally exposed to either 75-Se NaSe or SeMeth in the drinking water over a 4-wk period. The water contained 3.0 mg Se/L and 3.7 MBq 75-Se/L drinking water. Four rats from each group were sacrificed after 7, 14, 21, and 28 d of exposure, and the selenium concentration estimated in the cerebral hemisphere, cerebellum, and spinal cord. No difference in water consumption was observed.
Experiment 2 Two groups of 40 rats were lightly anesthetized with pentobarbital at t -- 5 min. At t = 0, they were injected ip with either 75-Se NaSe or SeMeth (1.7 mg Se and 740 kBq 75-Se/kg body wt). Within 10 rain, each rat was whole body counted for the first time. Two hours later, four rats from each of the two groups were recounted, sacrificed, and the organs (the cerebral hemisphere, cerebellum, and the spinal cord) removed. This was also done 24 and 48 h later, and further 3, 6, 9, 14, 21, 40, and 80 Biological Trace Element Research
Vol. 35, 1992
Se in the Central Nervous System
121
d after the injection. The selenium concentrations observed in the individual organs 2 h after the injection were used as the 100% reference value. The content of labeled selenium in the cerebral hemisphere, cerebellum, and the spinal cord was estimated. The procedures for acquiring the individual parts of the central nervous system were equal for all rats. Before the sacrifice, each rat was whole body counted for determining the whole body content of labeled selenium. Afterward, the rats were anesthetized by an overdose of pentobarbital, exsanguinated by v. porta puncture, and the organs removed. The wet weights of the organs were recorded before the selenium content was determined using a well counter. The organs were counted together with standards containing known concentrations of labeled selenium to correct for background irradiation and physical decay of the isotope. The standards were prepared from the initial drinking or injection solutions. Counting values < 10,000 were not accepted, which assures an SE below 1%. The slope of the elimination curve for the organs assembling a first-order equation was estimated by linear logarithmic regression and T1/2 determined. All statistics were performed using BMDP (3) and P-values estimated by a Mann-Whitney test.
RESULTS Experiment 1 Selenium accumulated in all parts of the CNS, and the contents in the individual parts are shown in Figs. 1A and B. Significantly more selenium accumulated in all parts of the CNS after exposure to SeMeth compared to NaSe after 1 wk (p < 0.001). This difference lasted for the rest of the period. After 14 d of exposure, the highest amount of selenium was observed in both groups in the cerebellum (1.6 • 0.5 [SD] lag/g vs 0.20 + 0.03 [SD] IJ,g/g wet wt) followed by the cerebral hemisphere (1.4 • 0.4 [SD]. ~g/g vs 0.18 • 0.07 SD lag/g) and the spinal cord (1.2 • 0.3 [SD] lag/g vs 0.15 _+ 0.03 [SD] tag/g).
Experiment 2 Distribution of Selenium in the CIXlS The selenium content in the individual parts of the CNS after an ip injection of SeMeth or NaSe is shown in Figs. 2A and B. In both groups, highest selenium concentrations were observed 2 h after the injection, and the group treated with SeMeth contained significantly more selenium compared to the NaSe group (p < 0.05). Two hours after the injection, the cerebellum contained 0.79 -- 0.12 (SD) lag/g vs 0.58 • 0.1 (SD) lag/g wet wt, the cerebral hemisphere 0.67 -- 0.08 lag/g vs 0.42 -40.07 ~g/g, and the spinal cord 0.75 • 0.07 lag/g vs 0.43 • 0.03 lag/g. Biological Trace Element Research
Vol. 35, 1992
Gronbaek and Thorlacius-Ussing
122
A pp;~
Se 9 V
2.5
days
[] s
days
[] 2 X
days
20
days
1,5
Q.5
~+++I Cepe}~{ I l u m
~ Cex~el~al
l~emispbe~e
E|
Spinal
iiiii co~,t
Fig. 1A. Selenium concentrations (l~g Se/g wet wt) in the CNS of rats orally exposed to 75-Se SeMeth (3.0 mg Se/L drinking water).
B ppt~
Se
2.5 9 ?
days
[] 14 d a y s [] 21 d a y s 28
days
1.5
0.5
ml]i~i~!i!i] Ce~e~ellu~
, Ce~e~ral
hemisphere
Spinal
cox,4
Fig. lB. Selenium concentrations (~g Se/g wet wt) in the CNS of rats orally exposed to 75-Se NaSe (3.0 mg Se/L drinking water). Biological Trace Element Research
Vol. 35, 1992
Se in the Central Nervous System
123
A Se
pp~
I-
0.8
9
Cer, e ~ e l
luM
B
Cere~x, al hel~i s p h e r e
~
Spinal
cord
i'i '0 2
24
48
3
6
9
14
R1
42
82
~ays
Fig. 2A. Selenium concentrations (~g Se/g wet wt) in the CNS after an ip injection of 75-Se SeMeth (1.7 mg Se/kg body wt).
