Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice

Free Radical Biology & Medicine, Vol. 28, No. 3, pp. 397– 402, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(99)00253-1

Original Contribution ANTIOXIDANT AND OXIDATIVE STATUS IN TISSUES OF MANGANESE SUPEROXIDE DISMUTASE TRANSGENIC MICE WISSAM IBRAHIM,* UNG-SOO LEE,*, HSIU-CHUAN YEN,† DAVET K. ST. CLAIR,†

and

CHING K. CHOW*,†

*Department of Nutrition and Food Science and †Graduate Center for Toxicology, University of Kentucky, Lexington, KY USA (Received 21 July 1999; Revised 4 November 1999; Accepted 22 November 1999)

Abstract—Manganese superoxide dismutase (Mn-SOD) plays an important role in attenuating free radical–induced oxidative damage. The purpose of this research was to determine if increased expression of Mn-SOD gene alters intracellular redox status. Twelve week old male B6C3 mice, engineered to express human Mn-SOD in multiple organs, and their nontransgenic littermates were assessed for oxidative stress and antioxidant status in heart, brain, lung, skeletal muscle, liver, and kidney. Relative to their nontransgenic littermates, transgenic mice had significantly (p ⬍ .01) higher activity of Mn-SOD in heart, skeletal muscle, lung, and brain. Copper, zinc (Cu,Zn)-SOD activity was significantly higher in kidney, whereas catalase activity was lower in brain and liver. The activities of selenium (Se)-GSH peroxidase and non-Se-GSH peroxidase, and levels of vitamin E, ascorbic acid and GSH were not significantly different in any tissues measured between Mn-SOD transgenic mice and their nontransgenic controls. The levels of malondialdehyde were significantly lower in the muscle and heart of Mn-SOD mice, and conjugated dienes and protein carbonyls were not altered in any tissues measured. The results obtained showed that expression of human SOD gene did not systematical alter antioxidant systems or adversely affect the redox state of the transgenic mice. The results also suggest that expression of human SOD gene confers protection against peroxidative damage to membrane lipids. © 2000 Elsevier Science Inc. Keywords—Mn-SOD, Transgenic mice, Antioxidant status, Oxidative stress, Free radical

INTRODUCTION

occur when antioxidant potential is decreased and/or when oxidative stress is increased. Free radical–induced oxidative damage has been implicated in the pathogenesis of a number of injury and diseases states [4 – 6]. As a large number of interacting antioxidant systems are involved in preventing/limiting oxidative damage, individual contribution of the multiple antioxidant systems in intact cell/organ is difficult to assess. SOD is considered as the first line of defense against oxygen toxicity [7]. It exists as a family of three metalloproteins with copper, zinc (Cu, Zn-SOD), manganese (Mn-SOD) and iron (Fe-SOD) forms. Mn-SOD is a critical antioxidant enzyme in aerobic organisms because the superoxide radical is mainly generated on the matrix side of the inner mitochondrial membrane [8]. Thus, it is conceivable that increases in Mn-SOD activity may provide increased protection against oxidative stress. Indeed, transgenic mice expressing Mn-SOD in pulmonary tissues are more resistant to oxygen toxicity [9], and those expressing Mn-SOD in the heart are protective against adriamycin-induced cardiac toxicity [10] or myo-

While constantly being subjected to oxidative stress, aerobic organisms are protected against oxidative damage by a variety of antioxidant systems under normal conditions. Major antioxidant mechanisms include: (i) interaction with oxidants and oxidizing agents by ascorbic acid and reduced glutathione (GSH); (ii) scavenging of free radicals and singlet oxygen by vitamin E, ascorbic acid, ␤-carotene, and superoxide dismutase (SOD); (iii) reduction of hydroperoxides by GSH peroxidases and catalase; (iv) binding of transition metals by various chelators; and (v) repair of resultant damage via metabolic activities [1–3]. Oxidative damage, however, may Address correspondence to: Dr. Ching K. Chow, Department of Nutrition and Food Science, University of Kentucky, 204 Funkhouser Building, Lexington, KY 40506-0054, USA; Tel: (606) 257-7783; Fax: (606) 257-3707; E-Mail: [email protected]. Current address: Dr. U.-S. Lee, Department of Food Engineering, Chung-Ju National University, Chung-Ju Si, Chung-Buk, Korea. Current address: Dr. H.-C. Yen, School of Medical Technology, Chang Gung Medical College, Kwei-san, Tao-Yuan, Taiwan. 397

