@Copyright 1985 bv The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/85/0706-0209$02.80
Developmental Changes Affected by Mn Deficiency Mn-Superoxide Dismutase, CuZn-Superoxide Dismutase, Mn, Cu, Fe, and Zn in Mouse Tissues SHER1 ZIDENBERG-CHERR, CARL L KEEN, SHARON M. CASEY, AND LUCILLE S. HURLEY* Department of Nutrition, University of California, Davis, California 95616 Received November 17, 1984; Accepted J a n u a r y 8, 1985
ABSTRACT The changes in tissue Mn, Cu, Fe, Zn, and superoxide dismutase (SOD) activity were studied in control and Mn-deficient mice during postnatal development. Mn levels were lower in tissues from Mn-deficient mice than in controls throughout development. By day 60, Mn concentration in tissues from Mn-deficient mice was at least 70% lower than that of controls. Cu levels in the two groups did not differ appreciably. Liver Cu concentration was highest at day 5, then decreased. Heart and kidney Cu increased throughout development. Fe concentration in heart and liver was similar in both groups at 1, 5, and 20 days of age, but at day 60, kidney Fe in the Mndeficient mice was 40% higher than in controls. The developmental pattern for MnSOD activity paralleled that of Mn concentration. At day 5, there were no differences in MnSOD activity between control and deficient mice. By day 60, MnSOD activity in most tissues was at least 50% lower than that of controls, possibly increasing the susceptibility of the Mn deficient animal to oxidative damage. These developmental patterns should help investigators to determine the tissues and time periods in which to study trace element metabolism. index Entries: Manganese deficiency, and development; MnSOD, and development; tissue Mn, and development; development;
~Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
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Vol. 7, I985
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Zidenberg-Cherr et aL and Mn deficiency; superoxide dismutase, in Mn-deficient mouse tissues; CuZn-SOD, and development; manganese, in developing mouse tissues; copper, in Mn-deficient mouse tissues; iron, in Mndeficient mouse tissues; zinc, in Mn-deficient mouse tissues.
INTRODUCTION Manganese, although widely distributed in the biosphere, occurs in only low amounts in animal tissues, with typical values less than 2 txg/g (1). Despite these low concentrations, the essentiality of Mn has been demonstrated in numerous species (2). Manifestations of m a n g a n e s e deficiency include high neonatal death, poor growth, abnormal skeletal development, congenital ataxia, and defects in carbohydrate and lipid metabolism (1,2). Although its essentiality was first demonstrated over 50 yr ago, the underlying mechanisms responsible for these diverse deficiency signs are poorly understood. Manganese can function both as an enzyme activator and as an integral part of metalloenzymes. Three manganese metalloenzymes that have been identified in mammals are arginase, pyruvate carboxylase, and Mn-superoxide dismutase (MnSOD). Most protein b o u n d Mn is thought to be contained in these enzymes. The superoxide dismutases (SODs) function to protect cells from free radical damage by catalyzing the following reaction (3): O~-
+
O2- + 2H + ---->02 + H202
Superoxide dismutase containing Cu and Zn is found in the cytosol (CuZnSOD; MW 32,000), while SOD containing Mn is localized in the mitochondria. MnSOD isolated from chicken liver has a molecular weight of 80,000 and contains four subunits of equal size each containing one atom of manganese (1). The activities of CuZnSOD and MnSOD have been s h o w n to be influenced by the availability of Cu or Mn, respectively. Inadequate levels of Cu in the diet have been associated with lower than normal levels of CuZnSOD activity in rats (4,5) and swine (6). Similarly, feeding a Mndeficient diet results in lower than normal levels of MnSOD activity in tissues of the rat (5,7), mouse (8), and chicken (8). The extent to which SOD activity responds to a dietary deficiency of Cu or Mn d e p e n d s on the severity of the deficiency, the absolute level of the element in the diet, the duration and timing of the deficiency, and the species and the tissues investigated. De Rosa et al. (8) reported that for mice, feeding a Mn-deficient diet (1 ~g Mn/g diet) both prenatally and postnatally resulted in liver MnSOD activity in adults that was 80% lower than similarly aged controls (fed 45 ~,g Mn/g diets). Brain MnSOD activity was also severely affected, as evidenced by levels that were 50% of those observed in controls. Using rats fed Mn-deficient diets (2 ~g Mrgg diet) for 10 wk beginning at weaning, Paynter noted that MnSOD activity was lower in Biological Trace Element Research
