Journal of Neurochemistry. 1975. Val. 24, pp. 59S595. Pergamon Press. Printed in Great Britain

SHORT COMMUNICATION ~~

The distribution of ''Se in brains of seleniumdeficient rats (Received 12 July 1974. Accepted 16 September 1974)

SELENIUM is an essential micronutrient for many animals of economic significance.Its deficiency states often occur along with deficiency of vitamin E, and have been implicated in fulminating hepatic necrosis in swine and rats, poor growth in fowl and also poor growth and failure to reproduce in sheep (UNDERWOOD, 1971). Failure of spermatogenesis of haircoat development in rats (McCoy & WESWIG,1969)' and also pancreatic atrophy in chicks (GRIES& SCOTT,1972) are specifically correctable by administration of selenite. The element is an integral part of two enzymes, GSH peroxidase (glutathione: hydrogen peroxide oxidoreductase, EC 1.11.1.9) of erythrocytes and bacterial formate dehydrogenase (formate: NADP oxidoreductase, EC 1.2.1.2) and also occurs in protein A of the clostridial glycine reductase system and in a muscle hemoprotein of unknown function (STADTMAN, 1974). The precise biochemical functions of selenium are not known except, perhaps, in the case of GSH peroxidase. GSH and its peroxidase may have roles in protecting membrane lipids from peroxidation, in maintenance of tissue sulphydryl and also in GSH-dependent amino acid transport (MEISTER,1973); the glycine reductase system generates ATP (STADTMAN, 1974). In a recent study of selenium in tissues of deficient rats, BROWN& BURK(1973) found a remarkable degree of 75Se retention in brain and in testis. The GSH peroxidase activity of brain in several species has been found to be low relative to liver and erythrocytes (DEMARCHENA et al., 1974; LAWRENCE et a!., 1974); testis contains GSH peroxidase, but is not a rich source (CHOW& TAPPEL,1974). These observations suggested to us the existence of biological functions for selenium other than participation in GSH peroxidase activity. This work is a portion of a study of the anatomical and subcellular distribution of selenium and of its chemical form in tissues.

and^

45 min. Isotope content of tissues was based on the blotted wet weight determined to + 0 3 m g on a Mettler H-6 balance. In the case of minute quantities of tissue, such as pineal gland, weighing was done on an Ainsworth type 24N balance with precision kO.01 mg. Na, 75Se03at 99% purity was obtained from Amersham/ Searle Corp. and had a specific activity of 8.5 mCi/mg. Radioactivity was determined over the range of 080@340 MeV in a Nuclear-Chicago well-type gamma counter, 1185 series. The S.D. of the count rate was 1 per cent of that rate or less, except for minute tissue samples of pineal gland and optic nerve; in these cases the coefficient of variation was 3 per cent. Counting rates were corrected for radioactive decay over the experimental period by the inclusion of a standard which represented 1.0 per cent of the injected activity. The soluble fraction of brain was prepared by homogenization of tissue in pH 7 1 Tris phosphate, 0.01 M, and 20 min centrifugation at 24,000 8.

EXPERIMENTAL PROCEDURES Weanling Sprague-Dawley male rats (Holtzman Co., Madison, WI) consumed a selenium-deficient diet supplied by ICN Nutritional Biochemicals, Cleveland, Ohio, which contained less than 10 pg/kg of selenium. The diet composition by weight was (a) Torula yeast, 30%, (b) sucrose, 58.8%, (c) stripped lard, 5%, (d) WilliamsBriggs salt mixture, 5%, (e) vitamin diet-fortification mixture (ICN Nutritional), 1% (f) D,L-methionine, 02% and (9) D,L-a-tocopherol, 250 i.u. per kg. After 12 weeks on the test ration, each rat received 4.2 pCi of Na, 75Se03 intraperitoneally in 1 ml isotonic saline. Rats anesthetized with methoxyflurane were quickly exsanguinated by cardiac puncture: dissection time was 30593

