Selenium and lead: Mutual detoxifying effects

Toxicology, 6 (1976) 377--388 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands

SELENIUM AND LEAD: MUTUAL DETOXIFYING EFFECTS

S.C. RASTOGI **, J. CLAUSEN * and K.C. SRIVASTAVA Institute of Hygiene, Preventive and Social Medicine, University of Odense and • Institute of Molecular Biology, Chemistry and Toxicology, University of Roskilde (Denmark) (Received March 2nd, 1976) (Revision received July 9th, 1976) (Accepted July 20th, 1976)

SUMMARY

Antagonistic toxic effects of selenium and lead were studied in growing rats. Chronic lead intoxication was produced by cutaneous application of lead naphthenate solution (80--200 mg Pb/kg body weight) for a period of 8 weeks and chronic selenium intoxication was induced by giving 5 ppm, 10 ppm and 15 ppm selenium in drinking water. The growth rate and food consumption of rats receiving selenium in addition to lead approached normal rate while animals treated with only one of them showed hampered growth rate and lower food consumption. The enzymatic activity of 5aminolevulinic acid dehydrase (ALA-D) in whole blood, liver and kidney and liver P-450 enzyme activity were normal in rats receiving both selenium and lead. The enzymic activities assayed were, however, depressed in the animals receiving either lead or selenium. Assay of lead and selenium in liver, brain, kidney and blood was carried out. Rats receiving both metals and higher concentrations of these metals in the organs studied, as compared to those only receiving one component. The data seem to indicate that the effect of selenium on the toxic effects of lead is similar to its protective role against methylmercury intoxication.

INTRODUCTION

Protection of cadmium (Cd)-induced testicular injury by selenium (Se) injections was observed by Kar et al. [1] and Mason and Young [2] dem** Present address: Neurochemical Institute, 58 R~dmandsgade, Copenhagen, Denmark. Abbreviations: ALA-D, ~-aminolevulinic acid dehydrase ; PBG, porphobilinogen; RBC, red blood cells.

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onstrated that chronic doses of Se could protect Cd-induced testicular injury. The protective role of Se seems also to cover intoxication with other heavy metals besides Cd, thus Parizek and Ostadalova [3] reported protection against sublimate intoxication by small doses of Se. Gunn et al. [4] proposed that Se makes complexes with Cd. These Se-Cd complexes may exert a lower toxicity than free Cd. Work by Pariken and co-workers [5--8] has demonstrated that animals intoxicated by mercury (Hg) and Cd were protected by long-term administration of Se and vice versa. These workers also proposed that the toxicity of silver (Ag) and I I B group metals of the periodic system could be prevented by Se [ 7,8]. Reports from Ganther et al. [9--11] indicate that Se offers protection against the toxic effects of Ag, methylmercury and Cd. Se toxicity in chicks has been shown to be modified by Ag and copper [12]. Besides antagonistic effects, the synergistic effects of long-term administration of inorganic Se on Hg intoxication has been described by Groth et al. [13]. Possibilities of Se protection against lead (Pb) toxicity have been indicated in one report [14]. This background p r o m p t e d us to study protection of chronic lead intoxication by chronically administered Se and vice versa. Absorption of Pb through the skin has been demonstrated by us previously [15], and this property was used to produce chronic lead intoxication in rats. It was, therefore, possible to introduce Se and Pb separately from different entrance routes, thus eliminating mutual interaction in the intestinal tract. Se was administered through drinking water. Results have shown that Se and Pb offer a definite protection against each other. MATERIALS AND METHODS Animal material 1-month-old conventional Wistar rats of both sexes, purchased from M~bllegaard Laboratory, Skensved, Denmark, were maintained on a diet optimal in all respects (Table I) in individual plastic cages free of heavy metal contamination. Rats were divided into five groups, A, B, C, D and E, each containing 10 animals. Group A served as control and was given distilled water for drinking. Rats in group B, C, and D were shaven (2 X 2 cm 2 area) at the back of the neck and were coated with lead naphthenate solution, in an ether--ethanol mixture (25 : 75, v/v), containing 5% (w/v) Pb. The first 3 weeks 0.2 ml of lead solution was applied every alternate day with disposable plastic syringes (by dripping on the shaven area). Thereafter the dose was increased to 0.5 ml, thus approx. 80--200 mg Pb/kg body weight (varied according to growth and body weight of the animals) was applied every time during the whole experimental period. Animals in group B received distilled water for drinking and in C and D groups, respectively, 5 ppm and 10 ppm Se as sodium selenite in distilled water. Rats in group E were given 15 p p m Se in distilled water. Body weight, the food consumed and water taken by rats were estimated once a week. After 8 weeks, four rats from each group were killed by de-