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heart and kidney than in controls (5). In contrast to the study by de Rosa et al., the tissue most affected by the deficiency was the heart, and no difference in liver MnSOD activity between the two groups was observed. We have recently obtained results which suggest that low levels of liver M n S O D activity result in higher than normal levels of mitochondrial lipid peroxidation by 60 d of age, and that this may lead to mitochondrial m e m b r a n e damage apparent by 9 mo of age (10). Aerobic metabolism increases from the time of birth through weaning as the i m m a t u r e animal relies on the oxidative metabolism of its reserves as a source of energy (11)~ Thus an increase in tissue SOD activity is predicted during this time period~ However, if the deficient animal is unable to maintain an adequate level of enzymatic protection from oxygen-derived radicals, detrimental consequences may occur. The high neonatal mortality observed in the Mn-deficient mouse may be the result of lower than normal levels of MnSOD activity. In this paper we present changes that occur from birth through maturity (day 60) in the activity of MnSOD, CuZnSOD, and the concentration of manganese (Mn), copper (Cu), iron (Fe), and zinc (Zn) in several tissues of control and m a n g a n e s e deficient mice.
MATERIALS AND METHODS Beginning at 21 d of age, weanling female Swiss-Webster mice (Simonsen Laboratories, Gilroy, CA) were randomly assigned to one of two dietary treatment groups and fed a purified diet containing either 45 ~g Mn/g diet (control) or 1 I~g Mn/g diet (deficient). The diet contained 30% casein, 54.5% cerelose, 8% corn oil, 6% salt mix, and 1.5% vitamin mix. The detailed composition of the vitamin and mineral mixes have been previously published (7). Food and distilled water were provided ad libitum. Mice were housed in stainless-steel cages in a temperature and light controlled room (22-23~ 12 h light/dark cycle). W h e n the animals reached sexual maturity (approximately 60 d of age), they were mated overnight with stock-fed (Purina Rat Chow, Ralston Purina Co., St. Louis, MO) males of the same strain. Offspring remained with their dams until day 25, at which time they were weaned. At weaning, the offspring continued to receive the same diets fed to their mothers. Offspring were killed by decapitation at 1, 5, 20, and 60 d postpartum; liver, kidney, heart, and brain samples were taken for measurement of SOD activity, and for determination of Mn, Cu, Fe, and Zn.
SOD Activity Tissue homogenates (10%) were prepared in cold 0.25M sucrose and sonicated for 2 rain (30 s with 30 s cooling) with an Insonator Model 500 (Savant Instruments Inc., Hicksvilte, NY). Following sonication, the Biological Trace Element Research
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h o m o g e n a t e s were centrifuged at 10,000g for 30 rain at 4~ Pellets w e r e discarded, and the assay was conducted on supernatants. Total SOD activity was d e t e r m i n e d by its ability to inhibit the autooxidation of pyrogallol (12). Total SOD activity was determined in 50 mM triscacodylic acid, 1 m M diethylenetriamine pentaacetic acid, p H 8.2 at 25~ M n S O D activity was m e a s u r e d u n d e r the same conditions with the addition to the assay buffer of i mM potassium cyanide, w h i c h inhibits CuZnSOD. C u Z n S O D activity was calculated by subtracting MnSOD activity from total activity. One unit of SOD was defined as the a m o u n t of e n z y m e n e e d e d to obtain 50% inhibition of pyrogallol autooxidation. The unit activity was c o m p u t e d by plotting the reciprocal of the slope of pyrogaltol autooxidation versus the reciprocal of the volume of sample used for the assay.
