- Email: [email protected]
Studies on the influence of sulfur compounds on molybdenum toxicity in rats
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
68,
l-8
(1956)
Studies on the Influence of Sulfur Compounds on Molybdenum Toxicity in Rats’ Robert Van Reen and Mary Ann Williams” ,3 From
the A1cCollunl-Pratt
Institute, I’he Johns Baltimore, Afaryland
Received
March
Hopkins
I:aiversit!y,
5, 1956
Ferguson, Lewis, and Watson (1) were the first to associate a scouring disease of cattle in certain areas of England with high molybdenum content of the pastures. Since then similar diseases in catt,le have been found in t,he United States and other parts of the world (2) and have been associated with copper insufficiency. The production of molybdenum t,oxicity in experimental animals has been reported also and copper salt shown to exert a protective effect (3-5). It has been suggested that molybdenum might cause the breakdown of labile phosphate esters in tissues (6,7) ; however, it has been observed that, the excretion of hippuric acid and acetylated p-aminobenxoic acid following the injection of benzoic acid and p-aminobenzoic acid, respectively, is approximately equal in control and molybdenum-toxic rats. This was interpreted to mean that adenosine triphosphate was still available for these reactions to occur in the toxic state.4 At, relatively high levels of dietary molybdenum, effects other than on copper metabolism have been reported in rats (8, 9).” In these studies liver alkaline phosphatase activity was found to increase, whereas kidney preparations decreased markedly in activity. These alterations were apparent in some animals who showed no external appearances of t’oxicity. 1 Contribution No. 140 of the McCollum-Pratt Institute, The Johns Hopkins University. This work supported in part by a contract No. AT(30-l)-1816 between the U. S. Atomic Energy Commission and The Johns Hopkins University. 2 Present address: Department of Home Economics, University of California, Berkeley 4, Calif. 3 With the technical assistance of Joyce Gile. 4 Williams, M. A., and Van Reen, R., in preparation for publication.
2
ROBERT VAN REEN AND MARY ANN WILLIAMS
Neilands, Strong, and Elvehjem (10) found that whole liver protected against molybdenum toxicity in rats when added to a purified type diet. Gray and Daniel (11) later reported that methionine had a similar effect in rats. The influence of methionine was extended to the enzymatic anomaly in rats and was shown to mitigate the effects of molybdenum. Dick (12) recently reported that sheep were more sensitive to molybdenum when fed a diet of chaffed lucerne hay than on a diet of chaffed oaten hay. The factor causing this difference was identified as inorganic sulfate (13). It seemed of interest, therefore, to re-examine the observations on rats and determine whether the influence of methionine could be explained on the basis of the conversion of its sulfur to inorganic sulfate. EXPERIMENTAL
PROCEDURE AND MATERIALS
The albino rats of the W&tar strain used in these studies were obtained from a commercial source at 4-5 weeks of age. The basal diet had the following percentage composition: casein, 20.0; glucose, 69.0; corn oil, 6.6; salt mixture U.S.P. XIV, 4.0; choline chloride, 0.1; 2500 international units (I.U.) vitamin A; 250 I.U. vitamin D; and 15 mg. a-tocopherol in corn oil, 0.3. Other vitamins were provided in the following quantities, mg./lOO g. diet: thiamine.HCl, 0.5; riboflavin, 0.8; niacin, 4.0; pyridoxine.HCl, 0.5; Ca pantothenate, 4.0; biotin, 0.04; folic acid, 0.2; menadione, 0.5; vitamin Brt , 0.003; inositol, 10.0; and p-aminobeneoic acid, 10.0. Sodium molybdate was added to the basal diet to provide the desired level of dietary molybdenum. The basal diet was also supplemented with 4 mg. copper/ 109 g. diet in the form of CuSOd.5Hz0 in addition to the 80 pg. copper in the salt mixture to provide sufficient copper to prevent anemia from occurring. The influence of methionine and inorganic sulfate on molybdenum toxicity was investigated in the first experiment. Molybdenum was fed