Modulations by dietary restriction on antioxidant. enzymes and lipid peroxidation in developing mice

Transcription

1 J Appl Physiol Articles in PresS. Published on November 8, 2002 as DOI /japplphysiol JAP R1 FINAL ACCEPTED VERSION Modulations by dietary restriction on antioxidant enzymes and lipid peroxidation in developing mice Aiguo Wu, 1 Xiufa Sun, 1 Fada Wan, 1 and Yugu Liu 2 1 Department of Nutrition and Food Hygiene and 2 Environmental Toxicology, Tongji Medical University, Wuhan, Hubei , China Running title: dietary restriction affects antioxidant systems Corresponding author: Aiguo Wu. Department of Physiological Science, University of California at Los Angeles, Los Angeles, CA 90095, USA. Phone: Fax: E- mail: agwu@ucla.edu Keywords: Dietary restriction; superoxide dismutase; catalase; glutathione peroxidase; lipid peroxidation 1 Copyright 2002 by the American Physiological Society.

2 Abstract The effects of dietary restriction (DR) on the activities of liver superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and the level of lipid peroxidation (LP) in developing mice were investigated in this study. Male and female Kunmin mice were fed a standard rodent diet ad libitum (AL); 80% of AL food intake (20% DR) or 65% of AL food intake (35% DR) for 12 or 24 wk. Both 12- and 24-wk of DR resulted in retarded body weight gain in male and female mice. The activities of SOD, CAT and GPx and content of LP in DR male and female mice were not different (P>0.05) from those in controls after 12 wk of DR. However, the SOD activity was increased at 24 wk in 20% DR (P<0.05) and 35% DR (P<0.01) male, but not in DR female mice. The CAT activity was elevated at 24 wk in both DR male (P<0.05 for 20% DR, P<0.01 for 35% DR) and female (P<0.01) mice with a greater increase in DR female (P<0.05) than in DR male animals. GPx activity was also increased at 24 wk in DR male (P<0.01) and female (P<0.01) mice with a greater elevation in DR female (P<0.05) than in DR males. Further, LP was decreased at 24 wk in both DR male (P<0.01) and female (P<0.01) animals with a greater reduction in DR females (P<0.01) compared to DR males. These findings indicated that 24-wk, but not 12-wk, of DR led to differential effects on liver SOD, CAT and GPx activity and LP content in male and female mice during development, suggesting sexassociated modulations of DR on antioxidant systems in developing animals. 2

3 Introduction It has been well known that reactive oxygen species (ROS) can damage proteins, lipids and DNA, playing a significant role in numerous diseases including atherosclerosis, cancer, diabetes, and neurodegenerative disorders (1,3,4). ROS also mediates cytotoxicity of many environment chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (38), arsenic (21), and cadmium (36). The generation of ROS involves normal cellular metabolism (17) and oxidation of a variety of cytotoxic agents such as bleomycin (9). The antioxidant systems including antioxidants and antioxidant enzymes can ameliorate the deleterious effects of ROS in vivo and in vitro. Antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) function by catalyzing the decomposition of oxidants and free radicals. Interventions to increase antioxidant capacity and reduce oxidative damage have been suggested as a potentially useful strategy to prevent or retard the adverse actions of ROS. It has been suggested that dietary restriction (DR) might extend life span, reduce incidence and degree of numerous pathologies, and increase resistance to environmental chemicals by limiting free radical production (10,25,30), improving detoxification of free radical (11,19,31) and inducing repair of oxidative damage (19). This hypothesis has been supported by many reports (5-7,18-20,25,26,29,31,36,40). It is known that liver is an important organ for fighting against toxic injury of xenobiotics and endogenous toxins, in which ROS might be involved. Keeping the balance of ROS production and free radical scavenging plays an important role in maintaining the normal function of liver. The majority of findings regarding the beneficial effects of DR on liver antioxidant systems were obtained in aged animals and the observations might vary in different species of animals. For example, Koizumi et al. (19) reported that long-term dietary restriction 3

4 increased liver CAT activity and decreases lipid peroxidation (LP) in 12- and 24-month female C3B10RF 1 mice. The study of Chipalkatti et al. (8) showed that 12-month-old mice fed half of the ad libitum intake showed lower LP level in liver and no effect on SOD activity. Ross (33) reported that the specific activity of liver CAT tended to be higher in old restricted rats, but no effects were found in younger rats. And more, the results of Rao et al. (31) showed that DR (40% restriction of energy intake) increased the activities of liver SOD and CAT at 21- and 28- month of age and GPx at 28-month of age in male Fischer F344 rats. These studies suggested that the beneficial effects of DR on liver antioxidant systems might be related to sex, age or species of tested animals. However, it remains unknown whether there are different modulations of DR on antioxidant systems in male and female developing animals. Therefore, the effects of DR on liver SOD, CAT, and GPx activity and LP level in developing mice were investigated in this study. Materials and methods Animals Sixty male and female Kunmin mice, all one month of age, were housed in individual cages and maintained in environmentally controlled rooms (22±2 C and 50±10% relative humidity) with a 12 h light/dark cycle. They were randomly divided into three groups: control, fed ad libitum (AL); 20% DR, fed 80% of AL food intake; 35% DR, fed 65% of AL food intake. Water was available ad libitum. Body weight was determined weekly. Animal care was consistent with the guidelines set by the Laboratory Animal Center of Tongji Medical University. All experimental procedures were approved by the Institutional Research Committee of Tongji Medical University. 4