RESULTS The CNS of sebnium deficient rats showed a n unusual pattern of continuous net uptake of 1 5 S e with only a slight decrease toward the end of the 36-day experimental period. Figure 1 illustrates the uptake into anatomically distinct regions of brain and spinal cord. Data for optic nerve, perirenal fat and liver are included for comparison; these show the exponential decrease typical of most tissues. The curves for cerebellum and cerebral hemispheres were significantly different by two-way analysis of variance ( P = 0.001). Figure 2 gives uptake curves for adrenal and pineal, nervous tissue derivatives which have a secretory function. Testis is included because of the similarity of its kinetic pattern to that of brain and because of its extremely high isotope content. The initial uptake of 6O,o,ooCrSO,OOO c.p.m./g of fresh gland ranks adrenal and pineal second only to testis among all tissues in 75Secontent on a weight basis. The difference between these glands and the liver and perirenal fat (representative of other tissues) is maintained even after 1015 days when tissue isotope levels have stabilized. Comparison of Figs. 1 and 2 shows that pineal contains 2-4 times more isotope per gram of wet weight than brain, and that pineal kinetics are very different. The pineal more resembles a typical peripheral tissue than it does CNS. When cerebellum was homogenized in Tris buffer, 59 per cent of the 75Sewas recovered in the soluble fraction. The isotope was not removed by dialysis at pH 7.

594

Short communication GSH

Cerncd Soinat Cord 1

GSSeSG

YI c E

0

V

5,000

'1

5

I0

15

20

25

30

35

Doys post Administration

I . Uptake of "Se0:- into central nervous tissue of selenium-deficient rat. Except for liver and fat where mean values only are shown, each point represents data for one animal.

FIG.

(2)

We doubt that this process accounts for the prolonged uptake and slow turnover observed for brain 75Se. Kinetics of other GSH-rich tissues were different from CNS; radioactivity in washed, packed red cells and in lens never exceeded 3000 c.p.m./g and both values decreased steadily to about 500 c.p.m./g in the final pair of rats. Further, selenotrisulphides are substrates for GSH reductase [NAD(P)H, :glutathione oxidoreductase, EC 1.6.4.21. The unstable selenopersulphide GSSeH in an intermediate in this reaction 1971). (GANTHER,

Cerebral Hemspheres

75se

+ GSSeSG + GSSeH + GSSG.

+ NAD(P)H, *-

2 GSH

+ Seo + NAD(P)

Accumulation of insoluble elemental selenium would be expected in tissues in which GSH selenotrisulphide is an important intermediate. The possibility of incorporation into brain protein must be taken most seriously. When cerebellum was extracted, much of the "Se was soluble in aqueous buffer in a nondialysable form. This suggests that Se0:- was not converted to Se'. Although most brain proteins have turnover half-times of IC-20 days, it is clear that some are degraded much more slowly. Half-lives of from 150 days to a lifetime have been reported for proteolipid and some non-protein compounds in CNS (LAITHA & TOTH,1966). The data for pineal gland suggest that transport across the blood-brain barrier may be an important factor in the accumulation of 7sSe in CNS. Although the brain and pineal are close anatomically, the two have been shown to

DISCUSSION Accumulation of "Se in brain confirms and extends the study of BROWN& BURK(1973). The result must be considered in terms of the very limited information available about the chemical forms of selenium as it occurs in tissues. Selenium is transported in association with plasma proteins (BURK,1973) and substantial quantities pass the bloodbrain barrier as early as 2 h after administration as SeO:-. Known metabolites include Se2- (DIPLOCKet al., 1971), dimethyl selenide (MCCONNELL & PORTMAN, 1952) and triet al., methyl selenonium selenonium derivatives (PALMER 1970). Prompt uptake and retention by brain is not easily explained by partition of the above metabolites in favour of brain lipid because body fat does not show corresponding uptake and retention. We considered the possibility that "Se may be covalently bound to GSH or to protein sulphhydryl as selenotrisulphide. GSH has been found in brain and erythrocytes at a 1958; BEUTLERet concentration of 2 mM (BERL& WAELSCH, 20,000 al., 1963)and in lens at 8 mM (ROSNER et al., 1938); in these & tissues it has a turnover half-time of 65-71 h (DOUGLAS I I I I 1 I I MORTENSEN, 1956; MORTENSEN et a[., 1959; McMlLLAN rt 5 10 15 20 25 30 35 al., 1959).There is evidence that selenium incorporation may 75% Doys post Administration occur in oiuo into some high cystine content proteins (JENKINS & HIDiRocLou, 1971)and a reaction sequence has been FIG.2. Uptake of ?SeO:- into testis, adrenal and pineal proposed (GANTHER, 1971). gland. Data points are mean values for 2-4 animals. The 4GSH +. H,SeO,+ range did not exceed 20 per cent of the mean for any set of - values and was usually less than 10 per cent. GSSeSG + GSSG + 3 H 2 0 ( I )

