9 1987 by The Humana Press Inc. All rights of any nature, whatsoever, reserved. 01634984/87/1300-0255502.00
Response of Hepatic Metailothionein to Iron Administration Lack of Correlation With Serum Corticosteronet ANN PETRO AND C. H. HILL* Department of Poultry Science, North Carolina State University, Raleigh, NC 27695- 7608 Received November 2, 1986; Accepted March 19, 1987
ABSTRACT Female broiler chicks receiving an ip injection of Fe (10 mg/kg bw) were found to have a greatly increased level of hepatic metallothionein. The increase in metallothionein was not correlated with changes in serum corticosterone. Attempts to vary serum corticosterone levels by feeding metyrapone, an 11-13 steroid hydroxylase inhibitor, were unsuccessful. Index Entries; Iron; corticosterone; metallothionein; metyrapone.
INTRODUCTION Metallothionein (MT), a low molecular weight (6,500 dalton) protein characterized by an unusually high cysteine content (26-33 mol%) and devoid of aromatic amino acids (1) was first isolated from equine renal cortex in 1957 (2). It is currently accepted that exposure to certain metals, most notably cadmium, zinc, and mercury, which have a high affinity for MT (3) also induces biosynthesis of the protein in liver and kidney. In addition, reports have shown that exposure to various physiological *Author to whom all correspondence and reprint requests should be addressed. tPaper No. 10743 of the Journal Series of the NC Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names implies neither endorsement of the products named n o r criticism of similar products not mentioned by the NCARS. Biological Trace Element Research
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stressors, such as heat and cold (5), food restriction (6), and bacterial infection (7), increase MT concentrations. As a result of such studies, adrenal cortical steroid involvement in MT induction has been implicated. Studies with synthetic and natural adrenal corticoids in intact (6) and adrenalectomized (8) animals have shown that the administration of these compounds results in the induction of MT. Recently, iron given intraperitoneally to chickens (4) was found to increase hepatic MT despite a low affinity for the protein (3). The suggestion (4) was made that an ip injection of iron could be sufficiently stressful to cause release of adrenal glucocorticoids that, in turn, could stimulate synthesis of MT. The purpose of the work presented here was to further examine the effect of injected iron on MT induction while simultaneously monitoring levels of endogenous corticosterone, serum zinc, liver zinc, and liver iron in chickens. An attempt was made to vary the endogenous corticosterone by administering metyrapone (2-methyl-l,2-di-3-pyridyl-L-propanone), an inhibitor of corticosterone production (9).
METHODS Three experiments using female broiler chicks (400-700 g) were carried out. The chicks were fed a soybean meal-corn diet supplemented with the required vitamins, manganese, calcium, phosphorus, and methionine. It was found by analysis to contain 12 ppm copper, 300 ppm iron, and 48 ppm zinc. Experiments 1 and 2 differed only in the amount of metyrapone added to the diet. Metyrapone was not included in the third experiment. In the first experiment, metyrapone (400 ppm) was incorporated into the feed for 6 d. In the second experiment, metyrapone was increased to 800 ppm for the same duration. Based on average feed consumption calculations, metyrapone was consumed at the rate of 33 mg/chick/day in the first experiment and 53 mg/chick/day in the second experiment. The remaining procedures for experiments 1 and 2 were identical. Methods for experiment 3 differed, and are described separately. On the sixth day of the experiment, blood was drawn from the wing vein of all chicks at 0, 2, 4, and 6 h after iron injection. The plasma collected was retained for corticosterone analysis by competitive protein binding radioassay (10). The assay, modified slightly, included a preliminary extraction with isooctane:ethyl acetate (100:1) to remove nonpolar steroids (11). Immediately following the blood sampling at time 0, 5 control and 5 metyrapone-treated chicks were injected with saline or iron. For the iron injection, 2 mg/mL of iron as ferrous sulfate was dissolved in .85% saline immediately before use and injected ip at the rate of 1 mg iron/100 g body wt. An equivalent volume of .85% saline was injected ip