5 Diets The control and restricted diets were prepared according the compositions as previously described (43) and shown in Table 1. The food consumption in the ad libitum (control) animals was measured daily. The pre-measured amount of restricted diets (relative to 80% and 65% of control mice daily consumption) was given daily to the 20% DR and 35% DR animals respectively. The contents of protein, fat, vitamins and minerals were adjusted so that intake of these nutrients would be constant in control and restricted mice (Table 1) to avoid malnutrition. Food was changed daily to avoid degradation problem. Tissue preparation After 12 wk or 24 wk on control or restricted diet, the mice were anesthetized, livers were then removed, immediately frozen in liquid nitrogen and stored at 70 o C until used for the following assays. Measurement of antioxidant enzyme activities The activity of SOD was determined by monitoring the inhibition of the autoxidation of pyrogallol (22). One unit of SOD activity was defined as the amount of enzyme required to inhibit pyrogallol autoxidation by 50%. The CAT activity was assayed according to the reported method (2). One unit of CAT activity was defined as the amount of enzyme required to decompose 1 µmol of hydrogen peroxide (H 2 O 2 ) per min. The GPx activity was determined by means of a coupled reaction system assay (39). One unit of GPx activity was defined as the amount of enzyme needed to catalyze the oxidation of 1 nmol NADPH per min. 5

6 Measurement of lipid peroxidation Lipid peroxidation level was quantified by measuring thiobarbituric-reactive substances (TBARS) production as previously described (12). The TBARS values were expressed as nmol of malondialdehyde (MDA) equivalents per g of tissue. Statistical analysis All data were expressed as mean ± SEM and presented in bar figures. The data were analyzed by a three-way ANOVA (diet, time and sex) followed by Bonferroni t-test. Differences were considered significant when P < Results Food consumption and body weight The food intake, in grams of food consumed, was 80% in 20% DR group and 65% in 35% DR group relative to control (AL) animals. Body weight gain of DR mice was reduced relative to control except that of females with no difference between 20% DR compared to control group at 12 wk (Fig. 1), suggesting that DR retarded the body weight gain in developing mice. There were no significant differences in food consumption per gram body weight in all groups of male and female mice (data not shown). SOD Activity The SOD activity in DR male and female mice was not significantly different (P>0.05) from that in control animals at 12 wk (Fig. 2A). However, the activity of SOD was significantly 6

7 increased at 24 wk in 20% DR (P<0.05) and 35% DR (P<0.01) male, but not in DR female mice, relative to control mice (Fig. 2B). CAT Activity The CAT activity in DR male and female mice was not significantly different (P>0.05) from that in control animals at 12 wk (Fig. 3A). However, The CAT activity was elevated at 24 wk in both DR male (P<0.05 for 20% DR, P<0.01 for 35% DR) and female (P<0.01) mice compared to respective controls (Fig. 3B). Further, the increase of CAT activity was greater in DR female (P<0.05) than in DR male animals (Fig. 3B). GPx Activity The GPx activity in DR male and female mice was not significantly different (P>0.05) from that in control animals at 12 wk (Fig. 4A). However, GPx activity was also increased at 24 wk in DR male (P<0.01) and female (P<0.01) mice relative to respective control animals (Fig. 4B). Moreover, the elevation of GPx activity was greater in DR female (P<0.05) than in DR males (Fig. 4B). LP Content The content of LP was not different (P>0.05) in DR male and female mice from that in controls at 12 wk (Fig. 5A), whereas it was decreased at 24 wk in both DR male (P<0.01) and female (P<0.01) animals compared to respective control animals (Fig. 5B). Furthermore, the reduction of LP content is greater in DR females (P<0.01) than in DR males (Fig. 5B). 7

8 Discussion In this study, we investigated the effects of DR on the activities of liver SOD, CAT, and GPx and the level of LP in developing mice. Our results demonstrated that 24-wk, but not 12- wk, of DR led to differential effects on liver SOD, CAT and GPx activity and LP content in male and female mice during development, suggesting sex-associated modulations of DR on antioxidant systems in developing animals. The majority of findings regarding the beneficial effects of DR on liver antio xidant systems were obtained in aged animals and the results may be related to sex and/or species of animals. For example, long-term DR has been reported to increase liver CAT activity and decrease LP in aged female C3B10RF 1 mice, but has no effect on SOD activity (12). Another report showed that DR increased the activities of liver SOD, CAT and GPx in aged male Fischer F344 rats (31). In this study we provide evidence for the first time that DR produced different effects on liver antioxidant systems in developing animals. Our results demonstrated that DR significantly increased SOD activity in male mice with no effect on SOD activity in female mice, but elicited a greater increase in activities of CAT and GPx in female mice (P<0.05) than in male animals. Further, DR triggered a greater reduction of LP content in female animals (P<0.01) compared to males. The sex-related effects of DR on antioxidant enzymes and LP in liver of developing animals were not extensively studied, hence the mechanisms underlying DR- induced different modulations on antioxidant systems in animals during development remain to be further investigated. The DR regimen in this study including different restriction level of energy intake (0% as control, 20% DR and 35% DR) with consistent intake of essential nutrients in all groups was based on previous evidence (41). The beneficial effects of DR appear to depend on restriction of 8