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capitation. Approx. 2 ml blood drawn from the heart in disposable plastic syringes containing 0.2 ml of 3.8% (w/v) citrate was transferred into plastic tubes and cooled immediately. Liver, brain and kidneys were removed, washed with cold saline, dried with filter paper, weighed and frozen till further analysis. Rats remaining in B, C, and D groups were given 6 subcutaneous injections of lead naphthenate solution (0.5 ml) every alternate day and their mortality was recorded during a period of 2 weeks. TABLE I COMPOSITION OF RAT DIET

(A) Crude mixture o f rat diet Wheat Oats Maize Fishmeal Soy meal Dry yeast Lucerne Skimmed milk powder Vitamin mixture Mineral mixture

(B) Mineral mixture Dicalcium phosphate Sodium chloride Iron sulfate Manganese oxide Copper sulfate Cobalt sulfate Zinc oxide Potassium iodide

(C) Vitamin mixture Oyster shell meal Ethoxyquin Wheat bran

% (w/w) 12.0 30.0 30.0 8.0 9.0 3.0 2.0 3.0 1.0 2.0 % (w/w) 83.0 15.0 1.4 0.3 0.2 0.06 0.03 0.01 % (w/w) 82.5 2.5 15.0

Vitamins were added in the following amounts (per g crude diet): Vitamin A 40 I.U. Vitamin D 3 10 I.U. Riboflavin 18/ag D-Panthothenic acid 27/ag Vitamin K 40 pg Niacinamide 50/dg Thiamine 6 pg Vitamin E 200/dg Choline chloride 1000/ag Vitamin B12 0.025/~g Diet mixed as indicated above contained 16% protein.

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Preparation o f liver microsomes L i v e r acid m i c r o s o m e s for c y t o c h r o m e P-450 assay were p r e p a r e d by the m e t h o d o f F r y and Bridges [ 1 6 ] . A c c o r d i n g to this m e t h o d a p p r o x . 1 g liver was h o m o g e n i z e d in 2 ml o f 1.15% (w/v) KC1 and c e n t r i f u g e d at 8 0 0 0 gmax f o r 15 min. T h e p o s t m i t o c h o n d r i a l s u p e r n a t a n t t h u s o b t a i n e d was adjusted t o pH 5.6 with sufficient 0.2 M s o d i u m acetate buffer. It was t h e n c e n t r i f u g e d at 10 000 g-max, for 10 min and the resulting pellet was washed once in 1.15% (w/v) KCl-glycerol (4 : 1, v/v) and recentrifuged. The final pellet was t e r m e d " a c i d m i c r o s o m e s " .

E n z y m e assays The e n z y m i c activity o f A L A - D , EC 4.2.1.24, was assayed in fresh b l o o d , liver and k i d n e y ; t h a t o f c y t o c h r o m e P-450 also in liver m i c r o s o m e s . A L A - D was assayed at p H 6.4 with A L A as substrate. T h e f o r m a t i o n o f PBG was e s t i m a t e d by the PBG-Ehrlich reagent c o m p l e x . M e t h o d s used for A L A - D assay in liver and k i d n e y [15] and in b l o o d [17] have been r e p o r t e d in detail elsewhere. A L A - D activity in liver and k i d n e y was expressed as n m o l e s o f P B G / h / g tissue, and for the b l o o d it was expressed as p m o l e s o f PBG/h/1 RBC.