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into control and metyrapone-treated chicks as a control. Twenty-four h after iron or saline injections the chicks were given intravenously 1 IxCi 109 Cd (sp. activity 1 #,Ci -- 0.5 ~g Cd). One hour later blood was drawn for zinc analysis, the chicks were killed, and the livers removed. One gram of liver from each chick was homogenized in 4 mL of 0.1 M Tris buffer, pH 8.0. The homogenate was centrifuged at 10,000 x g for 10 min, and the supernatant fluid subjected to gel column chromatography using G-75 Sephadex. The column size was 35 by 2.5 cm, the eluting buffer was 0.1 M Tris, pH 8.0, and the flow rate was 3 mL/min. Six mL fractions were collected and monitored for protein by UV spectroscopy at 280 nm and 260 nm, and for radioactivity by scintillation spectroscopy. Cadmium was eluted from the column in two peaks. One peak was within the void volume representing proteins greater than 80,000 daltons and the other in a Ve/Vo of ca 2 representing proteins of about 6,000-7,000 daltons. The latter fraction would contain metallothionein. The proportion of total radioactivity eluted from the column that fell within the second peak was considered a relative measure of MT. Recovery of radioactive cadmium was 85-95% in these studies. Serum zinc was determined by flame atomic absorption spectroscopy after 1:4 dilution. In the third experiment, 20 chicks were injected with saline or iron as described above. Twenty-four h after iron or saline injections, blood was drawn for zinc analysis, the chicks were killed, and the livers removed. Livers were analyzed for zinc, iron, and MT. To determine zinc and iron content, one g of liver was digested with two treatments of 10 mL concentrated nitric acid and one treatment of nitric/perchloric acid (3:1 by volume). Liver zinc and liver iron concentration as well as serum zinc were determined by flame atomic absorption spectroscopy. Samples were prepared for gel column chromatography as follows. Two grams of liver from each chick were homogenized in 8 mL of 0.1 M Tris buffer, pH 8.0. The homogenate was centrifuged at 100,000 x g for 60 min. Prior to application on the column, a 4 mL aliquot of the supernatant was incubated for l0 min at room temperature with 1 mL of a solution containing 2 p,g Cd/mL and radioactivity of .5 txCi/mL. The remaining gel column chromatography procedure was identical to that described above. The hepatic MT content was quantified following the cadmiumhemoglobin assay of Eaton and Toal (12) with minor modifications. A single addition of hemoglobin or buffer and 1 heat treatment of 4 min were used. After heating, the content of the tubes was diluted 1:2 and filtered through spun glass prior to removing a 200 p~L aliquot for radioactivity determination. The data in all three experiments were compared by one-way analysis of variance. Differences in means were determined using an F-test.
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RESULTS The level of w9Cd eluted from the Sephadex columns in a Ve/Vo of about 2, characteristic of MT, was dramatically increased by the injection of iron in all three experiments (Tables 1, 2, and 3). In addition, the Cdhemoglobin assay for MT also indicated a large increase in MT as a result of iron administration (Table 3). The increase in hepatic MT was not reflected by a change in the serum zinc concentration in the first two experiments (Tables 1 and 2), but was accompanied by a significant decrease in serum zinc in the third experiment (Table 3). Liver zinc, determined in the third experiment, tended to increase in concert with the decreasing serum zinc values and increasing hepatic MT concentrations (Table 3). Hepatic iron levels, also determined in experiment 3, rose significantly in iron treated birds (Table 3). These increases, however, did not correlate quantitatively with increases in MT. Plasma corticosterone levels were followed for 6 h (Figs. 1 and 2) starting immediately prior to the iron or saline injection. Corticosterone was present in the plasma of all chicks at all time points regardless of metyrapone status. There were no significant differences statistically among the treatments at any time point in either experiment. However, in both experiments an increase in corticosterone occurred between 0 and 2 h. In some cases these increases were significant--metyrapone group in the second experiment and saline group in both experiments. This increase in corticosterone was attributed to a handling effect, most likely the ip injection rather than blood drawing. In general, the corticosterone levels declined between 2 and 4 h, and this trend continued to 6 h despite repeated blood drawing procedures. Considering the serum zinc and liver zinc levels in experiment 3, there was a trend of lower serum levels and increased liver levels with a concomitant increase in MT in chicks given iron. Table 1 Effect of Iron and Metyrapone on Hepatic Metallothionein and Serum Zinc Concentrations 0 Iron
%l~ Eluted at Ve/Vo ~ 2 52.1 _+ 22.7'~ 85.5 -+ 8.4" 38.2 + 13.3" 79.8 _+ 12.4" Serum zinc p,g/dL 119 _+ 52 133 + 37 86 _+ 13 78 +- 16
0 Metyrapone + Metyraponeb 0 Metyrapone + Metyrapone ~ nag Fe/kg b w b400 p p m in diet 'Mean _+ S.E.M. qVleans followed
+ Iron"
as FeSO4.7H20 injected ip. for 6 d. of 5 observations/treatment. by different letters are significantly different at p = .05.