B ppM
Se
9
Ce~e~ellu~
hemisphere
Cerebral
0.8-
Spinal
coma
0.6-
0,4-
0.2-
2
24
48
4
10
20
3~
40
60
80
d~ys
Fig. 2B. Selenium concentrations (p~g Se/g wet wt) in the CNS after an ip injection of 75-Se NaSe (1.7 mg Se/kg body wt). Biological Trace Element Research
Vol. 35, 1992
124
Grenbaek and Thorlacius-Ussing
The initial insignificant difference in selenium concentration between the different parts of the CNS lasted for the first 3 wk of the experiment. Thereafter, the selenium concentrations were nearly equal in all parts of the CNS.
Retention of Selenium Retention of selenium in the CNS of rats injected with SeMeth is shown in a semilogarithmic plot in Fig. 3A. Within 48 h after the injection, 20-30% of selenium disappeared from the organs investigated. The spinal cord had an initial faster excretion compared to cerebellum and the cerebral hemisphere, though at the end of the study, the organs retained almost the same amount of selenium (6%). Generally, the excretion of selenium was characterized by an initial fast disappearance followed by a slow excretion phase. The slow excretion phase started at day 6, and T1/2 was almost the same for the three parts of the CNS investigated: Cerebellum (T1/2 = 25.5 + 4.3 d), the cerebral hemisphere (TI/2 = 25.3 -+ 4.4 d), and the spinal cord (T1/2 = 29.2 _+ 4.3 d). The retention of selenium in the group injected with NaSe is shown in a semilogarithmic plot in Fig. 3B. A very fast disappearance of selenium was observed from both cerebellum and the cerebral hemisphere in that approx 70% disappeared within 48 h. Practically no labeled selenium was retained after 60 d (1-2%). Both organs showed a biphasic excretion pattern with a slow phase starting at day 6. Tin was estimated to 12.9 + 1.9 d for cerebellum and 13.6 _+ 1.9 d for the cerebral hemisphere. Selenium disappeared more slowly from the spinal cord during the first period after the injection. Tl/2 after 6 d was, however, practically identical to the others (Tin -- 12.9 + 0.8 d), and nearly all selenium had disappeared after 60 d.
DISCUSSION Our results show that selenium accumulates in the CNS of the rat independent of the chemical form or the route of administration. In both experiments, the cerebellum contained the highest level followed by nearly identical levels in the cerebral hemisphere and the spinal cord. This distribution in the CNS seems to be a general phenomenon, since the present results are in accordance with those of others (4,5), although they used only trace amounts of NaSe. The distribution of selenium in the CNS is generally fairly homogeneous, and the difference observed between different parts of the CNS seems to reflect the content of gray matter compared to white matter as observed in the rat brain (5) and human brain (6). In our study, the CNS generally contained substantially smaller amounts of exogenous selenium compared to whole-body as well as fullblood content (7,8). This might indicate a regulatory mechanism that is Biological Trace Element Research
Vol. 35, 1992
Se in the Central Nervous System
125
A 1s
~"~
"
.
--I
10
i ZO
I
I
i
I
30
4s
5s
6~
I 7~
Cer, e~ratl
he~i sP}lePe
i
80
!, 90
~ays
Fig. 3A. Retention of selenium in the CNS after an ip injection of 75-Se SeMeth.