at 1200 p.p.m. and methionine at 0.3 g./lOO g. diet. Sodium sulfate was mixed in the diet at 0.29 g./lOO g. These levels provided 1.2 mmoles molybdenum and 2.0 mmoles of sulfur per 100 g. diet. Male weanling rats were employed. The’ second experiment extended the studies to the influence of cystine, sodium thiosulfate, and intraperitoneally injected sodium sulfate. Both male and female rats were used with equal numbers of each in the groups. Ad Zibitum feeding was employed since previous experiments indicated the same alteration in alkaline phosphatase activity in ad Zibitum and pair-fed animals. Determinations were made of liver and kidney alkaline phosphatase activity after 4-5 weeks on the experimental regimen. The tissues were prepared for assay in the following manner: the animals were lightly anesthetized with ether, decapitated, and exsanguinated. The visceral tissues were perfused with cold, distilled water until pale in color. The tissues were removed rapidly, cooled, and immediately homogenized in a Waring blendor with ten times their weight of cold water. Alkaline phosphatase activities were determined by the method of Bessey, Lowry, and Brock (14) in which the breakdown of p-nitrophenyl phosphate to
MOLYBDESUM
TOXICITY
STUDIES
3
p-nitrophenol is measured, Each tissue was assayed at two concentrations and each concentration run in duplicate. Activities are expressed as micromoles of phosphate released in 30 min./mg. protein. The protein content of the homogenates was determined by the method of Lowry et al. (15). The enzyme data and the weight values were analyzed st)atistically and values are reported wit,h standard errors of the means. RESULTS
Tissue Alkaline
Phosphatase Activities During Molybdenum
Toxicity
The previous reports t,hat rat liver alkaline phosphatase activity is markedly increased by the feeding of excessive molybdenum salts was confirmed in the present study (Table I). The further observat,ion that dietary methionine partially mitigates the enzymatic anomaly during toxicity was also substantiated. The inclusion of sodium molybdate in t)he diet resulted in a mean alkaline phosphatase activity which was more than two times the mean activity of control animals. The inclusion of 2.0 mmoles of methionine along with the I .2 mmoles of molybdenum per 100 g. diet resulted in a 42% reduction of the phosphatase values from the toxic level. The inclusion of sodium sulfate in the diet at the same molar level as methionine was effective in reducing the phosphatase level by 33 % from the toxic Icwl. ;\ statistical analysis of the dat#a indicated that, the inrrease in act,ivity over the control value of 0.069 pmole phosphate released/SO min.,jmg. protein due to the sodium molybdate was highly significant (P < 0.01). T,ikrwisr, the correction of the TBBLE Rat Lioer Alkaline
Phosphatase
Supplement in basal diet
None ?u’a&IoO~ .2H& Xa~~IoO4.2H~O
methionine Sa&IoOa.2H&) NnrSO4 1fethioninr Na&O,
Activities
Quantity, mmoles/lOO g. diet
1.2 +
+
I Dwing
Xolybdenum
Toxicity
Liver alkaline phosphatase, pm&s phosphate released/.?0 min./mg. protein
0.069 f
0.005
w
0.17s
,014
(IO,
.OllC
(101
f
1.2
2.0
0.103 *
1.2 2.0 2.0 2.0
0.117 &
.014
(9?
0.065 f 0.070 f
.ooi ,010
(0) (S)
a Figures in parentheses give number of animals assayed. h Significantly greater than control group P < 0.01. c Significantly less than molybdenum-toxic group P < 0.01.
in basal
diet
1.2 1.2 1.0 1.2 1.0 1.2 0.2 mM twice 1.0 1.0 0.2 mM twice
mmoles/100
Quantity
a week
a week
8. dirt
of Sulfur-Containing
f f f f
.Ollb (9) .002 (10) .002 (10) .004 (10)
.005c (10)
0.082 f 0.161 0.056 0.054 0.060
.OlO” (10)
0.097 f
pmoles
.003 (10)s O.lOb (10)
0.715 1.83 1.21 1.44
f f * f
f
1.37
.12b .34 .lO .09
(5) (5) (5) (5)
.18 (5)
.22 (5)
*
1.21
min./mg.
activities
.12 (5) .13b (5)
releosea/30
Male
phosphatase
Toxicity
1.80 f 0.804 f
phos@ate
Alkaline
on M.olybdenum
0.059 f 0.144 f
Liver
Compounds
II
5 Figures in parentheses give number of animals assayed. b Significantly different from control group P < 0.01. c Significantly different from molybdenum toxic group P < 0.01.