TABLE II SURVIVAL OF RATS AFTER Pb AND Se TREATMENT Treatment of rats

Number of rats

Rats survived after 8 weeks

% survival

10 10 10 10 10

10 10 10 9 9

100 100 100 90 90

Remarks

Cutaneous Pb Control Pb Pb+5ppmSe Pb + 10 ppm Se 15 ppm Se

1 rat died in the 4th week 1 rat died in the 4th week

Rats survived after 2 weeks

Subcutaneous lead Control Pb

6 6

6 2

100 33.3

Pb + 5 ppm Se

6

4

66.7

Pb + 10 ppm Se

5

1

20

15 ppm Se

5

5

100

380

2 rats died after the 4th injection; 1 after the 5th and 1 after the 6th 1 rat died after the 4th injection and another after the 6th 2 rats died after the 4th injection, 1 after the 5th and 1 after the 6th

Cytochrome P-450 activity in the acid microsomes was assayed by the demethylation of para-nitroanisole to para-nitrophenol [18]. Microsomal protein was determined by the m e t h o d of Lowry et al. [19]. Enzyme activity is expressed as pmoles of p-nitrophenol/min/mg protein. Pb and Se determinations Pb in blood, brain, kidney and liver was determined by flameless atomic absorption s p e c t r o p h o t o m e t r y at 283.3 nm [15,17]. Se was assayed, after the same pretreatment of samples used for Pb assay, using Se-hollow cathode lamp at 196 nm. Ch e mica Is Commercial lead naphthenate containing 30% w/v Pb, a gift from Corn. Van Loocke, Brugge, Belgium, was used without any purification. All other chemicals were of highest purity available from E. Merck, Darmstadt, W. Germany. Statistical analysis Student's t-test was used for assay of level of significance. Differences were estimated of significance for p-values ~< 1%. RESULTS

Behavioural and physical parameters Cutaneous application of lead solution resulted in hyperactivity and aggressiveness in the rats after one week in B, C and D groups. Weakness was first observed in these rats after the 3rd subcutaneous injection of lead naphthenate solution. Mortality o f rats is described in Table II. Incoordina-

TABLE III BODY WEIGHT OF RATS Week

0 1 2 3 4 5 6 7 8

Treatment of rats Control g

Pb g

Pb + 5 p p m Se g

Pb + 10 p p m Se g

15 p p m Se g

57-+ 9 9 6 + 11 1 4 8 _+ 14 1 8 9 +- 24 2 1 8 + 35 2 6 2 -+ 4 5 278 ± 49 3 1 2 ± 21 3 2 9 ± 23

57+- 8 105+- 11 1 5 0 ± 13 1 8 2 +- 14 1 9 9 + 20 2 4 3 + 27 2 6 0 ± 17 267 -+ 14 271 ± 16

49+ 9 96+ 7 1 4 0 +- 10 1 7 8 +- 15 2 0 5 ± 20 2 4 5 +- 21 2 5 3 ± 23 2 7 0 -+ 16 2 9 0 -+ 15

42-+ 7 77± 9 1 1 3 + 13 1 4 3 -+ 22 1 3 4 _+ 32 1 9 3 +_ 4 0 211 ± 30 2 2 8 ± 14 2 5 9 ± 18

39-+ 5 58± 7 77 ± 9 94 ± 15 8 5 ± 28 107 ± 37 1 2 4 ± 41 1 2 9 _+ 53 135 + 48

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TABLE IV FOOD CONSUMED/RAT/WEEK Week

Treatment of rats Control g

1

77±

Pb g

6

2

113± 13

3 4 5 6 7 8

130± 124 ± 140± 141 ± 155± 149±

80± 118+133± 118 ± 133± 124 ± 125± 129±

20 23 16 22 11 6

7 6 11 26 9 38 13 4

Pb + 5 p p m Se g

Pb + 10 p p m Se g

71± 3 105± 6 129± 8 123 ± 20 132± 10 1 5 1 ± 21 152± 5 147± 5

59± 83± 102± 98 ± 108_+ 132 ± 144± 144±

7 9 11 18 19 11 3 5

15 p p m Se g

40± 55-+ 64± 66 ± 51± 6 5 -+ 71± 72±

3 5 4 10 11 21 23 25

tion of legs followed by paralysis was observed in rats who died in groups B and D, after subcutaneous administration of Pb. These rats could n o t eat or drink for 24 h because they were not able to walk to the food and water supply. This was, however, not observed with the rats in group C. After 2nd subcutaneous injection of lead solution, rats in group D had blood in their urine (visually evaluated).

