9 1985 by The Humana Press Inc. Atl rights of any nature whatsoever reserved. 0163-4984/85/0809-0137502.00

The Effects of Dietary Sulfur on Selenium Utilization by the Rat AHMED G. A B D E L - R A H I M , t J O H N Ro ARTHUR,* AND COLIN F. MILLS

Rower Research Institute, Bucksburn, Aberdeen, AB2 9SB, UK Received April 20, 1985; Accepted May 2, 1985

ABSTRACT The antagonistic effects of S on Se utilization were studied by measurement of tissue Se concentrations and glutathione peroxidase (GSHpx, E.C.1.11.1.9) activities in rats offered Torula yeast-based diets supplemented with sodium sulfate to provide 1.5, 3, or 5 g S/kg diet. Increasing sulfur concentrations in the diet caused small but significant decreases in GSHpx activity of liver, spleen, heart, lung, small intestine, and skeletal muscle. Also significantly decreased were Se concentrations in liver and hair. Dietary sulfur content had no significant effect on body weight gain of the rats. With an adequate supply of Se in the diet, there are probably no major deleterious effects of dietary S on Se metabolism in the rat. lndex Entries: Selenium, effect of dietary S on; sulfur, effect on Se of dietary; selenium deficiency, and glutathione peroxidase; gluthathione peroxidase, and dietary sulfur; liver Se concentrations, and dietary sulfur.

INTRODUCTION Interactions b e t w e e n dietary S a n d Se in the r u m i n a n t have b e e n the subject of several investigations since it was first suggested (1) that h i g h dietary intakes of S m a y increase the incidence of Se-responsive m y o p a *Author to whom all correspondence and reprint requests should be addressed. tPresent address: Department of Physiology and Biochemistry, Faculty of Veterinary Science, University of Khartoum, Sudan. Biological Trace Element Research

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thy in grazing sheep. However, subsequent studies have indicated that, in sheep, the effects of dietary S on Se metabolism are not always demonstrable and, when present, are unlikely to have pathological relevance (2,3). Investigations of the effects of S on Se metabolism in the rat have been more limited than those in the ruminant. Sulfate added to the diets of rats has been shown to counteract the toxic effects of excess dietary selenate (4-6), possibly by increasing the urinary excretion of Se rather than by inhibiting its intestinal absorption. Lane et al. (7) have demonstrated that the supplementation of corn-soya or Torula yeast-based diets with sulfate will affect the activity of the selenoenzyme glutathione peroxidase (GSHpx E.C.1.11.1.9) in small intestine, colon, and liver of rats. Tissue GSHpx activity was decreased, with the exception of colonic tissue, where activity was increased when Se-deficient diets were supplemented with sulfate. The possible antagonistic effect of dietary S on Se utilization was monitored by comparing responses in tissue Se and Se-dependent GSHpx of rats recovering from Se deficiency on diets differing in S content.

MATERIALS AND METHODS Forty-four female Hooded Lister rats (37-39 g) of the Rowett Institute strain were housed in groups of five or six in plastic cages with stainless-steel grid floors. To enhance sensitivity of effects of S on Se metabolism, the rats were first depleted of Se. Selenium-deficient basal diet (Table 1), containing 0.007-0.01 mg Se/kg, and deionized water were available to the rats ad libitum for a period of 8 wk (pre-experimental period). At the end of this period, five of the rats were killed for measurement of tissue GSHpx activities. Five female rats that had consumed diet (Table 1) supplemented with Na2SeO4"10H20 (final concentration 0.06 mg/kg Se) were also killed and tissue GSHpx activity was determined. The remaining Se-deficient rats were then randomized into three groups (13 animals/group) and the basal diet (Table 1) was modified as indicated to give the following S concentrations: Group A (low S, 1.5 g S/kg), MgO replaced MgSO4.7H20; Group B (intermediate S, 3.0 g S/kg), diet as Table 1; Group C (high S, 5.0 g S/kg). The S source, NaaSO4, was added in place of an equivalent weight of sucrose. All diets were modified to provide 0.06 mg Se/kg with Na2SeO4.10H20; allowance was made for the small amounts of Se (1.2 mg/kg) introduced in the Na2SO4 supplements added. Seven rats from each group were killed after 3 wk repletion with Se (repletion phase 1) and the remaining six after 6 wk of treatment (repletion phase 2). Animals were anesthetised with ether and blood removed by cardiac puncture into acid-washed heparinized test tubes, plasma was obtained after centrifugation of blood at 4~ Blood and plasma were stored at 4~ before enzyme analysis, within 1 d of Biological Trace Element Research