None NazMoOa. 2HzO NazMoOc.2H20 + cystine Na*MoOa .2HzO + NakLOa. 5HzO NatMoOd. 2H~0 + injected NazSOa Cystine Nakh03.5H~O Injected Na2S04
Supplement
Influence
TAHLE
Female
1.05 2.78 3.30 3.24
2.11
1.93
f f f f
f
f
.18” .22 .46 .56
(4) (5) (5) (5)
.3Sc (5)
.27c (5)
3.73 xk .15 (5) 0.772 1. .07b (5)
protein
Kidney
MOLYBDENUM
TOXICITY
STUDIES
5
molybdenum effect by methionine and sodium sulfate was analyzed. The mean alkaline phosphatase values for t’hese groups differed significantly (P < 0.01) from the value obtained in the molybdenum-toxic group. When the basal diet was supplemented wit)h only methionine or sodium sulfate there was no measurable effect on liver alkaline phosphat#aseactivities. This is interpreted to mean that these substances do not have a direct effect on the enzyme but rather have some influence on the metabolism of molybdenum. In the second experiment reported in Table II, equal numbers of male and female rats were used and both liver and kidney tissues were assayed. There was no sex difference noted in liver assay values; therefore, the values were combined for the table. It was observed that in the case of liver bot,h cystine and sodium thiosulfate feeding t’ended to correct the molybdenum effect. Intraperitoneal injection of sodium sulfate, on the other hand, was ineffective. Cyst,ine or sodium thiosulfate added alone to the basal diet had no effect on the liver alkaline phosphatase act’ivities. A marked sex difference was noted when the kidneys were assayed (Table II); therefore, separat’e group means are presented. Kidneys from female rats showed alkaline phosphat’ase values which were more than t,wice those of male rats. Dietary molybdenum caused a marked decrease in kidney phosphatase activity, while bot,h cystine and sodium thiosulfate tended to correct the condition. Injected sodium sulfate again was ineffective in altering the effect of molybdenum. The decrease in kidney alkaline phosphatase activity in molybdenum toxicity was stat)&tically significant (P < 0.01) for both sexes although t’he percentage decrease was greater in the females. The action of cystine and t,hiosulfatr was significant (P < 0.01) in female rats but had a less significant influence in the males. Met’hionine, sodium sulfate, cystine, and sodium thiosulfate when fed to rats as supplement’s to Dhe basal diet had little or no effect on the level of liver and kidney alkaline phosphatase act,ivit’ies. Exceptions to this were some depression of female kidney activity when cystine was administered, and the male liver activity during thiosulfate administration. To assure that there was no direct effect of these materials on tho assay system, alkaline phosphatase activity was determined on normal and toxic, liver and kidney tissues in t,he presence of the various sulfur compounds. Results of these assays, presented in Table III, show little or no influence of these compounds on the assay system. Cystine could
6
ROBERT
VAN
REEN
AND
TABLE Lack
of Injluence
MARY
ANN
WILLIAMS
III
of Sulfur Compounds on Phosphatase Assay Alkaline phosphatase activity
Additions
Kidney Control
Liver Toxic
imoles )hosjhatc released/30 min./ml.
None 10-3 M lo-3 M 1O-3M 10-s M
methionine
1.036 1.022
0.938 0.858
N&04
0.965
0.935
NazSzOa.5H20 cystine
0.935 0.967
0.865 0.922
Toxic
Control
homogenate
1.504 1.474 1.464 1.420 1.390
0.519 0.508 0.510 0.499 0.512
be studied at only the low concentration because of its poor solubility. Methionine, sodium sulfate, and sodium thiosulfate were also tested at 1O-4 and 1O-5 M and were found to be without effect. It has previously been shown that under the conditions of the assay sodium molybdate up to 1OP M has no effect on the enzyme values. Weight Responses During Molybdenum
Toxicity
In the two experiments described above, the inclusion of sodium molybdate in the diet caused a pronounced retardation of body growth (Table IV). Supplementation of the toxic diet with methionine, sodium TABLE
IV
Weight Responses Supplement
None Molybdenum MO + methionine MO + NazSOc Methionine Nak304 MO + cystine MO + Na&Oa MO + intraperitoneal N&l01 Cystine Nakh03 Intraperitoneal NazSOd
Body weights after four weeks Experiment Experiment I, g.
153 f 9 (1O)a 89 f 6* (10) 136 f 5” (10) 131 f 4” (10) 166 f 17 (10) 158 f 13 (10)
II, g.
177 i 95 f
7 (10) 3 (10)
118 f 138 f 98 f 184 f 176 f 184 f
5” (10) 10” (9) 6b (10) 13 (10) 16 (10) 12 (10)
a Numbers in parentheses give the number of animals in each group. * Values significantly less than control group P < 0.01. c Values significantly greater than molybdenum-toxic group P < 0.01.