B
~,
9 CePeb~al hemisphere
+ Ce~Pe~,ellum )K ~ p i n a l
0
10
20
30
4~
50
60
70
80
cord
90
days
Fig. 3B. Retention of selenium in the CNS after an ip injection of 75-Se NaSe. Biological Trace Element Research
Vol. 35, 1992
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Gr~nbaek and Thorlacius-Ussing
able to protect the CNS from selenium intoxication. Indeed, this mechanism seems to be more effective against selenite intoxication compared to organic selenium, since earlier results (7,8) demonstrated a greater gradient from blood to the CNS in animals exposed to NaSe compared to SeMeth. Ammar and Couri (2) have earlier demonstrated NaSe to be much more toxic after intracranial injection compared to iv injection, and a protection from selenite may therefore be important. They also demonstrated that the total amount of selenium in the CNS was rather unimportant, whereas the chemical form was important, since the minimal lethal dose of NaSe was 200 times that for SeMeth after intracranial administration. Indeed, the selenium content in the CNS seems to be strictly regulated, in that the selenium concentration in the CNS remained relatively high during depletion studies (9). In experiment 1, the selenium concentration in the CNS of NaSe-treated animals reached a maximal concentration within 2 wk and stayed nearly constant thereafter. It is indeed remarkable that the concentration reached a level only two times higher than the normal CNS level estimated by Prohaska and Ganther (5). In the SeMeth-treated animals, maximal concentrations also appeared after 2 wk, but this level was seven times the estimated normal level. Our results also demonstrate for the first time a significantly higher content of selenium in the CNS after exposure to organic selenium compared to inorganic selenium in rats exposed to high amounts of selenium. This difference was especially remarkable after long-term exposure at which the cerebellum content in the SeMetb-exposed animals exceeded the content in the NaSe-treated animals by a factor of 8 compared to a factor of 1.4 2 h after the ip injection. This result may indicate that either SeMeth is incorporated in more stable complexes than selenite or the transport over the blood-brain barrier may be more favorable for SeMeth. Both mechanisms seem to operate in the case of SeMeth, since the CNS contained higher amounts of selenium just after the injection of a single dose. On the other hand, the disappearance of selenium from the CNS of SeMeth-treated animals was much slower than in the NaSetreated rats. The concentration of the selenoenzyme glutathionperoxidase (GSHpx) in the CNS is generally low (10), although Prohaska and Ganther (5) observed a higher concentration compared to that observed by DeMarchena et al. (10). In other organs of the rat, SeMeth has been demonstrated (11,12) to be as potent as NaSe to increase the amount of GSHpx. During supplementation with SeMeth, however, there was observed a larger proportion of selenium in other proteins. Thus, the high selenium concentration in the CNS after exposure to SeMeth could not solely be explained by an increase in GSHpx, but more likely by a general incorporation of SeMeth into specific and unspecific selenium-containing proteins (for review, see 13). After administration of SeMeth, selenium Biological Trace Element Research
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seems to be incorporated in specific selenium-containing proteins w h e n available in smaller doses, but incorporated in the general protein pool w h e n available in excess (14). An earlier study has demonstrated four selenium-containing proteins in the CNS (5) and a recent study at least seven selenium-containing proteins (9) after administration of selenite. In conclusion, our study demonstrates that selenium accumulates in the CNS of rats exposed to high levels of either organic or inorganic selenium. The level was, however, lower in the CNS compared to most other organs, which may reflect a protective mechanism against the extreme neurotoxicity of selenium.
ACKNOWLEDGMENTS The authors gratefully acknowledge the skillful technical assistance rendered by Else Maria S. Hansen, Karin Wiedemann, and Thorkild Nielsen. We further want to express our thanks to Prof. C. Frederickson, Dallas, for valuable comments on the manuscript. This study was supported by grants from NOVO's Foundation, Fonden at 1870; Konsul Johannes Fogh-Nielsen og hustru Ella Fogh-Nielsens Fond, and Jemo Pharm. Ltd., Hillerod, Denmark.
REFERENCES 1. A. L. Moxon, Saulh. Dak. Agric. Expt. Sta. Bull. 311, 1-91 (1937). 2. E. M. Ammar and D. Couri, Neurotoxicology 2, 383--386 (1981). 3. W. J. Dixon, BMDP Statistical Software. University of California Press, Berkeley, 1983. 4. G. A. Trapp and J. Millam, ]. Neurochem. 24, 593-595 (1975). 5. J. R. Prohaska and H. E. Ganther, ]. Neurochem. 27, 1379-1387 (1976). 6. A. H6ch, V. Demmel, H. Schicha, K. Kasparek, and L. E. Feinendegen, Brain 98, 49-64 (1975). 7. O. Thorlacius-Ussing and F. T. Jensen, Biol. Trace Element Res. 15, 277-287 (1988). 8. H. Gronb~ek and O. Thorlacius-Ussing, in Selenium in Biology and Medicine, A. Wendel, ed., Springer Verlag, Heidelberg, 1989, pp. 130-132. 9. D. Behne, H. Hilmert, S. Scheid, H. Gessner, and W. Elger, Biochem. Biophys. Acta 966, 12-21 (1988). 10. O. DeMarchena, M. Guarnieri, and G. McKhann, J. Neurochem. 22, 773-776 (1974). 11. J. T. Deagen, J. A. Butler, M. A. Beilstein, and P. D. Whanger, J. Nutr. 117, 91-98 (1987). 12. M. A. Beilstein and P. D. Whanger, J. Inorg. Biochem. 33, 31-46 (1988). 13. R. A. Sunde, Annu. Rev. Nutr. 10, 451-474 (1990). 14. C. Ip, J. Nat. Cancer Inst. 80, 258-262 (1988).
Biological Trace Element Research
Vol. 35, 1992