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TABLE 1 Composition of Semisynthetic Diets ~ Component Torula yeast Sucrose Lard Major minerals Trace minerals Trace mineral mix Vitamin mix Choline HCI Vitamin B12 R-Tocopherol acetate Retinyl acetate Calciferol Menadione (Vit. K3) DL-Methionine

Weight used, g/kg 300 596 50 b c d e 1.0 25 200 8.0 0.025 5.0 4.0

(P~g) (rag) (mg) (rag) (rag)

"Torula yeast-Type B was obtained from Chas. Tennant and Co., Glasgow. Vitamins were obtained from Sigma (London) except vitamin B12 (Cytamen, Glaxo Laboratories Ltd., containing 250 ~g cyanocobalarnin/mL). All other chemicals obtained from BDH were of 'Analar' grade. ~Minerals added (g/kg): CaCO3, 15; Na2HPO4, 6.6; KH2PO4, 15.7; KC1, 1.1; MgSO4"7H20, 5.1; Na2SiOy5H20, 0.76. q~race minerals added (mg/kg): CuSO4"5H2O, 19.7; FeSO4"7H20, 249; MnSO4"4H20, 203; ZnSO4"7H20, 176. ~Trace mineral mix (5 g of mix added per kg diet) contains g/250 g: KIO3, 0.84; NaF, 0.277; NH4VO3, 0.023; NiCI2"6H20, 0.203; Cr2(SO4)3 K2SO4"24H20, 2.400; SnC14-5H20, 0.296; sucrose, 246.717. ~Vitamin mix (I g of mix added per kg diet) contains g/50 g: thiamine, 0.5; pyridoxine, 0.5; riboflavin, 0.5; p-aminobenzoic acid, 0.5; nicotinic acid, 1.5; Ca pantothenate, 1.00; folic acid, 0.25; biotin, 0.25; inositol, 20.00; sucrose, 25.00. sampling; tissue s a m p l e s w e r e r e m o v e d , w a s h e d with glass-distilled water, b l o t t e d with filter paper, a n d stored at - 2 0 ~ until a n a l y z e d . Tissues w e r e h o m o g e n i z e d in a teflon pestle/glass b o d y h o m o genizer in 10 vol of 0.1M p o t a s s i u m p h o s p h a t e buffer, p H 7.4. A f t e r centrifugation at 18,000g for 20 min, the s u p e r n a t a n t s were u s e d for enz y m e analysis. S e l e n i u m a n a l y s e s w e r e carried o u t using the m e t h o d of O l s o n et al. (8) with s o m e modifications (9). W h o l e blood, p l a s m a , a n d

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tissue GSHpx activities were determined by the method of Paglia and Valentine (10) using H202 as substrate. Protein was determined by the Biuret method. Statistical analysis was carried out by analysis of variance and Student's t-test.

RESULTS Table 2 shows the GSHpx activity of the rat tissues after the preexperimental Se-depletion period and 3 and 6 wk Se-repletion. Increased concentrations of dietary sulfur (high vs low or intermediate) caused significant decreases in spleen, lung, small intestinal, heart, and skeletal muscle GSHpx activity after 3 wk Se-repletion. After 6 wk Se-repletion, the GSHpx activity was significantly lower in liver, spleen, lung, small intestine, heart, and skeletal muscle. Changes in kidney GSHpx activity did not attain statistical significance. Also shown on Table 2, for comparison, are the tissue GSHpx activities of rats maintained on the Se-supplemented diet for the preexperimental period. These animals had significantly higher GSHpx activities than the Se-depleted group in all tissues examined. Table 3 shows the blood and plasma GSHpx activities of the rats after phases 1 and 2 of the experiment. Although GSHpx activity tended to be ret~ited inversely to dietary S, treatment differences were not statistically significant. The liver and hair Se concentrations of the rats are given in Table 4. Liver Se was significantly decreased by addition of sulfate S to the diets after 6 wk Se-repletion, but not after 3 wk repletion. Hair Se concentrations were significantly lower in the rats consuming the high S diets after 3 and 6 wk Se-repletion. The differing amounts of sulfur in the diets did not affect the growth rates of the animals (Table 5).

DISCUSSION The experiments described here were designed to favor the expression of any metabolic interactions between Se and S. Thus, the corresponding analogs SO 2- and Se042- were added to the diets for the following reasons, (i) SO 2- and SeO 2- are mutually competitive during absorption by ~olants (11) and by loops of rat intestine (12), (ii) the oxyanions SeO$- and SO 2- exhibit a greater diversity of structural and chemical properties than Se04 and SOa (13) and interactions between 2 2 SeOsand S03are less strong during absorption by plants (11), (iii) the importance of analagous chemical speciation has been demonstrated in previous studies of the differing potencies of a range of S compounds in protecting against Se toxicity (6). 9

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