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Table 2
Effect of Iron and Metyrapone on Hepatic Metallothionein and Serum Zinc Concentrations (Exp. 2) 0 Iron
%'~ Eluted at Ve/Vo = 2 40.5 _+ 9.5" 80.2 + 11.3" 39.3 _+ 13.1' 70.3 _+ 15.9~ Serum zinc ~.g/dL 103 +_ 9 92 _+ 18 110 + 12 129 _+ 32
0 Metyrapone + Metyrapone ~' 0 Metyrapone + Metyrapone "10 mg Fe/kg bw "800 ppm in diet '"Means followed 'Mean + S.E.M.
+ Iron"
as FeSO4.7H20 injected ip. for 6 d. by different letters are significantly different at p = .05. of 5 observations/treatment.
Table 3 Effect of Iron on Hepatic Metallothionein, Iron and Zinc and Serum Zinc Concentrations (Exp. 3) 0 Iron Metallothionein ~g/g liver~' "~Cd Eluted at Ve/Vo =- 2 ~tg Zinc/g liver ~g Zinc/dL serum ~,g Iron/g liver
6.0 9.08 16.4 118 36
+_ 1.6' • 6.9' _• 6.6 +_ 9' _+ 5'
+ Iron" 89.7 61.68 24.6 82 56
_+ 50.3 't + 20.46" _+ 15.7 +_ 9" _+ 7"
"10 mg Fe/kg bw as FeSO4-7H20 injected ip. "Determined by the Cd-hemoglobin assay of Eaton and Toal (12). " M e a n s followed by different letters are significantly different at p = .05.
Liver iron concentration was increased significantly following iron injections.
DISCUSSION The p r e s e n t s t u d y d e m o n s t r a t e d that hepatic MT is increased by ip injections of iron. The combination of the experiments dispelled s o m e of the uncertainties i n h e r e n t in using either the gel p e r m e a t i o n or the Cdh e m o g l o b i n m e t h o d alone to d e t e r m i n e increases in MT. The m e t h o d e m p l o y e d in the first two e x p e r i m e n t s indicated an increase in MT. Peak 1 represents high molecular w e i g h t proteins (>80,000 daltons) that bind l~ If these proteins also b o u n d iron but were not saturated prior to iron injection, the shift in % c p m to the second peak m i g h t have reflected d i s p l a c e m e n t of Cd from Peak 1 to Peak 2 by iron. The i m p r e s s i o n of an increase in MT w o u l d result. Sample t r e a t m e n t in the competitive binding assay in the third e x p e r i m e n t precipitates high molecular weight, Biological Trace Element Research
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Petro and Hill EFFECT OF IRON AND METYRAPONE ON PLASMA CORTICOSTERONE C9 0 N
C E N
T R
A T I
O N
/\ \
/ J ~
O .....
"
2 Saline Metyrapone
\
/
h ....... _ _
J
HOUR
6
Iron 10 mg/kg b.w. Iron IO mg/kg b.w. and Metyrapone 400 ppm
*Concentration relative to hour 0 determined by normalizing values for each bird using hour 0 as the base value. Points on the graphs are the mean of ]-5 observations.
TRT Saline Metyrapone Iron Iron + Metyrapone
Actual corticosterone concentrations (ng/ml) as determined by competitive protein binding radloassay (lO,ll). Each value represents the mean • S.E.M. of 3-5 observations. Hour 0 2 4 6
2.5• 5.4• 5.6• 2.5•
2.5• 7.4• 8.7• 3.1tl.2
5.1• 5.1• 6.2• 3.1•
1.6• 8.4• 4.1• 2.3•
FIGURE I
heat labile proteins leaving low molecular weight, heat stable, soluble ~09roteinsthat bind cadmium. The possibility of mistaking displacement of Cd from one peak to the other for an increase in MT was thus eliminated. However, any Cd-binding proteins remaining after heat treatment would appear as MT in the Cd-hemoglobin assay. Proteins with a molecular weight not characteristic of MT would appear in the elution profile as an additional peak. The absence of a first peak in elution profiles following heat treatment has been observed (unpublished) indicating high Biological Trace Element Research
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EFFECT OF IRON AND METYRAPONE ON PLASMA CORTICOSTERONE C~ 0 N C E N T R A T I 0 N
.
\ //,. \ ////.. " ,, \
0
2
........
4
Saline Metyrapone
.....