'+

Short communication differ with respect to the dye-transport experiments which operationally define the blood-brain barrier. It is possible that the brain form, but not the pineal form of selenium, is trapped with limited access to the general circulation. There is a small decrease in cerebellum radioactivity near the end of our experimental period which suggests that this is not the case. It is reasonable to assume, without proof for the present, that there exists some mechanism, however slow, for eliminating selenium from the CNS; the rate of that process could be a determining factor in the development of prominent CNS signs in acute selenium toxicity (UNDERWOOD, 1971). Selenium content of central nervous system was highest in regions containing the most gray matter. Optic nerve contained less than half the radioactivity per gram of tissue found in cerebellum; peripheral nerve, lower spinal cord, and cauda equina (not shown) were even lower than optic nerve. Regional anatomical localization of the isotope also suggests that the data reflect the distribution of a specific chemical compound. The pattern found, cerebellum > cerebral hemisphere > spinal cord, is not that described for Vitamin E (DIPLOCK et al., 1967), GSH and GABA (BERL& WAELSCH,I958), enzymes of catecholamine synthesis 1967). Cerebellum does contain, in some spe(GOLDSTEIN. cies, higher concentrations of the following than spinal cord: ATP:L-methionine S-adenosyl transferase (EC 2.4.2.13).cystathionine sythase (VOLPE& LASTER, 1970). D-amino-acid oxidase D-amino-acid oxygen oxidoreductase (deaminating), 1.4.3.3 (NEIMSer al., 1966). The initial uptake of 60,OOO-80,OOOc.p.m./g of fresh pineal ranks this organ with only testis and adrenal as the most radioactive tissues on a wet weight basis. Pineal uptake of \ has been reported in a single study of turkeys (McFARLAND et al., 1970). The difference between these glands and the liver and perirenal fat, representatives of other organs, is maintained even after tissue isotope levels have stabilized at 10-15 days. Pineal contains 2-4 times the "Se found in brain, and pineal kinetics are also very different; turnover of the isotope in pineal more resembles that of a peripheral tissue than it the turnover of brain, to which pineal is intimately related anatomically. This finding gives added weight to the evidence for specificity of the processes involved in selenium metabolism. The data presented indicate unique patterns of selenium uptake and distribution in the brains of deficient animals but they do not define the precise physiological relevance of the element at this dosage. We used moderately seleniumdeficient animals because retention in most tissues of a tracer dose is inversely related to dietary load (BURKet al., 1973). The '% retained in brain was about 2.5 ng/g brain tissue; the use of a nutritionally adequate in selenium would have reduced brain content to near the detection limits. Brain has been generally overlooked in most other studies of selenium metabolism and of GSH peroxidase. Recently DE MARCHENA et al. (1974) have shown that brain and endrocrine tissue levels of this enzyme are low; these low levels are less sensitive to the effects of selenium deficiency than are the higher levels found in liver and erythrocytes (LAWRENCE ut al., 1974). We would suggest that selenium

595

has biological functions not yet described and that these may be of neurochemical importance. Department of Psychiatry and Psychiatry Research Unit, University of Minnesota, Health Sciences Center, Minneapolis, M N 55455, U.S.A.

G. A. TRAPP J. MILLAM

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