Body weight, food and Se consumption Growth rate of rats (Table III) during an 8-week experimental period was in the following order, A > C > B > D > E. The growth rate of group E was the lowest among all groups. As shown in Table IV, the order of food intake was A > C > D > B > E. Table V shows Se consumed by rats per week. It is evident that rats in group D had a higher growth rate (Table III) and consumed more food (Table IV) compared to rats in group E, though the first group consumed more Se (Table V). TABLE V Se C O N S U M E D / R A T / W E E K Week

Treatment of rats Control

1 2 3 4 5 6 7

. . -------

8

--

382

.

Pb

Pb + 5 p p m Se Pg

P b + 1 0 p p m Se Pg

-------

470+ 560-+ 625 ± 655 ± 650 ± 675 ± 650±

690-+ 800-+ 970-+ 850 ± 1060 ± 1090 ± 1310+

--

5 9 0 ± 85

.

20 30 30 50 45 85 35

60 60 140 170 230 230 90

1 1 9 0 _+ 190

15 p p m S e ~g 645+ 690-+ 765 ± 795 ± 615 ± 630 ± 1185±

30 60 105 150 180 120 630

9 0 0 ± 180

TABLE VI ORGAN WEIGHT/BODY Organ

W E I G H T • 10 4

Treatment of rats Control

Brain

Pb

91 ± 2 8

Liver Kidney

P b + 5 p p m Se

65 ± 1

474±

6

414±

7

76±

5

92± 1 p < 0.005

P b + 1 0 p p m Se

1 5 p p m Se

76 ± 4

7 8 ± 15

1 2 2 -+ 3 9 p < 0.005

5265 9 p < 0.001

572± 12 p < 0.001

447+

95+- 1 p < 0.005

98± 8 p < 0.005

112± 5 p < 0.001

114

p-values concern comparisons with control values.

Ratio of organ to b o d y weight (relative growth) was calculated for the killed rats (Table VI). Brain and liver weights of rats treated with lead (B) were significantly lower than in the control group and the decrease was restored in groups C and D. Group E of rats, however, had the highest brain weight (p < 0.005). There was no change in the liver weight of group E animals as compared to the control group. The kidney weight was significantly higher in the experimental group than in group A, having a maximum value in group E.

Enzyme activities Significantly depressed ALA-D, an indicator of Pb intoxication, was observed in blood, liver, and kidney 09 < 0.001) of rats in group B (Table VII). This change was, however, n o t found in groups C and D except for kidney in group D (p < 0.005). Se also reduced ALA-D activity in all the studied tissues (p < 0.001). TABLE VII ALA-D ACTIVITY

Tissue

Blood a Liver b Kidney b

IN VARIOUS

TISSUES (ASSAYED

IN TRIPLICATE)

T r e a t m e n t o f rats Control

Pb

P b + 5 p p m Se

P b + 1 0 p p m Se

15 p p m Se

683 + 36

469 ± 77 p < 0.001

6 9 2 ± 22

635 + 39

320 ± 41 p < 0.001

2214 ± 53

1720 ± 67 p < 0.001

2 1 3 9 +- 3 8

2021 ± 88 p < 0.005

1688 ± 86 p < 0.001

7 4 2 ± 54

505 ± 26 p < 0.001

677 ± 68

666 ± 78

4 0 9 -+ 6 7 p < 0.001

a pmoles of PBG/h/I RBC.

b n m o l e s o f P B G / h / g tissue.