MOLYBDENUM
TOXICITY
STUDIES
7
sulfate, or sodium t’hiosulfate resulted in a statistically significant (P < 0.01) remission of the growth repression of molybdate. Cystine, while giving some improvement from the toxicity, did not demonstrate as pronounced an effect as methionine. As in the case of the enzymatic anomaly of molybdenum toxicity, the intraperitoneal injection of sodium sulfate was ineffective in improving weight responses. Kane of the sulfurcontaining compounds tested had any significant, effect on rat growt)h when added alone to the basal diet. DISCUSSIOS Molybdenum toxicity in rats has been investigated in a number of laboratories. Neilands et al. (10) fed rats a diet containing 400 p.p.m. molybdenum and found that the effects could largely be overcome by feeding an additional 20 p.pm. copper to the diet already containing 7’7.3 p.p.m. Comar et al. (3) reported that rats fed a diet containing 2 p.p.m. copper and 80 p.p.m. molybdenum grew poorer then rats receiving the same level of molybdenum but 35 p.p.m. copper. Both workers have reported that copper does not affect the distribution of molybdenum in rats. We have consist,ently observed an alterat’ion in liver and kidney alkaline phosphatase values in rats receiving high doses of molybdenum, whereas those substances which are usually decreased in copper deficiency, that is, hemoglobin concentrations, catalase activity, and rytorhrome oxidase activity remained essentially normal (8).4 In attempting to determine how added dietary methionine corrects the enzymatic alteration, it has been observed that cystine, sodium thiosulfate, and sodium sulfate on an equal molar sulfur basis will all to some degree reduce the liver and increase the kidney alkaline phosphat,ase activities of molybdenum-toxic rats. It thus appears probable that’ the substance responsible for t)his action is inorganic sulfate. The conversion of the sulfur of methionine and cystine to inorganic sulfate has recently been reviewed (16). It has also been reported that’ thiosulfat’e gives rise to sulfate (17). Dick (2), in experiments with sheep, has demonst’rated hhat at a given dietary level of molybdenum increasing the sulfate int’ake results in a lowering of the proportion of molybdenum recovered in the urine. This would suggest an influence of sulfate on the absorption of molybdenum from the gastrointest,inal tract. It was also shown that 6.3 mg./day of inorganic sulfate reduced t.he content of molybdenum in most tissues when the level is 0.3 or 20.8 mg. molybdenum/day. If similar effects of
8
ROBERT
VAN
REEN
AND MARY
ANN
WILLIAMS
sulfate occur in rats, the results of the above studies could be due to decreased absorption of molybdenum and reduced tissue molybdenum under the influence of sulfate or sulfate-yielding substances. It does seem clear that the sulfur-containing compounds do not directly influence the enzyme level of liver and kidney nor do they have a direct effect on the assay system. SUMMARY
1. The influence of methionine, cystine, sodium thiosulfate, and sodium sulfate on molybdenum toxicity in rats was studied. 2. When the sulfur-containing compounds were included in the toxic diet, there was a definite improvement of the enzymatic anomaly observed in molybdenum toxicity. 3. Growth was also improved by the incorporation of these compounds in the toxic diet. REFERENCES 1. FERGUSON, W. S., LEWIS, A. H., AND WATSON, S. T., Nature 141,553 (1938). 2. DICK, A. T., in “Inorganic Nitrogen Metabolism” (W. D. McElroy and B. Glass, eds.). The JohnsHopkins Press,Baltimore, Md., 1956. 3. COMAR, C. L., SINGER, L., AND DAVIS, G. K., J. Biol. Chem. 180,913 (1949). 4. JETER, M. A., AND DAVIS, G. K., J. Animal Sci. 9, 660 (1950). 61, 295 (1953). 5. ARRINGTON, L. R., AND DAVIS, G. K., J. Nutrition 6. LUTWAK, L., AND SACKS, J., J. Biol. Chem. 200,565 (1953). 7. DANIEL, L. J., AND GRAY, L. F., Proc. Sot. Exptl. Biol. Med. 83, 487 (1953). 8. VAN REEN, R., AND PEARSON, P. B., Federation Proc. 13, 314 (1954). 9. VAN REEN, R., Arch. Biochem. and Biophys. 63, 77 (1954). 10. NEILANDS, J. B., STRONG, F. M., AND ELVEHJEM, C. A., J. Biol. Chem. 173, 431 (1948). 11. GRAY, L. F., AND DANIEL, L. J., J. Nutrition 63,43 (1954). 12. DICK, A. T., Australian Vet. J. 28,30 (1952). 13. DICK, A. T., Australian Vet. J. 29, 18 (1953). 14. BESSEY, 0. A., LOWRY, 0. H., AND BROCK, M. J., J. Biol. Chem. 164,321 (1946). 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 16. SINGER, T. P., AND KEARNEY, E. B., in “Amino Acid Metabolism” (W. D. McElroy, and B. Glass, eds.). The Johns Hopkins Press, Baltimore, Md., 1955. 17. PIRIE, N. W., Biochem. J. 28, 1063 (1934).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