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cardial ischemia-reperfusion injury [11]. Also, overexpression of Mn-SOD in the lungs of mice prior to irradiation prevents irradiation-induced acute and chronic damage [12] and increased hepatic mitochondrial oxidative damage has been shown to closely correlate with altered mitochondrial function in heterozygous Mn-SOD knockout mice with 50% decrease in Mn-SOD activity and no change in GSH peroxidase or Cu, Zn-SOD activities [13]. However, Mn-SOD transgenic mice with approximately 50% of normal pulmonary Mn-SOD activity and normal levels of lung Cu, Zn-SOD, catalase and GSH peroxidase activities survived to 100% oxygen as well as their normal littermates, and those with 50% of normal heart Mn-SOD do not develop any ultrastructural abnormality in the myocardium after 100% oxygen exposure for 90 h [14]. These findings suggest that a certain level of Mn-SOD is essential for animal survival, and increased expression could provide protection under certain pathogenic conditions. The use of genetic manipulation to construct cells/ organs that specifically enrich with or lack of a single antioxidant enzyme represents a physiological avenue to understand its role in situ. It has been speculated that increased (or decreased) expression of one antioxidant enzyme may shift the balance and alter the status of other antioxidant systems. In order to determine if increased expression of one antioxidant enzyme shifts the balance or alters the status of other antioxidant systems, we assessed the status of important antioxidant systems in tissues of transgenic mice expressing human Mn-SOD gene and in those of their nontransgenic littermates. Additionally, whether higher rate of hydrogen peroxide generation that may result from increased expression of human Mn-SOD gene leads to increased oxidative damage was determined. The results obtained showed that expression of human SOD gene did not systematically alter the antioxidant or adversely affect the oxidative stress status of transgenic mice. MATERIALS AND METHODS

Production and identification of transgenic mice The mice used for producing transgenic mice were the F1 progeny of C57BL/6 x C3H hybrids (B6C3), which were purchased from Harlan Sprague Dawley (Indianapolis, IN, USA). Mn-SOD transgenic mice were prepared at the transgenic animal facility of the University of Kentucky using standard procedures [15]. Human MnSOD cDNA under the transcriptional control of human beta-actin were used for the expression of Mn-SOD mRNA in transgenic mice. The DNA was introduced into pronuclei of fertilized mouse eggs by microinjection. Mice with stable integrated human Mn-SOD trans-

genes identified by Southern analysis from tail DNA were selected as founders. All transgenic mice were propagated as heterozygous transgenic mice. At sexual maturity (6 – 8 weeks after birth), the line of transgenic mice expressing human Mn-SOD were bred with nontransgenic mice to produce transgenic and nontransgenic offspring. Two weeks after birth, the pups were marked for identification and genomic DNA was prepared from a small piece of tail. Southern blot analysis [16] was used to detect the presence of human Mn-SOD transgene after isolation of genomic DNA from mouse tail, and the levels of the human Mn-SOD mRNA in each tissue were detected by Northern analysis after isolation of total RNA by the guanidine isothiocyanate method [17] as described previously [10]. Approximately 50% of the animals produced is transgenic. After identification of each mouse to confirm the presence of human Mn-SOD gene, 12 male transgenic mice and 12 nontransgenic male littermates, were maintained at the transgenic animal facility of the University of Kentucky until they were 12 weeks old. Measurement of antioxidant systems and oxidation products Immediately after killing the mice, heart, brain, kidney, liver, lung and portions of skeletal muscle were removed, trimmed and homogenized with 1.15% KCl in 0.05 M phosphate buffer, pH 7.4. Portions of homogenates were immediately pipetted for measuring the levels of oxidation products, malondialdehyde (MDA), conjugated dienes and protein carbonyls, and small-molecular weight antioxidants, GSH, ascorbic acid and vitamin E. Another portion of homogenate was employed for measuring the activities of GSH peroxidases, catalase and superoxide dismutases. The levels of ascorbic acid in homogenate were measured after reacting with 2,4-dinitrophenylhydrazine at 515 nm [18], and of GSH after reaction with 5,5⬘-dithiobis(2-nitrobenzoic acid) at 412 nm [19]. After extraction with hexane, the levels of vitamin E (␣-tocopherol) were measured by high-performance liquid chromatography using a C18 reverse phase column with fluorescence detection [20]. The activity of Mn-SOD was assayed by monitoring nitroblue tetrazonium reduction in the presence of 5 mM NaCN, which inhibits Cu, Zn-SOD, as described by Spitz and Oberley [21] with some modifications. The activity was confirmed by the ferricytochrome c reduction assay [22]. In the absence of NaCN, total SOD activity was measured, and the activity of Cu, Zn-SOD was calculated. Catalase activity was measured spectrophotometrically at 240 nm [23]. The activities of GSH peroxidases were measured by monitoring NADPH ox-