Trace Mineral Analysis Tissue samples were ;vet ashed with 16N nitric acid, concentrated by evaporation, and diluted with distilled deionized water (13). Liver Cu, Fe, and Zn concentrations were determined by flame atomic absorption spectrophotometry (IL551, Instrumentation Laboratories, Wilmington, MA). Mn concentration was determined by t a m e l e s s atomic absorption spectrophotometry (IL 551 in conjunctiion with the IL 55B t a m e l e s s atomizer) (14). Using these methods, recovery of a d d e d metal is 98 + 2%. Data w e r e analyzed by student's t-test comparing control and deficient animals at each time point.
RESULTS Postnatal Growth and Survival of the Offspring There were no differences in the birth weight or n u m b e r of offspring of Mn deficient or control dams (Table 1, Fig. 1). Similarly, there was no difference in the postnatal weight gain between the groups (Fig. 1). In contrast to weight gain, survival to day 14 was lower in offspring of Mn deficient dams than of control dams. Approximately 94% of the offspring born to control dams survived to day 14 whereas only 80% of those born to Mn deficient dams survived to this time (Table 1).
Trace Element Concentration
Manganese Concentration LrVER. In deficient animals, Mn concentration was lower than that
of controls at each time measured; the concentration at maturity (day 60) was (mean -+ SEM) 0.262 -+ 0.01 ~g/g liver, which was 80% lower than control values. Mn concentration increased in both groups from day 5 to
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02' 0!:
0
2
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AGE ( D A Y S )
Fig. 1. Body weight of offspring from control (Q) and Mn deficient (A) mice from birth through 14 d of age. Each point represents the mean weight per pup -+ SEM in 13 litters for controls and 13 litters for deficient mice. day 20; h o w e v e r , at day 60 tissue Mn was 50% lower than at day 20 (p
Developmental Changes Affected by Mn Deficiency Mn-Superoxide Dismutase, CuZn-Superoxide Dismutase, Mn, Cu, Fe, and Zn in Mouse Tissues SHER1 ZIDENBERG-CHERR, CARL L KEEN, SHARON M. CASEY, AND LUCILLE S. HURLEY* Department of Nutrition, University of California, Davis, California 95616 Received November 17, 1984; Accepted J a n u a r y 8, 1985
ABSTRACT The changes in tissue Mn, Cu, Fe, Zn, and superoxide dismutase (SOD) activity were studied in control and Mn-deficient mice during postnatal development. Mn levels were lower in tissues from Mn-deficient mice than in controls throughout development. By day 60, Mn concentration in tissues from Mn-deficient mice was at least 70% lower than that of controls. Cu levels in the two groups did not differ appreciably. Liver Cu concentration was highest at day 5, then decreased. Heart and kidney Cu increased throughout development. Fe concentration in heart and liver was similar in both groups at 1, 5, and 20 days of age, but at day 60, kidney Fe in the Mndeficient mice was 40% higher than in controls. The developmental pattern for MnSOD activity paralleled that of Mn concentration. At day 5, there were no differences in MnSOD activity between control and deficient mice. By day 60, MnSOD activity in most tissues was at least 50% lower than that of controls, possibly increasing the susceptibility of the Mn deficient animal to oxidative damage. These developmental patterns should help investigators to determine the tissues and time periods in which to study trace element metabolism. index Entries: Manganese deficiency, and development; MnSOD, and development; tissue Mn, and development; development;
~Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
209
Vol. 7, I985
210
Zidenberg-Cherr et aL and Mn deficiency; superoxide dismutase, in Mn-deficient mouse tissues; CuZn-SOD, and development; manganese, in developing mouse tissues; copper, in Mn-deficient mouse tissues; iron, in Mndeficient mouse tissues; zinc, in Mn-deficient mouse tissues.