68,
l-8
(1956)
Studies on the Influence of Sulfur Compounds on Molybdenum Toxicity in Rats’ Robert Van Reen and Mary Ann Williams” ,3 From
the A1cCollunl-Pratt
Institute, I’he Johns Baltimore, Afaryland
Received
March
Hopkins
I:aiversit!y,
5, 1956
Ferguson, Lewis, and Watson (1) were the first to associate a scouring disease of cattle in certain areas of England with high molybdenum content of the pastures. Since then similar diseases in catt,le have been found in t,he United States and other parts of the world (2) and have been associated with copper insufficiency. The production of molybdenum t,oxicity in experimental animals has been reported also and copper salt shown to exert a protective effect (3-5). It has been suggested that molybdenum might cause the breakdown of labile phosphate esters in tissues (6,7) ; however, it has been observed that, the excretion of hippuric acid and acetylated p-aminobenxoic acid following the injection of benzoic acid and p-aminobenzoic acid, respectively, is approximately equal in control and molybdenum-toxic rats. This was interpreted to mean that adenosine triphosphate was still available for these reactions to occur in the toxic state.4 At, relatively high levels of dietary molybdenum, effects other than on copper metabolism have been reported in rats (8, 9).” In these studies liver alkaline phosphatase activity was found to increase, whereas kidney preparations decreased markedly in activity. These alterations were apparent in some animals who showed no external appearances of t’oxicity. 1 Contribution No. 140 of the McCollum-Pratt Institute, The Johns Hopkins University. This work supported in part by a contract No. AT(30-l)-1816 between the U. S. Atomic Energy Commission and The Johns Hopkins University. 2 Present address: Department of Home Economics, University of California, Berkeley 4, Calif. 3 With the technical assistance of Joyce Gile. 4 Williams, M. A., and Van Reen, R., in preparation for publication.
2
ROBERT VAN REEN AND MARY ANN WILLIAMS
Neilands, Strong, and Elvehjem (10) found that whole liver protected against molybdenum toxicity in rats when added to a purified type diet. Gray and Daniel (11) later reported that methionine had a similar effect in rats. The influence of methionine was extended to the enzymatic anomaly in rats and was shown to mitigate the effects of molybdenum. Dick (12) recently reported that sheep were more sensitive to molybdenum when fed a diet of chaffed lucerne hay than on a diet of chaffed oaten hay. The factor causing this difference was identified as inorganic sulfate (13). It seemed of interest, therefore, to re-examine the observations on rats and determine whether the influence of methionine could be explained on the basis of the conversion of its sulfur to inorganic sulfate. EXPERIMENTAL
PROCEDURE AND MATERIALS
The albino rats of the W&tar strain used in these studies were obtained from a commercial source at 4-5 weeks of age. The basal diet had the following percentage composition: casein, 20.0; glucose, 69.0; corn oil, 6.6; salt mixture U.S.P. XIV, 4.0; choline chloride, 0.1; 2500 international units (I.U.) vitamin A; 250 I.U. vitamin D; and 15 mg. a-tocopherol in corn oil, 0.3. Other vitamins were provided in the following quantities, mg./lOO g. diet: thiamine.HCl, 0.5; riboflavin, 0.8; niacin, 4.0; pyridoxine.HCl, 0.5; Ca pantothenate, 4.0; biotin, 0.04; folic acid, 0.2; menadione, 0.5; vitamin Brt , 0.003; inositol, 10.0; and p-aminobeneoic acid, 10.0. Sodium molybdate was added to the basal diet to provide the desired level of dietary molybdenum. The basal diet was also supplemented with 4 mg. copper/ 109 g. diet in the form of CuSOd.5Hz0 in addition to the 80 pg. copper in the salt mixture to provide sufficient copper to prevent anemia