Antioxidant status of Mn-SOD transgenic mice

399

Table 1. Enzymic Antioxidants in the Tissues of Mn-SOD Transgenic Mice Mn-SOD (U/mg protein) Tissue Brain Kidney Lung Muscle Liver Heart

Control

Cu-Zn-SOD (U/mg protein)

Transgenic

23.2 ⫾ 2.0a 30.3 ⫾ 4.0* 38.1 ⫾ 4.8 43.0 ⫾ 6.9 9.3 ⫾ 1.7 15.2 ⫾ 2.3* 10.0 ⫾ 2.6 19.7 ⫾ 2.1* 28.3 ⫾ 3.2 30.7 ⫾ 4.2 77.3 ⫾ 9.0 135.0 ⫾ 15.8*

Control

Catalase (␮mol/min/mg protein)

Transgenic

55.6 ⫾ 3.9 76.3 ⫾ 6.4 23.5 ⫾ 3.7 22.0 ⫾ 3.1 69.1 ⫾ 7.6 62.6 ⫾ 6.8

Control

Se-GSH Px (nmol/min/mg protein)

Transgenic

Control

Transgenic

61.7 ⫾ 6.0 8.9 ⫾ 0.8 6.7 ⫾ 0.9* 48.5 ⫾ 3.6 45.0 ⫾ 4.3 94.6 ⫾ 10.4* 377 ⫾ 22 396 ⫾ 11 580 ⫾ 41 553 ⫾ 45 26.3 ⫾ 4.3 116 ⫾ 6 104 ⫾ 14 117 ⫾ 14 130 ⫾ 16 20.5 ⫾ 3.7 7.0 ⫾ 1.5 8.3 ⫾ 1.4 31.2 ⫾ 4.1 27.4 ⫾ 3.3 75.7 ⫾ 6.9 883 ⫾ 68 801 ⫾ 60* 805 ⫾ 68 794 ⫾ 64 68.7 ⫾ 9.8 62.6 ⫾ 6.8 68.7 ⫾ 9.8 44.0 ⫾ 5.0 38.4 ⫾ 4.2

Non-Se-GSH Px (nmol/min/mg protein) Control

Transgenic

14.4 ⫾ 0.6 94.6 ⫾ 14.1 82.0 ⫾ 13.8 8.2 ⫾ 1.4 447 ⫾ 85 11.2 ⫾ 1.8

13.3 ⫾ 2.2 89.2 ⫾ 15.6 80.1 ⫾ 8.5 9.0 ⫾ 1.5 434 ⫾ 75 12.0 ⫾ 2.1

a Data are expressed as mean ⫾ standard deviation (n ⫽ 12). *Significant difference (p ⬍ .01) was relative to the corresponding nontransgenic littermates.

idation at 340 nm using hydrogen peroxide and cumene hydroperoxide as substrates [24]. The latter substrate measures total GSH peroxidase whereas Se-GSH peroxidase activity was measured using hydrogen peroxide as substrate. Protein was measured using Folin’s reagent at 540 nm [25]. The levels of lipid oxidation products, mainly MDA, were determined fluorometrically with excitation at 515 nm and emission at 550 nm after reaction with thiobarbituric acid and extraction with isobutanol according to the modified method of Li and Chow [26]. The levels of conjugated dienes, another indicator of lipid oxidation, were measured spectrophotometrically at 234 nm [27]. The content of protein-bound carbonyls, an indicator of protein oxidation, was determined by the 2,4-dinitrophenylhydrazine method [28]. Data obtained were analyzed using Student’s t-test for significant differences (p ⬍ .01) between two sample means.

sion than other organs. Also, lung and brain had appreciable amount of expression, while the expression in kidney and liver was rather limited. The activity of Mn-SOD was significantly higher (p ⬍ .01) in the heart, brain, lung and skeletal muscle and not in liver and kidney of Mn-SOD transgenic mice (Table 1). The increases of Mn-SOD activity, relative to their nontransgenic littermates, were greater in the heart and skeletal muscle than in the lung and brain. The integration, expression and localization of the human Mn-SOD gene in the transgenic mice have been reported previously [10]. The activity of Cu, Zn-SOD was significantly higher in the kidney, whereas catalase activity was significantly lower in the brain and liver, of transgenic mice than those of their nontransgenic littermates (Table 1). The activities of Se-GSH peroxidase and non-Se-GSH peroxidase were not significantly different between transgenic and nontransgenic mice in all tissues analyzed. As is shown in Table 2, increased expression of human Mn-SOD genes did not significantly alter the levels of small molecular weight antioxidants GSH, ascorbic acid or ␣-tocopherol in all tissues measured. The effect of expressing human Mn-SOD gene on the levels of oxidation products is shown in Table 3. The levels of MDA were significantly lower in the muscle and heart, but not in other organs, of Mn-SOD transgenic mice than their nontrans-