6
HOUR
I r o n 10 mg/kg b 9 Iron lO mg/kg b.w. and Metyrapone B00 ppm
*Concentration r e l a t i v e to hour 0 determined by normalizing values f o r each blrd using hour 0 as the base value. Points on the graphs are the mean o f 3-5 observations. Actual corticosterone concentrations (ng/ml) as determined by competitive protein binding radloassay ( 1 0 , 1 ] ) . Each value
represents the mean • S.E.M. of 3-5 observations. Hour 2
4
7.1•
27,2•
17.6•
Metyrapone 5.0• Iron 8.4• Iron + 4.2• Metyrapone
17.9• 12.7• 13.4•
10.1• 4.8• 6.8•
TRT Saline
0
6 7 9177149
II.9•149 7.l•149 4.6•
FIGURE 2
molecular weight proteins were removed and did not contribute erroneously to calculation of MT concentration in the Cd-hemoglobin assay. Cadmium-binding proteins with a molecular weight lower than MT would appear as a third peak in the elution profile9 This occurred occasionally and the percentage contribution of this peak attributed to MT concentration was deducted. Both procedures were used to show 15-fold increase in MT concentration subsequent to a single ip administration of iron in experiment 3. Biological Trace Element Research
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Petro and Hill
The increase in MT by iron administration was not accompanied by a greater increase in plasma corticosterone than was observed in control animals. This finding is in contrast to the finding of McCormick (4). In the latter case, two injections of iron were given, whereas in the present study one injection was used. This might have accounted for the differences in results between the two investigations. The attempt to lower corticosterone levels by the use of metyrapone was unsuccessful in these experiments. There are at least two explanations for this. In the first place, the assay may have been misleading. It is k n o w n that 11 deoxycortisol, a precursor of corticosterone, binds to the corticosteroid binding globulin (13,14). Generally, the precursor is present in very low quantities. Blocking the enzymatic pathway may have allowed sufficient quantities to accumulate, resulting in an apparent level of corticosterone higher than the true level. Second, although the level of 400 and 800 p p m in the feed was sufficient to lower plasma corticosterone in White Leghorns, broiler chickens may be much less susceptible to metyrapone. In any case, metyrapone treatment had no effect on hepatic MT concentration. The mechanism by which injected iron results in higher levels of hepatic MT observed here and by others is unknown. Unlike other metals (Cd, Zn, Hg) that induce biosynthesis of MT, it has been shown (3) that hepatic MT has a low affinity for iron. The results of the present experiments indicate that the mechanism is not necessarily mediated by corticosterone.
REFERENCES 1. J. H. R. Kagi, S. Himmelhoch, P. Whanger, J. Bethune, and B. Vallee, J. Biol. Chem. 249, 3537 (1974). 2. M. Margoshes, and B. Vallee, J. Amer. Chem. Soc. 79, 4813 (1957). 3. M. P. Waalkes, M. J. Harvey, and C. D. Klaassen, Toxicol. Lett. 20, 33 (1984). 4. C. McCormack, Proc. Soc. Exp. Biol. and Med. 176, 392 (1984). 5. S. H. Oh, J. T. Deagen, P. D. Whanger, and P. H. Weswig, Am. J. Physiol. 234, E282 (1978). 6. C. D. Klaassen, Toxicol. 20, 275 (1981). 7. P. Z. Sobocinski, W. J. Canterbury, C. A. Mapes, and R. E. Dinterman, Am. I. Physiol. 234, E399 (1978). 8. K. R. Etzel, S. G. Shapiro, and R. J. Cousins, Biochem. and Biophys. Res. Commun. 89{4), 1120 (1979). 9. J. A. Thomas, and M. G. Mawhinney, Synopsis of Endocrine Pharmacology, Univ. Park Press, Baltimore, MD, 1973. 10. B. E. P. Murphy, J. Clin. Endocrin. 27, 973 (1967). 11. H. S. Siegel, N. R. Gould, and J. W. Latimer, Proc. Soc. Exp. Biol. and Med. 178, 523 (1985). 12. D. L. Eaton, and B. F. Toal, Toxicol. Appl. Pharmacol. 66, 134 (1982). 13. A. B. Myles, and J. R. Daley, Corticosterone and ACTH Treatment. Principles and Problems, Edward Arnold, London, 1974.
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14. L. E. Edqvist, and E. D. B. Johansson, "Radioimmunoassay and Competitive Binding Protein for Measurement of Certain Steroid Hormones in Farm Animals," in Isotope Studies on the Physiology of Domestic Animals (Athens, Greece International Atomic Energy Agency, Vienna, 1972), pp. 245-258. 15. D. L. Thompson, K. D. Elgert, W. B. Gross, and P. B. Siegel, Am. J. Vet. Res. 41(1), 91 (1980). 16. C. McCormack, Fed. Proc. 44, 1853 (1985).