INTRODUCTION Manganese, although widely distributed in the biosphere, occurs in only low amounts in animal tissues, with typical values less than 2 txg/g (1). Despite these low concentrations, the essentiality of Mn has been demonstrated in numerous species (2). Manifestations of m a n g a n e s e deficiency include high neonatal death, poor growth, abnormal skeletal development, congenital ataxia, and defects in carbohydrate and lipid metabolism (1,2). Although its essentiality was first demonstrated over 50 yr ago, the underlying mechanisms responsible for these diverse deficiency signs are poorly understood. Manganese can function both as an enzyme activator and as an integral part of metalloenzymes. Three manganese metalloenzymes that have been identified in mammals are arginase, pyruvate carboxylase, and Mn-superoxide dismutase (MnSOD). Most protein b o u n d Mn is thought to be contained in these enzymes. The superoxide dismutases (SODs) function to protect cells from free radical damage by catalyzing the following reaction (3): O~-
+
O2- + 2H + ---->02 + H202
Superoxide dismutase containing Cu and Zn is found in the cytosol (CuZnSOD; MW 32,000), while SOD containing Mn is localized in the mitochondria. MnSOD isolated from chicken liver has a molecular weight of 80,000 and contains four subunits of equal size each containing one atom of manganese (1). The activities of CuZnSOD and MnSOD have been s h o w n to be influenced by the availability of Cu or Mn, respectively. Inadequate levels of Cu in the diet have been associated with lower than normal levels of CuZnSOD activity in rats (4,5) and swine (6). Similarly, feeding a Mndeficient diet results in lower than normal levels of MnSOD activity in tissues of the rat (5,7), mouse (8), and chicken (8). The extent to which SOD activity responds to a dietary deficiency of Cu or Mn d e p e n d s on the severity of the deficiency, the absolute level of the element in the diet, the duration and timing of the deficiency, and the species and the tissues investigated. De Rosa et al. (8) reported that for mice, feeding a Mn-deficient diet (1 ~g Mn/g diet) both prenatally and postnatally resulted in liver MnSOD activity in adults that was 80% lower than similarly aged controls (fed 45 ~,g Mn/g diets). Brain MnSOD activity was also severely affected, as evidenced by levels that were 50% of those observed in controls. Using rats fed Mn-deficient diets (2 ~g Mrgg diet) for 10 wk beginning at weaning, Paynter noted that MnSOD activity was lower in Biological Trace Element Research
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21 1
heart and kidney than in controls (5). In contrast to the study by de Rosa et al., the tissue most affected by the deficiency was the heart, and no difference in liver MnSOD activity between the two groups was observed. We have recently obtained results which suggest that low levels of liver M n S O D activity result in higher than normal levels of mitochondrial lipid peroxidation by 60 d of age, and that this may lead to mitochondrial m e m b r a n e damage apparent by 9 mo of age (10). Aerobic metabolism increases from the time of birth through weaning as the i m m a t u r e animal relies on the oxidative metabolism of its reserves as a source of energy (11)~ Thus an increase in tissue SOD activity is predicted during this time period~ However, if the deficient animal is unable to maintain an adequate level of enzymatic protection from oxygen-derived radicals, detrimental consequences may occur. The high neonatal mortality observed in the Mn-deficient mouse may be the result of lower than normal levels of MnSOD activity. In this paper we present changes that occur from birth through maturity (day 60) in the activity of MnSOD, CuZnSOD, and the concentration of manganese (Mn), copper (Cu), iron (Fe), and zinc (Zn) in several tissues of control and m a n g a n e s e deficient mice.