RESULTS

Northern analysis confirmed the expression of the steady state mRNA from human Mn-SOD in various issues of transgenic mice. Heart and skeletal muscle had the most significant amount of human Mn-SOD expres-

Table 2. Nonenzymic Antioxidants in the Tissues of Mn-SOD Transgenic Mice GSH (␮moles/g) Tissue

Control

Brain Kidney Lung Muscle Liver Heart

2.5 ⫾ 0.2 6.0 ⫾ 0.5 1.2 ⫾ 0.2 0.9 ⫾ 0.1 6.6 ⫾ 0.4 1.4 ⫾ 0.1

a

Ascorbic acid (␮moles/g)

Transgenic

Control

Transgenic

Control

Transgenic

2.3 ⫾ 0.2 5.8 ⫾ 1.0 1.1 ⫾ 0.1 1.0 ⫾ 0.1 6.6 ⫾ 0.5 1.4 ⫾ 0.2

3.0 ⫾ 0.2 1.4 ⫾ 0.1 3.0 ⫾ 0.2 0.9 ⫾ 0.1 1.7 ⫾ 0.3 1.4 ⫾ 0.1

3.1 ⫾ 0.2 1.5 ⫾ 0.2 3.0 ⫾ 0.2 0.8 ⫾ 0.1 1.8 ⫾ 0.1 1.2 ⫾ 0.1

3.7 ⫾ 0.5 6.7 ⫾ 0.6 4.9 ⫾ 0.5 2.9 ⫾ 0.4 9.8 ⫾ 1.4 7.3 ⫾ 1.3

4.1 ⫾0.6 7.3 ⫾ 0.8 4.4 ⫾ 1.0 3.4 ⫾ 0.5 11.2 ⫾ 1.6 7.5 ⫾ 1.6

Data are expressed as mean ⫾ standard deviation (n ⫽ 12).

a

␣-Tocopherol (␮g/g)

400

W. IBRAHIM et al. Table 3. Oxidation Products in the Tissues of Mn-SOD Transgenic Mice Malondialdehyde (nmoles/g)

Conjugated dienes (nmoles/g)

Protein carbonyls (nmoles/g)

Tissue

Control

Transgenic

Control

Transgenic

Control

Transgenic

Brain Kidney Lung Muscle Liver Heart

31.0 ⫾ 4.5a 39.5 ⫾ 5.5 29.0 ⫾ 3.5 33.5 ⫾ 4.4 12.8 ⫾ 2.0 19.3 ⫾ 1.6

30.5 ⫾ 2.5 41.4 ⫾ 5.2 27.0 ⫾ 3.2 22.5 ⫾ 3.6* 10.5 ⫾ 1.5 15.1 ⫾ 1.2*

208 ⫾ 18 192 ⫾ 24 404 ⫾ 42 363 ⫾ 24 354 ⫾ 29 239 ⫾ 33

196 ⫾ 30 212 ⫾ 28 372 ⫾ 38 336 ⫾ 31 360 ⫾ 41 221 ⫾ 26

3.0 ⫾ 0.4 2.2 ⫾ 0.3 2.5 ⫾ 0.6 2.6 ⫾ 0.6 1.0 ⫾ 0.2 1.6 ⫾ 0.2

2.9 ⫾ 0.5 2.3 ⫾ 0.4 2.3 ⫾ 0.5 2.4 ⫾ 0.5 1.1 ⫾ 0.2 1.5 ⫾ 0.3

a Data are expressed as mean ⫾ standard deviation (n ⫽ 12). *Significant difference (p ⬍ .01) was relative to the corresponding nontransgenic littermates.

genic littermates. The levels of conjugated dienes and protein carbonyls were not significantly different between Mn-SOD transgenic mice and their nontransgenic littermates in any organs examined.