Biological Trace Element Research
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Response of Hepatic Metailothionein to Iron Administration Lack of Correlation With Serum Corticosteronet ANN PETRO AND C. H. HILL* Department of Poultry Science, North Carolina State University, Raleigh, NC 27695- 7608 Received November 2, 1986; Accepted March 19, 1987
ABSTRACT Female broiler chicks receiving an ip injection of Fe (10 mg/kg bw) were found to have a greatly increased level of hepatic metallothionein. The increase in metallothionein was not correlated with changes in serum corticosterone. Attempts to vary serum corticosterone levels by feeding metyrapone, an 11-13 steroid hydroxylase inhibitor, were unsuccessful. Index Entries; Iron; corticosterone; metallothionein; metyrapone.
INTRODUCTION Metallothionein (MT), a low molecular weight (6,500 dalton) protein characterized by an unusually high cysteine content (26-33 mol%) and devoid of aromatic amino acids (1) was first isolated from equine renal cortex in 1957 (2). It is currently accepted that exposure to certain metals, most notably cadmium, zinc, and mercury, which have a high affinity for MT (3) also induces biosynthesis of the protein in liver and kidney. In addition, reports have shown that exposure to various physiological *Author to whom all correspondence and reprint requests should be addressed. tPaper No. 10743 of the Journal Series of the NC Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names implies neither endorsement of the products named n o r criticism of similar products not mentioned by the NCARS. Biological Trace Element Research
255
VoL 14, 1987
256
Petro and Hill
stressors, such as heat and cold (5), food restriction (6), and bacterial infection (7), increase MT concentrations. As a result of such studies, adrenal cortical steroid involvement in MT induction has been implicated. Studies with synthetic and natural adrenal corticoids in intact (6) and adrenalectomized (8) animals have shown that the administration of these compounds results in the induction of MT. Recently, iron given intraperitoneally to chickens (4) was found to increase hepatic MT despite a low affinity for the protein (3). The suggestion (4) was made that an ip injection of iron could be sufficiently stressful to cause release of adrenal glucocorticoids that, in turn, could stimulate synthesis of MT. The purpose of the work presented here was to further examine the effect of injected iron on MT induction while simultaneously monitoring levels of endogenous corticosterone, serum zinc, liver zinc, and liver iron in chickens. An attempt was made to vary the endogenous corticosterone by administering metyrapone (2-methyl-l,2-di-3-pyridyl-L-propanone), an inhibitor of corticosterone production (9).
METHODS Three experiments using female broiler chicks (400-700 g) were carried out. The chicks were fed a soybean meal-corn diet supplemented with the required vitamins, manganese, calcium, phosphorus, and methionine. It was found by analysis to contain 12 ppm copper, 300 ppm iron, and 48 ppm zinc. Experiments 1 and 2 differed only in the amount of metyrapone added to the diet. Metyrapone was not included in the third experiment. In the first experiment, metyrapone (400 ppm) was incorporated into the feed for 6 d. In the second experiment, metyrapone was increased to 800 ppm for the same duration. Based on average feed consumption calculations, metyrapone was consumed at the rate of 33 mg/chick/day in the first experiment and 53 mg/chick/day in the second experiment. The remaining procedures for experiments 1 and 2 were identical. Methods for experiment 3 differed, and are described separately. On the sixth day of the experiment, blood was drawn from the wing vein of all chicks at 0, 2, 4, and 6 h after iron injection. The plasma collected was retained for corticosterone analysis by competitive protein binding radioassay (10). The assay, modified slightly, included a preliminary extraction with isooctane:ethyl acetate (100:1) to remove nonpolar steroids (11). Immediately following the blood sampling at time 0, 5 control and 5 metyrapone-treated chicks were injected with saline or iron. For the iron injection, 2 mg/mL of iron as ferrous sulfate was dissolved in .85% saline immediately before use and injected ip at the rate of 1 mg iron/100 g body wt. An equivalent volume of .85% saline was injected ip
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2.57