from occurring. The influence of methionine and inorganic sulfate on molybdenum toxicity was investigated in the first experiment. Molybdenum was fed at 1200 p.p.m. and methionine at 0.3 g./lOO g. diet. Sodium sulfate was mixed in the diet at 0.29 g./lOO g. These levels provided 1.2 mmoles molybdenum and 2.0 mmoles of sulfur per 100 g. diet. Male weanling rats were employed. The’ second experiment extended the studies to the influence of cystine, sodium thiosulfate, and intraperitoneally injected sodium sulfate. Both male and female rats were used with equal numbers of each in the groups. Ad Zibitum feeding was employed since previous experiments indicated the same alteration in alkaline phosphatase activity in ad Zibitum and pair-fed animals. Determinations were made of liver and kidney alkaline phosphatase activity after 4-5 weeks on the experimental regimen. The tissues were prepared for assay in the following manner: the animals were lightly anesthetized with ether, decapitated, and exsanguinated. The visceral tissues were perfused with cold, distilled water until pale in color. The tissues were removed rapidly, cooled, and immediately homogenized in a Waring blendor with ten times their weight of cold water. Alkaline phosphatase activities were determined by the method of Bessey, Lowry, and Brock (14) in which the breakdown of p-nitrophenyl phosphate to
MOLYBDESUM
TOXICITY
STUDIES
3
p-nitrophenol is measured, Each tissue was assayed at two concentrations and each concentration run in duplicate. Activities are expressed as micromoles of phosphate released in 30 min./mg. protein. The protein content of the homogenates was determined by the method of Lowry et al. (15). The enzyme data and the weight values were analyzed st)atistically and values are reported wit,h standard errors of the means. RESULTS
Tissue Alkaline
Phosphatase Activities During Molybdenum
Toxicity
The previous reports t,hat rat liver alkaline phosphatase activity is markedly increased by the feeding of excessive molybdenum salts was confirmed in the present study (Table I). The further observat,ion that dietary methionine partially mitigates the enzymatic anomaly during toxicity was also substantiated. The inclusion of sodium molybdate in t)he diet resulted in a mean alkaline phosphatase activity which was more than two times the mean activity of control animals. The inclusion of 2.0 mmoles of methionine along with the I .2 mmoles of molybdenum per 100 g. diet resulted in a 42% reduction of the phosphatase values from the toxic level. The inclusion of sodium sulfate in the diet at the same molar level as methionine was effective in reducing the phosphatase level by 33 % from the toxic Icwl. ;\ statistical analysis of the dat#a indicated that, the inrrease in act,ivity over the control value of 0.069 pmole phosphate released/SO min.,jmg. protein due to the sodium molybdate was highly significant (P < 0.01). T,ikrwisr, the correction of the TBBLE Rat Lioer Alkaline
Phosphatase
Supplement in basal diet
None ?u’a&IoO~ .2H& Xa~~IoO4.2H~O
methionine Sa&IoOa.2H&) NnrSO4 1fethioninr Na&O,
Activities
Quantity, mmoles/lOO g. diet
1.2 +
+
I Dwing
Xolybdenum
Toxicity
Liver alkaline phosphatase, pm&s phosphate released/.?0 min./mg. protein
0.069 f
0.005
w
0.17s
,014
(IO,
.OllC
(101
f
1.2
2.0
0.103 *
1.2 2.0 2.0 2.0
0.117 &
.014
(9?
0.065 f 0.070 f
.ooi ,010
(0) (S)
a Figures in parentheses give number of animals assayed. h Significantly greater than control group P < 0.01. c Significantly less than molybdenum-toxic group P < 0.01.
in basal
diet
1.2 1.2 1.0 1.2 1.0 1.2 0.2 mM twice 1.0 1.0 0.2 mM twice
mmoles/100
Quantity
a week
a week
8. dirt
of Sulfur-Containing
f f f f
.Ollb (9) .002 (10) .002 (10) .004 (10)
.005c (10)
0.082 f 0.161 0.056 0.054 0.060
.OlO” (10)
0.097 f
pmoles
.003 (10)s O.lOb (10)
0.715 1.83 1.21 1.44
f f * f
f
1.37
.12b .34 .lO .09
(5) (5) (5) (5)
.18 (5)
.22 (5)
*
1.21
min./mg.
activities
.12 (5) .13b (5)
releosea/30
Male
phosphatase
Toxicity
1.80 f 0.804 f
phos@ate
Alkaline
on M.olybdenum
0.059 f 0.144 f
Liver
Compounds
II
5 Figures in parentheses give number of animals assayed. b Significantly different from control group P < 0.01. c Significantly different from molybdenum toxic group P < 0.01.