383

TABLE VIII LIVER P°450 ENZYME ACTIVITY a IN RATS IN VARIOUS GROUPS Control

1.5 +- 0.2

Treatment of rats Pb

Pb + 5 ppm Se

Pb + 10 ppm Se

15 ppm Se

2.3 -+ 0.5 p < 0.05

2.0 ± 0.9 p < 0.10

1.8 +- 0.6

1.1 _+ 0.4 p < 0.10

a pmoles of p-nitrophenol]min/mg protein, assayed in triplicate. L i v e r c y t o c h r o m e P - 4 5 0 e n z y m e a c t i v i t y was s t i m u l a t e d b y t h e t r e a t m e n t w i t h l e a d n a p h t h e n a t e (p < 0 . 0 5 ) ( T a b l e V I I I ) . T h i s was, h o w e v e r , r e s t o r e d t o a n o r m a l level b y Se i n t a k e . O n l y Se (E g r o u p ) i n h i b i t e d t h e a c t i v i t y o f l i v e r P - 4 5 0 e n z y m e . P b a n d Se d i s t r i b u t i o n .

Pb and Se distribution T a b l e I X s h o w s d i s t r i b u t i o n o f P b i n b l o o d , b r a i n , k i d n e y , a n d liver o f all t h e g r o u p s . T h e r e is a n i n c r e a s e ~o f P b c o n t e n t o f k i d n e y a n d b l o o d (p 0 . 0 0 1 ) f r o m A t o D. I n t h e livers, t h e i n c r e a s e o f l e a d f r o m A t o B was s i g n i f i c a n t (p < 0 . 0 0 1 ) , a n d t h e r e was a f u r t h e r i n c r e a s e i n C. P b c o n t e n t o f b r a i n was i n c r e a s e d f o r B as c o m p a r e d t o A (p < 0 . 0 0 1 ) a l t h o u g h it was s l i g h t l y l o w e r i n g r o u p s C a n d D, b u t still s i g n i f i c a n t . P b c o n t e n t o f s t u d i e d t i s s u e s o f g r o u p E a n i m a l s w e r e c o m p a r a b l e w i t h g r o u p A. Se d i s t r i b u t i o n i n b l o o d , k i d n e y , liver, a n d b r a i n is d e s c r i b e d i n T a b l e X. T h e r e was v e r y l i t t l e Se p r e s e n t i n all t h e s t u d i e d o r g a n s o f g r o u p s A a n d B. Se c o n t e n t was h i g h e r i n g r o u p s C, D a n d E. T h o s e r a t s r e c e i v i n g 5 p p m Se

TABLE IX DISTRIBUTION OF Pb IN VARIOUS TISSUES (DETERMINED IN DUPLICATE) Tissue

T r e a t m e n t o f rats

Control

Pb

Pb + 5 ppm Se

Pb + 10 ppm Se

15 ppm Se

Blood a

9.0 -+ 1.2

36.0 ~ 1.2 p < 0.001

53.5 + 2.9 p < 0.001

85.5 ± 2.9 p < 0.001

11.0 -+ 1.4

Liver b

5.5 -+ 0.3

27.4 ± 1.4 p < 0.001

32.5 -+ 2.3 p < 0.001

30.9 + 1.1 p < 0.001

5.5 -+ 0.6

Kidney b

8.4 +- 0.3

61.6 -+ 2.0 p < 0.001

135.7 -+ 5.0 p < 0.001

169.7 ± 2.1 p < 0.001

9.9 -+ 1.0

Brain b

7.3 -+ 0.7

25.1 -* 2.9 p < 0.001

19.8 + 0.9 p < 0.001

20.8 -+ 0.2 p < 0.001

8.6 + 0.4

a ng Pb/ml blood. b pg Pb/g tissue.

384

TABLE X DISTRIBUTION

O F Se I N V A R I O U S

Treatment

TISSUES (DETERMINED

IN DUPLICATE)

of rats

Control

Pb

P b + 5 p p m Se

P b + 1 0 p p m Se

1 5 p p m Se

Blooda

17-+

6

19± 1

93± 6 p < 0.001

205± 4 p < 0.001

84± 8 p < 0.001

Liver b

17 ±

4

19 ± 6

328 ± 40 p < 0.001

690 ± 150 p < 0.001

518 ± 112 p < 0.001

Kidney b

45 ± 10

54 ± 4

941 + 67 p < 0.001

1220 ± 114 p < 0.001

875 ± 48 p < 0.001

7± 2

61± 3 p < 0.001

112± 9 p < 0.001

10± 2 p < 0.005

Brainb

6±

1

a ng Se/ml blood. b 10-2 pg Se/g wet tissue. .