into control and metyrapone-treated chicks as a control. Twenty-four h after iron or saline injections the chicks were given intravenously 1 IxCi 109 Cd (sp. activity 1 #,Ci -- 0.5 ~g Cd). One hour later blood was drawn for zinc analysis, the chicks were killed, and the livers removed. One gram of liver from each chick was homogenized in 4 mL of 0.1 M Tris buffer, pH 8.0. The homogenate was centrifuged at 10,000 x g for 10 min, and the supernatant fluid subjected to gel column chromatography using G-75 Sephadex. The column size was 35 by 2.5 cm, the eluting buffer was 0.1 M Tris, pH 8.0, and the flow rate was 3 mL/min. Six mL fractions were collected and monitored for protein by UV spectroscopy at 280 nm and 260 nm, and for radioactivity by scintillation spectroscopy. Cadmium was eluted from the column in two peaks. One peak was within the void volume representing proteins greater than 80,000 daltons and the other in a Ve/Vo of ca 2 representing proteins of about 6,000-7,000 daltons. The latter fraction would contain metallothionein. The proportion of total radioactivity eluted from the column that fell within the second peak was considered a relative measure of MT. Recovery of radioactive cadmium was 85-95% in these studies. Serum zinc was determined by flame atomic absorption spectroscopy after 1:4 dilution. In the third experiment, 20 chicks were injected with saline or iron as described above. Twenty-four h after iron or saline injections, blood was drawn for zinc analysis, the chicks were killed, and the livers removed. Livers were analyzed for zinc, iron, and MT. To determine zinc and iron content, one g of liver was digested with two treatments of 10 mL concentrated nitric acid and one treatment of nitric/perchloric acid (3:1 by volume). Liver zinc and liver iron concentration as well as serum zinc were determined by flame atomic absorption spectroscopy. Samples were prepared for gel column chromatography as follows. Two grams of liver from each chick were homogenized in 8 mL of 0.1 M Tris buffer, pH 8.0. The homogenate was centrifuged at 100,000 x g for 60 min. Prior to application on the column, a 4 mL aliquot of the supernatant was incubated for l0 min at room temperature with 1 mL of a solution containing 2 p,g Cd/mL and radioactivity of .5 txCi/mL. The remaining gel column chromatography procedure was identical to that described above. The hepatic MT content was quantified following the cadmiumhemoglobin assay of Eaton and Toal (12) with minor modifications. A single addition of hemoglobin or buffer and 1 heat treatment of 4 min were used. After heating, the content of the tubes was diluted 1:2 and filtered through spun glass prior to removing a 200 p~L aliquot for radioactivity determination. The data in all three experiments were compared by one-way analysis of variance. Differences in means were determined using an F-test.
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RESULTS The level of w9Cd eluted from the Sephadex columns in a Ve/Vo of about 2, characteristic of MT, was dramatically increased by the injection of iron in all three experiments (Tables 1, 2, and 3). In addition, the Cdhemoglobin assay for MT also indicated a large increase in MT as a result of iron administration (Table 3). The increase in hepatic MT was not reflected by a change in the serum zinc concentration in the first two experiments (Tables 1 and 2), but was accompanied by a significant decrease in serum zinc in the third experiment (Table 3). Liver zinc, determined in the third experiment, tended to increase in concert with the decreasing serum zinc values and increasing hepatic MT concentrations (Table 3). Hepatic iron levels, also determined in experiment 3, rose significantly in iron treated birds (Table 3). These increases, however, did not correlate quantitatively with increases in MT. Plasma corticosterone levels were followed for 6 h (Figs. 1 and 2) starting immediately prior to the iron or saline injection. Corticosterone was present in the plasma of all chicks at all time points regardless of metyrapone status. There were no significant differences statistically among the treatments at any time point in either experiment. However, in both experiments an increase in corticosterone occurred between 0 and 2 h. In some cases these increases were significant--metyrapone group in the second experiment and saline group in both experiments. This increase in corticosterone was attributed to a handling effect, most likely the ip injection rather than blood drawing. In general, the corticosterone levels declined between 2 and 4 h, and this trend continued to 6 h despite repeated blood drawing procedures. Considering the serum zinc and liver zinc levels in experiment 3, there was a trend of lower serum levels and increased liver levels with a concomitant increase in MT in chicks given iron. Table 1 Effect of Iron and Metyrapone on Hepatic Metallothionein and Serum Zinc Concentrations 0 Iron
%l~ Eluted at Ve/Vo ~ 2 52.1 _+ 22.7'~ 85.5 -+ 8.4" 38.2 + 13.3" 79.8 _+ 12.4" Serum zinc p,g/dL 119 _+ 52 133 + 37 86 _+ 13 78 +- 16
0 Metyrapone + Metyraponeb 0 Metyrapone + Metyrapone ~ nag Fe/kg b w b400 p p m in diet 'Mean _+ S.E.M. qVleans followed
+ Iron"
as FeSO4.7H20 injected ip. for 6 d. of 5 observations/treatment. by different letters are significantly different at p = .05.