None NazMoOa. 2HzO NazMoOc.2H20 + cystine Na*MoOa .2HzO + NakLOa. 5HzO NatMoOd. 2H~0 + injected NazSOa Cystine Nakh03.5H~O Injected Na2S04
Supplement
Influence
TAHLE
Female
1.05 2.78 3.30 3.24
2.11
1.93
f f f f
f
f
.18” .22 .46 .56
(4) (5) (5) (5)
.3Sc (5)
.27c (5)
3.73 xk .15 (5) 0.772 1. .07b (5)
protein
Kidney
MOLYBDENUM
TOXICITY
STUDIES
5
molybdenum effect by methionine and sodium sulfate was analyzed. The mean alkaline phosphatase values for t’hese groups differed significantly (P < 0.01) from the value obtained in the molybdenum-toxic group. When the basal diet was supplemented wit)h only methionine or sodium sulfate there was no measurable effect on liver alkaline phosphat#aseactivities. This is interpreted to mean that these substances do not have a direct effect on the enzyme but rather have some influence on the metabolism of molybdenum. In the second experiment reported in Table II, equal numbers of male and female rats were used and both liver and kidney tissues were assayed. There was no sex difference noted in liver assay values; therefore, the values were combined for the table. It was observed that in the case of liver bot,h cystine and sodium thiosulfate feeding t’ended to correct the molybdenum effect. Intraperitoneal injection of sodium sulfate, on the other hand, was ineffective. Cyst,ine or sodium thiosulfate added alone to the basal diet had no effect on the liver alkaline phosphatase act’ivities. A marked sex difference was noted when the kidneys were assayed (Table II); therefore, separat’e group means are presented. Kidneys from female rats showed alkaline phosphat’ase values which were more than t,wice those of male rats. Dietary molybdenum caused a marked decrease in kidney phosphatase activity, while bot,h cystine and sodium thiosulfate tended to correct the condition. Injected sodium sulfate again was ineffective in altering the effect of molybdenum. The decrease in kidney alkaline phosphatase activity in molybdenum toxicity was stat)&tically significant (P < 0.01) for both sexes although t’he percentage decrease was greater in the females. The action of cystine and t,hiosulfatr was significant (P < 0.01) in female rats but had a less significant influence in the males. Met’hionine, sodium sulfate, cystine, and sodium thiosulfate when fed to rats as supplement’s to Dhe basal diet had little or no effect on the level of liver and kidney alkaline phosphatase act,ivit’ies. Exceptions to this were some depression of female kidney activity when cystine was administered, and the male liver activity during thiosulfate administration. To assure that there was no direct effect of these materials on tho assay system, alkaline phosphatase activity was determined on normal and toxic, liver and kidney tissues in t,he presence of the various sulfur compounds. Results of these assays, presented in Table III, show little or no influence of these compounds on the assay system. Cystine could
6
ROBERT
VAN
REEN
AND
TABLE Lack
of Injluence
MARY
ANN
WILLIAMS
III
of Sulfur Compounds on Phosphatase Assay Alkaline phosphatase activity
Additions
Kidney Control
Liver Toxic
imoles )hosjhatc released/30 min./ml.
None 10-3 M lo-3 M 1O-3M 10-s M
methionine
1.036 1.022
0.938 0.858
N&04
0.965
0.935
NazSzOa.5H20 cystine
0.935 0.967
0.865 0.922
Toxic
Control
homogenate
1.504 1.474 1.464 1.420 1.390
0.519 0.508 0.510 0.499 0.512
be studied at only the low concentration because of its poor solubility. Methionine, sodium sulfate, and sodium thiosulfate were also tested at 1O-4 and 1O-5 M and were found to be without effect. It has previously been shown that under the conditions of the assay sodium molybdate up to 1OP M has no effect on the enzyme values. Weight Responses During Molybdenum
Toxicity
In the two experiments described above, the inclusion of sodium molybdate in the diet caused a pronounced retardation of body growth (Table IV). Supplementation of the toxic diet with methionine, sodium TABLE
IV
Weight Responses Supplement
None Molybdenum MO + methionine MO + NazSOc Methionine Nak304 MO + cystine MO + Na&Oa MO + intraperitoneal N&l01 Cystine Nakh03 Intraperitoneal NazSOd
Body weights after four weeks Experiment Experiment I, g.
153 f 9 (1O)a 89 f 6* (10) 136 f 5” (10) 131 f 4” (10) 166 f 17 (10) 158 f 13 (10)
II, g.
177 i 95 f
7 (10) 3 (10)
118 f 138 f 98 f 184 f 176 f 184 f
5” (10) 10” (9) 6b (10) 13 (10) 16 (10) 12 (10)
a Numbers in parentheses give the number of animals in each group. * Values significantly less than control group P < 0.01. c Values significantly greater than molybdenum-toxic group P < 0.01.