DISCUSSION

Induction of antioxidant enzymes in mammalian cells and tissues are generally accompanied by an increased tolerance to the toxicity of agents that cause oxidative stress [29 –31]. However, whether increased expression of a single antioxidant enzyme, especially SOD, is beneficial or not, is controversial. On the one hand, transgenic mice expressing Mn-SOD in pulmonary tissues are more resistant to oxygen toxicity [9], and transgenic mice expressing Mn-SOD in the heart are protective against adriamycin-induced cardiac toxicity[10] or myocardial ischemia–reperfusion injury [11]. On the other hand, overexpression of Cu, Zn-SOD has been implicated in the pathogenesis of rapid aging featuring in the brain of patients with Down’s syndrome [32,33]. Cu, Zn-SOD is also known to be a locus of mutation in familial amyotrophic lateral sclerosis (ALS), which causes the degeneration of motor neurons in cortex, brain stem, and spinal cord with consequent progressive paralysis and death. The motor neuron disease is linked to the gain-of-function mutation in the human Cu, Zn-SOD gene [34]. It has been suggested that an enhancement of free radical formation as measured by the spin trapping method at low hydrogen peroxide concentration, due to a decrease in Km for hydrogen peroxide, may induce a gain-of-function of the ALS-associated Cu, Zn-SOD mutant [35,36]. Similarly, Ditelberg et al. [37] have shown that brain injury after perinatal hypoxia-ischemia is exacerbated in transgenic mice expressing Cu, Zn-SOD gene, and suggested that excessive hydrogen peroxide and nitric oxide production may be responsible. Also, 24 h after perinatal hypoxic-ischemic injury, greater hydrogen peroxide accumulation is found in the brains of

Cu, Zn-SOD transgenic mice than their wild-type nontransgenic littermates [38]. As GSH peroxidase activity was decreased significantly 24 h after hypoxia-ischemia, it is suggestive that developmentally low activities of the hydrogen peroxide metabolizing enzymes may play a role in the increased brain injury in neonatal Cu, Zn-SOD transgenic mice [38]. Additionally, the incidence and number of skin carcinogenesis, initiated by 7,12-dimethylbenz(a)anthracene and promoted by 12-O-tetradecaneoylphorbol-13-acetate, are higher in transgenic mice with high expression of GSH peroxidase or both GSH peroxidase and SOD [39]. Although expression of Cu, Zn-SOD gene in mice is associated with increased generation of reactive oxygen species and oxidative damage [35–38], expression of human Mn-SOD gene does not adversely affect the oxidative stress status [9]. In the present study, none of the oxidation products measured was significantly increased in any tissues that express the human Mn-SOD gene (heart, muscle, brain and lung) or those of the nonexpressed tissues (liver and kidney). Furthermore, transgenic mice had significantly lower levels of MDA in heart and muscle of Mn-SOD than their nontransgenic littermates. The finding suggests that small changes in the activities of GSH peroxidase and catalase in various tissues of Mn-SOD–transgenic mice do not adversely affect their ability to handle hydrogen peroxide generated. It further suggests that expression of human MnSOD gene not only does not adversely influence the oxidative stress status but also provides some protection against peroxidative damage to membrane lipids. While the reason of the contrast outcome between the expression of Cu, Zn-SOD and Mn-SOD gene is not clear, the location where oxidant is generated and antioxidant enzyme is present may be critical in determining whether the effect is beneficial or detrimental [8]. In addition to Mn-SOD, the activity of Cu, Zn-SOD was significantly increased in the kidney, while catalase activity was decreased in the liver and brain, of Mn-SOD

Antioxidant status of Mn-SOD transgenic mice

transgenic mice. However, since oxidation products formed were not significantly increased, changes of Cu, Zn-SOD and catalase activities in those organs appear to have no negative impact on the oxidative status. Also, since catalase is never saturated under normal physiological condition, a small change in the enzyme activity is not expected to affect the intracellular steady state concentration of hydrogen peroxide. Additionally, it is possible that the significant alteration of Cu, Zn-SOD in kidney observed may be resulting from the methodological inaccuracies [21,22]. In summary, the results obtained showed that expression of human Mn-SOD gene in mice resulted in small but significant alteration in the activities of Cu, Zn-SOD and catalase in several tissues. Also, no significant changes were found in the activities of GSH peroxidases, or the levels of small molecular antioxidants in the tissues of Mn-SOD transgenic mice examined. Except for the lower levels of MDA in muscle and heart in Mn-SOD transgenic mice, no other indices of oxidative damages were significantly altered. The results obtained from the present study showed that expression of human Mn-SOD gene did not systematically alter antioxidant systems or adversely affect the redox state. The results also suggest that expression of human SOD gene confers protection against peroxidative damage to membrane lipids. Acknowledgments — Supported by Kentucky Agricultural Experiment station, and NIH grants.

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