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Table 2
Effect of Iron and Metyrapone on Hepatic Metallothionein and Serum Zinc Concentrations (Exp. 2) 0 Iron
%'~ Eluted at Ve/Vo = 2 40.5 _+ 9.5" 80.2 + 11.3" 39.3 _+ 13.1' 70.3 _+ 15.9~ Serum zinc ~.g/dL 103 +_ 9 92 _+ 18 110 + 12 129 _+ 32
0 Metyrapone + Metyrapone ~' 0 Metyrapone + Metyrapone "10 mg Fe/kg bw "800 ppm in diet '"Means followed 'Mean + S.E.M.
+ Iron"
as FeSO4.7H20 injected ip. for 6 d. by different letters are significantly different at p = .05. of 5 observations/treatment.
Table 3 Effect of Iron on Hepatic Metallothionein, Iron and Zinc and Serum Zinc Concentrations (Exp. 3) 0 Iron Metallothionein ~g/g liver~' "~Cd Eluted at Ve/Vo =- 2 ~tg Zinc/g liver ~g Zinc/dL serum ~,g Iron/g liver
6.0 9.08 16.4 118 36
+_ 1.6' • 6.9' _• 6.6 +_ 9' _+ 5'
+ Iron" 89.7 61.68 24.6 82 56
_+ 50.3 't + 20.46" _+ 15.7 +_ 9" _+ 7"
"10 mg Fe/kg bw as FeSO4-7H20 injected ip. "Determined by the Cd-hemoglobin assay of Eaton and Toal (12). " M e a n s followed by different letters are significantly different at p = .05.
Liver iron concentration was increased significantly following iron injections.
DISCUSSION The p r e s e n t s t u d y d e m o n s t r a t e d that hepatic MT is increased by ip injections of iron. The combination of the experiments dispelled s o m e of the uncertainties i n h e r e n t in using either the gel p e r m e a t i o n or the Cdh e m o g l o b i n m e t h o d alone to d e t e r m i n e increases in MT. The m e t h o d e m p l o y e d in the first two e x p e r i m e n t s indicated an increase in MT. Peak 1 represents high molecular w e i g h t proteins (>80,000 daltons) that bind l~ If these proteins also b o u n d iron but were not saturated prior to iron injection, the shift in % c p m to the second peak m i g h t have reflected d i s p l a c e m e n t of Cd from Peak 1 to Peak 2 by iron. The i m p r e s s i o n of an increase in MT w o u l d result. Sample t r e a t m e n t in the competitive binding assay in the third e x p e r i m e n t precipitates high molecular weight, Biological Trace Element Research
Vol. 14, 1987
260
Petro and Hill EFFECT OF IRON AND METYRAPONE ON PLASMA CORTICOSTERONE C9 0 N
C E N
T R
A T I
O N
/\ \
/ J ~
O .....
"
2 Saline Metyrapone
\
/
h ....... _ _
J
HOUR
6
Iron 10 mg/kg b.w. Iron IO mg/kg b.w. and Metyrapone 400 ppm
*Concentration relative to hour 0 determined by normalizing values for each bird using hour 0 as the base value. Points on the graphs are the mean of ]-5 observations.
TRT Saline Metyrapone Iron Iron + Metyrapone
Actual corticosterone concentrations (ng/ml) as determined by competitive protein binding radloassay (lO,ll). Each value represents the mean • S.E.M. of 3-5 observations. Hour 0 2 4 6
2.5• 5.4• 5.6• 2.5•
2.5• 7.4• 8.7• 3.1tl.2
5.1• 5.1• 6.2• 3.1•
1.6• 8.4• 4.1• 2.3•
FIGURE I
heat labile proteins leaving low molecular weight, heat stable, soluble ~09roteinsthat bind cadmium. The possibility of mistaking displacement of Cd from one peak to the other for an increase in MT was thus eliminated. However, any Cd-binding proteins remaining after heat treatment would appear as MT in the Cd-hemoglobin assay. Proteins with a molecular weight not characteristic of MT would appear in the elution profile as an additional peak. The absence of a first peak in elution profiles following heat treatment has been observed (unpublished) indicating high Biological Trace Element Research
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iron, Hepatic/Hetallothionen, and Corticosterone
261
EFFECT OF IRON AND METYRAPONE ON PLASMA CORTICOSTERONE C~ 0 N C E N T R A T I 0 N
.
\ //,. \ ////.. " ,, \
0
2
........
4
Saline Metyrapone
.....