MOLYBDENUM
TOXICITY
STUDIES
7
sulfate, or sodium t’hiosulfate resulted in a statistically significant (P < 0.01) remission of the growth repression of molybdate. Cystine, while giving some improvement from the toxicity, did not demonstrate as pronounced an effect as methionine. As in the case of the enzymatic anomaly of molybdenum toxicity, the intraperitoneal injection of sodium sulfate was ineffective in improving weight responses. Kane of the sulfurcontaining compounds tested had any significant, effect on rat growt)h when added alone to the basal diet. DISCUSSIOS Molybdenum toxicity in rats has been investigated in a number of laboratories. Neilands et al. (10) fed rats a diet containing 400 p.p.m. molybdenum and found that the effects could largely be overcome by feeding an additional 20 p.pm. copper to the diet already containing 7’7.3 p.p.m. Comar et al. (3) reported that rats fed a diet containing 2 p.p.m. copper and 80 p.p.m. molybdenum grew poorer then rats receiving the same level of molybdenum but 35 p.p.m. copper. Both workers have reported that copper does not affect the distribution of molybdenum in rats. We have consist,ently observed an alterat’ion in liver and kidney alkaline phosphatase values in rats receiving high doses of molybdenum, whereas those substances which are usually decreased in copper deficiency, that is, hemoglobin concentrations, catalase activity, and rytorhrome oxidase activity remained essentially normal (8).4 In attempting to determine how added dietary methionine corrects the enzymatic alteration, it has been observed that cystine, sodium thiosulfate, and sodium sulfate on an equal molar sulfur basis will all to some degree reduce the liver and increase the kidney alkaline phosphat,ase activities of molybdenum-toxic rats. It thus appears probable that’ the substance responsible for t)his action is inorganic sulfate. The conversion of the sulfur of methionine and cystine to inorganic sulfate has recently been reviewed (16). It has also been reported that’ thiosulfat’e gives rise to sulfate (17). Dick (2), in experiments with sheep, has demonst’rated hhat at a given dietary level of molybdenum increasing the sulfate int’ake results in a lowering of the proportion of molybdenum recovered in the urine. This would suggest an influence of sulfate on the absorption of molybdenum from the gastrointest,inal tract. It was also shown that 6.3 mg./day of inorganic sulfate reduced t.he content of molybdenum in most tissues when the level is 0.3 or 20.8 mg. molybdenum/day. If similar effects of
8
ROBERT
VAN
REEN
AND MARY
ANN
WILLIAMS
sulfate occur in rats, the results of the above studies could be due to decreased absorption of molybdenum and reduced tissue molybdenum under the influence of sulfate or sulfate-yielding substances. It does seem clear that the sulfur-containing compounds do not directly influence the enzyme level of liver and kidney nor do they have a direct effect on the assay system. SUMMARY
1. The influence of methionine, cystine, sodium thiosulfate, and sodium sulfate on molybdenum toxicity in rats was studied. 2. When the sulfur-containing compounds were included in the toxic diet, there was a definite improvement of the enzymatic anomaly observed in molybdenum toxicity. 3. Growth was also improved by the incorporation of these compounds in the toxic diet. REFERENCES 1. FERGUSON, W. S., LEWIS, A. H., AND WATSON, S. T., Nature 141,553 (1938). 2. DICK, A. T., in “Inorganic Nitrogen Metabolism” (W. D. McElroy and B. Glass, eds.). The JohnsHopkins Press,Baltimore, Md., 1956. 3. COMAR, C. L., SINGER, L., AND DAVIS, G. K., J. Biol. Chem. 180,913 (1949). 4. JETER, M. A., AND DAVIS, G. K., J. Animal Sci. 9, 660 (1950). 61, 295 (1953). 5. ARRINGTON, L. R., AND DAVIS, G. K., J. Nutrition 6. LUTWAK, L., AND SACKS, J., J. Biol. Chem. 200,565 (1953). 7. DANIEL, L. J., AND GRAY, L. F., Proc. Sot. Exptl. Biol. Med. 83, 487 (1953). 8. VAN REEN, R., AND PEARSON, P. B., Federation Proc. 13, 314 (1954). 9. VAN REEN, R., Arch. Biochem. and Biophys. 63, 77 (1954). 10. NEILANDS, J. B., STRONG, F. M., AND ELVEHJEM, C. A., J. Biol. Chem. 173, 431 (1948). 11. GRAY, L. F., AND DANIEL, L. J., J. Nutrition 63,43 (1954). 12. DICK, A. T., Australian Vet. J. 28,30 (1952). 13. DICK, A. T., Australian Vet. J. 29, 18 (1953). 14. BESSEY, 0. A., LOWRY, 0. H., AND BROCK, M. J., J. Biol. Chem. 164,321 (1946). 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 16. SINGER, T. P., AND KEARNEY, E. B., in “Amino Acid Metabolism” (W. D. McElroy, and B. Glass, eds.). The Johns Hopkins Press, Baltimore, Md., 1955. 17. PIRIE, N. W., Biochem. J. 28, 1063 (1934).