MATERIALS AND METHODS Beginning at 21 d of age, weanling female Swiss-Webster mice (Simonsen Laboratories, Gilroy, CA) were randomly assigned to one of two dietary treatment groups and fed a purified diet containing either 45 ~g Mn/g diet (control) or 1 I~g Mn/g diet (deficient). The diet contained 30% casein, 54.5% cerelose, 8% corn oil, 6% salt mix, and 1.5% vitamin mix. The detailed composition of the vitamin and mineral mixes have been previously published (7). Food and distilled water were provided ad libitum. Mice were housed in stainless-steel cages in a temperature and light controlled room (22-23~ 12 h light/dark cycle). W h e n the animals reached sexual maturity (approximately 60 d of age), they were mated overnight with stock-fed (Purina Rat Chow, Ralston Purina Co., St. Louis, MO) males of the same strain. Offspring remained with their dams until day 25, at which time they were weaned. At weaning, the offspring continued to receive the same diets fed to their mothers. Offspring were killed by decapitation at 1, 5, 20, and 60 d postpartum; liver, kidney, heart, and brain samples were taken for measurement of SOD activity, and for determination of Mn, Cu, Fe, and Zn.
SOD Activity Tissue homogenates (10%) were prepared in cold 0.25M sucrose and sonicated for 2 rain (30 s with 30 s cooling) with an Insonator Model 500 (Savant Instruments Inc., Hicksvilte, NY). Following sonication, the Biological Trace Element Research
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h o m o g e n a t e s were centrifuged at 10,000g for 30 rain at 4~ Pellets w e r e discarded, and the assay was conducted on supernatants. Total SOD activity was d e t e r m i n e d by its ability to inhibit the autooxidation of pyrogallol (12). Total SOD activity was determined in 50 mM triscacodylic acid, 1 m M diethylenetriamine pentaacetic acid, p H 8.2 at 25~ M n S O D activity was m e a s u r e d u n d e r the same conditions with the addition to the assay buffer of i mM potassium cyanide, w h i c h inhibits CuZnSOD. C u Z n S O D activity was calculated by subtracting MnSOD activity from total activity. One unit of SOD was defined as the a m o u n t of e n z y m e n e e d e d to obtain 50% inhibition of pyrogallol autooxidation. The unit activity was c o m p u t e d by plotting the reciprocal of the slope of pyrogaltol autooxidation versus the reciprocal of the volume of sample used for the assay.
Trace Mineral Analysis Tissue samples were ;vet ashed with 16N nitric acid, concentrated by evaporation, and diluted with distilled deionized water (13). Liver Cu, Fe, and Zn concentrations were determined by flame atomic absorption spectrophotometry (IL551, Instrumentation Laboratories, Wilmington, MA). Mn concentration was determined by t a m e l e s s atomic absorption spectrophotometry (IL 551 in conjunctiion with the IL 55B t a m e l e s s atomizer) (14). Using these methods, recovery of a d d e d metal is 98 + 2%. Data w e r e analyzed by student's t-test comparing control and deficient animals at each time point.
RESULTS Postnatal Growth and Survival of the Offspring There were no differences in the birth weight or n u m b e r of offspring of Mn deficient or control dams (Table 1, Fig. 1). Similarly, there was no difference in the postnatal weight gain between the groups (Fig. 1). In contrast to weight gain, survival to day 14 was lower in offspring of Mn deficient dams than of control dams. Approximately 94% of the offspring born to control dams survived to day 14 whereas only 80% of those born to Mn deficient dams survived to this time (Table 1).
Trace Element Concentration
Manganese Concentration LrVER. In deficient animals, Mn concentration was lower than that
of controls at each time measured; the concentration at maturity (day 60) was (mean -+ SEM) 0.262 -+ 0.01 ~g/g liver, which was 80% lower than control values. Mn concentration increased in both groups from day 5 to
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Fig. 1. Body weight of offspring from control (Q) and Mn deficient (A) mice from birth through 14 d of age. Each point represents the mean weight per pup -+ SEM in 13 litters for controls and 13 litters for deficient mice. day 20; h o w e v e r , at day 60 tissue Mn was 50% lower than at day 20 (p