6
HOUR
I r o n 10 mg/kg b 9 Iron lO mg/kg b.w. and Metyrapone B00 ppm
*Concentration r e l a t i v e to hour 0 determined by normalizing values f o r each blrd using hour 0 as the base value. Points on the graphs are the mean o f 3-5 observations. Actual corticosterone concentrations (ng/ml) as determined by competitive protein binding radloassay ( 1 0 , 1 ] ) . Each value
represents the mean • S.E.M. of 3-5 observations. Hour 2
4
7.1•
27,2•
17.6•
Metyrapone 5.0• Iron 8.4• Iron + 4.2• Metyrapone
17.9• 12.7• 13.4•
10.1• 4.8• 6.8•
TRT Saline
0
6 7 9177149
II.9•149 7.l•149 4.6•
FIGURE 2
molecular weight proteins were removed and did not contribute erroneously to calculation of MT concentration in the Cd-hemoglobin assay. Cadmium-binding proteins with a molecular weight lower than MT would appear as a third peak in the elution profile9 This occurred occasionally and the percentage contribution of this peak attributed to MT concentration was deducted. Both procedures were used to show 15-fold increase in MT concentration subsequent to a single ip administration of iron in experiment 3. Biological Trace Element Research
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262
Petro and Hill
The increase in MT by iron administration was not accompanied by a greater increase in plasma corticosterone than was observed in control animals. This finding is in contrast to the finding of McCormick (4). In the latter case, two injections of iron were given, whereas in the present study one injection was used. This might have accounted for the differences in results between the two investigations. The attempt to lower corticosterone levels by the use of metyrapone was unsuccessful in these experiments. There are at least two explanations for this. In the first place, the assay may have been misleading. It is k n o w n that 11 deoxycortisol, a precursor of corticosterone, binds to the corticosteroid binding globulin (13,14). Generally, the precursor is present in very low quantities. Blocking the enzymatic pathway may have allowed sufficient quantities to accumulate, resulting in an apparent level of corticosterone higher than the true level. Second, although the level of 400 and 800 p p m in the feed was sufficient to lower plasma corticosterone in White Leghorns, broiler chickens may be much less susceptible to metyrapone. In any case, metyrapone treatment had no effect on hepatic MT concentration. The mechanism by which injected iron results in higher levels of hepatic MT observed here and by others is unknown. Unlike other metals (Cd, Zn, Hg) that induce biosynthesis of MT, it has been shown (3) that hepatic MT has a low affinity for iron. The results of the present experiments indicate that the mechanism is not necessarily mediated by corticosterone.
REFERENCES 1. J. H. R. Kagi, S. Himmelhoch, P. Whanger, J. Bethune, and B. Vallee, J. Biol. Chem. 249, 3537 (1974). 2. M. Margoshes, and B. Vallee, J. Amer. Chem. Soc. 79, 4813 (1957). 3. M. P. Waalkes, M. J. Harvey, and C. D. Klaassen, Toxicol. Lett. 20, 33 (1984). 4. C. McCormack, Proc. Soc. Exp. Biol. and Med. 176, 392 (1984). 5. S. H. Oh, J. T. Deagen, P. D. Whanger, and P. H. Weswig, Am. J. Physiol. 234, E282 (1978). 6. C. D. Klaassen, Toxicol. 20, 275 (1981). 7. P. Z. Sobocinski, W. J. Canterbury, C. A. Mapes, and R. E. Dinterman, Am. I. Physiol. 234, E399 (1978). 8. K. R. Etzel, S. G. Shapiro, and R. J. Cousins, Biochem. and Biophys. Res. Commun. 89{4), 1120 (1979). 9. J. A. Thomas, and M. G. Mawhinney, Synopsis of Endocrine Pharmacology, Univ. Park Press, Baltimore, MD, 1973. 10. B. E. P. Murphy, J. Clin. Endocrin. 27, 973 (1967). 11. H. S. Siegel, N. R. Gould, and J. W. Latimer, Proc. Soc. Exp. Biol. and Med. 178, 523 (1985). 12. D. L. Eaton, and B. F. Toal, Toxicol. Appl. Pharmacol. 66, 134 (1982). 13. A. B. Myles, and J. R. Daley, Corticosterone and ACTH Treatment. Principles and Problems, Edward Arnold, London, 1974.
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14. L. E. Edqvist, and E. D. B. Johansson, "Radioimmunoassay and Competitive Binding Protein for Measurement of Certain Steroid Hormones in Farm Animals," in Isotope Studies on the Physiology of Domestic Animals (Athens, Greece International Atomic Energy Agency, Vienna, 1972), pp. 245-258. 15. D. L. Thompson, K. D. Elgert, W. B. Gross, and P. B. Siegel, Am. J. Vet. Res. 41(1), 91 (1980). 16. C. McCormack, Fed. Proc. 44, 1853 (1985).
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