in addition to the Pb treatment (C) showed Se values that were similar to or less than the values for rats receiving only 15 ppm Se (E). This, however, was not the case in brain tissue for E. Se c o n t e n t in all the tissues studied in group D was greater than those for group E. Se level in the brain of E group animals, although higher than A and B, was very much below C and D. •

DISCUSSION

it

~,-

Lead naphthenate, a c o m m o n additive of various lubricating oils and greases, may be absorbed through the skin and t h e absorbed lead may be distributed in the b o d y [15]. We therefore selected lead naphthenate for the present study since it made it possible to administer Pb and Se using t w o different routes, thus avoiding their interaction before the ~ptake in the b o d y . Although we have n o t studied the effect on the rats when they received 5 p p m and 10 ppm Se in drinking water, it is known that 3 p p m Se given in drinking water produces chronic Se toxicity in these animals [20,21]. Cutaneous application of lead naphthenate solution (80--200 mg Pb/kg b o d y weight) produced chronic lead intoxication in rats, indicated by reduced growth rate, less food consumption and reduced ALA-D activity in blood, liver, and kidney and P-450 enzyme activity during 8 weeks of experimental period. The growth rate, f o o d consumption and the ALA-D and P-450 enzymic activities of rats treated with both Se and Pb were comparable with control rats; Pb and Se levels in the studied organs of these rats were higher as compared to those treated with either of them. Thus it is evident that if Pb and 8e are given simultaneously, their toxicity will be counterbalanced. Therefore, it may be argued that the high concentration of one of these metals in

385

various organs can not be accepted as the only factor responsible when metal toxicity is expressed. Dennis [14] has indicated that Se may decrease the toxicity of lead shot in water fowls and Frost [25] has suggested that Pb, like many other heavy metals [9,22--24], may form a complex with Se. Sodium selinate has been used in the present communication and the complex formed may be PbSeO3. Since this c o m p o n e n t is slightly soluble in water, it may be the u n k n o w n complex [26]. Results of the present study support such a theory. The complex may be released from tissue to blood and accumulated in the kidneys, where the concentration of these materials is found to be highest. More Pb was absorbed in rats treated with Se, as indicated by concentration of Pb in the tissues under study (Table IX). When the Se dose was increased from group C to D there was a decrease in the liver Pb level, followed by increased Pb concentration in the blood and kidneys. The Se level in brains of rats receiving 15 ppm Se in the drinking water was higher compared to the levels in normal rats, although it was much lower than the Se level for brains of rats treated both with Se and Pb (Table X). Smith [ 27] has indicated that Se does n o t normally accumulate in the brain. Therefore, an explanation for the findings mentioned above, may be Pb damage to the blood-brain barrier which makes it possible for Se to reach the brain. Ohi et al. [ 28] suggested that Se accumulated in the brain of rats treated both with methylmercury and selenite. Impairment of the blood-brain barrier in methylmercury poisoning, causing plasma exudation and disturbed blood-brain exchange of nutrients, has been reported by Steinwall and Olsson [29]. Once Se has reached the brain it may start complexing Pb in the brain, analogous to the findings of Iwata et al. [30]. The elimination of methylmercury by Se may afford protection against acute methylmercury poisoning. Thus, it may account for a lower Pb-level in the brain of rats with a higher Se-level (Tables IX and X). ALA-D depression has been regarded as an optimal indicator for lead toxicity [31--39]. Our findings, that selenium also produced such an effect at a toxic level may show that the evaluation of lead toxicity is more complex than previously supposed. However, the exact relationship between ALA-D activity and the Se-level remains to be clarified. It will be necessary to evaluate whether in vivo inhibition of ALA-D takes place at a certain threshold concentration of Se. ACKNOWLEDGEMENT S. C. Rastogi is supported by DANIDA grant 104.P3.Ind.408. We are thankful to Mr. Erik Ostergaard for his help in animal work and to Mrs. Ranja Andersen for the assay of P-450 enzyme activity.

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