9 energy intake with adequate intake of essential nutrients (41). Therefore, optimal nutrient composition and feeding strategies for DR experiments such as levels of restriction, intake of essential nutrients, and term of restriction are very important for designing experiments associated with DR. Based on these considerations, we introduced three levels of energy intake (0%, 20% and 35% DR) with equal intake of fat, protein, vitamins and minerals to developing mice. Thus, our DR regimen (restriction of energy intake without malnutrition) is different from short-term fasting or food deprivation, which may lead to malnutrition, subsequently causing pathological or dysfunctional status in mammalian systems such as reduced detoxification in liver (14,23,37). In this study, we found that DR affect the body weight of both male and female mice in a restriction level-dependent way except that of females with no difference between 20% DR compared to control group at 12 wk (Fig. 1). The reason is unknown in this study. Furthermore, DR also modulated SOD activity in male mice (Fig. 2B), CAT activity and LP content in both male and female mice in a restriction level-dependent way (Fig. 3B,5B). The reduction of LP observed in liver of male and female mice may be related to the increase of SOD, CAT or GPx activity. It is known that SOD converts superoxide anion into H 2 O 2 and O 2 (28), while CAT and GPx reduce H 2 O 2 to H 2 O (13,42), resulting in the detoxification of free radicals. Thus, elevation of SOD, CAT and GPx activity may contribute to the decrease of LP. Another mechanism may be DR-induced reduction of energy expenditure, consequently leading to lowered ROS (30). Reduced LP products suggest decreased formation of fatty acid epoxide and subsequent free radical damage to cellular macromolecules. In addition, DR may lead to reduced mitochondrial oxyradical production and/or increased expression of cytoprotective stress proteins, which may suppress oxyradical production and stabilize cellular homeostasis (25). 9

10 SOD, CAT and GPx are main detoxifying enzymes in cells. SOD appears as a important enzyme for the prevention of aging and mutation by oxidative stresses and hazardous effects from environmental factors (34). CAT plays an important role in the acquisition of tolerance to oxidative stress in the adaptive response of cells (16). GPx also plays important role in protecting mammalian cells against oxidative damage (24). Thus, the beneficial effects of DR on these enzymes may promote the capacity of liver to protect against toxic actions of ROS, maintaining normal function. However, the mechanisms underlying elevation of antioxidant enzymes by DR in developing mice need further investigations. It was reported that increase of antioxidant enzyme activities might arise from changes of mrna (31). It has been described that the expression of these enzymes is regulated by transcriptional control (35), and genetic regulatory elements and promoter sequences for antioxidant enzyme genes have been recognized (27). Further, the evidence of post-transcriptional controls was also presented for antioxidant enzymes (15). In conclusion, our results ind icated that DR (restriction of energy intake with maintenance of essential nutrients) produced differential effects on liver SOD, CAT and GPx activity and LP level in male and female mice during development, suggesting sex-associated modulations of DR on antioxidant systems in developing animals. These modulations include elevation of SOD activity in male, and increase of CAT, GPx activity and reduction of LP in both male and female mice with a stronger effect in female animals compared to males. Since DR is considered to be potentially useful in extending lifespan of experimental animals, improving outcome of neurodegenerative diseases and producing protections against environmental chemical-induced toxicity, it is mandatory to investigate all possible physiological effects including those in developing animals. Our findings for the first time provide important 10

11 insights into the understanding of DR-related physiological implications in modulating antioxidant systems in developing animals and call for further investigations to address the mechanisms underlying DR-induced sex-associated effects on antioxidant systems. Furthermore, more attention should be paid to the potential different influence of DR on antioxidant systems when designing DR-related experiments for basic research, bioassays and/or toxicity studies by using developing animals, otherwise DR-induced different effects may lead to variable results in male and female developing animals, resulting in complicated interpretation of data. Acknowledgements This work was supported by funding from National Natural Science Foundation of China. 11

12 References 1. Adams JD Jr, and Odunze IN. Oxygen free radicals and Parkinson's disease. Free Radic Biol Med 10: , Aebi H. Catalase in vitro. Methods Enzymol 105: , Ames BN, Shigenaga MK, and Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 90: , Bachowski S, Kolaja KL, Xu Y, Ketcham CA, Stevenson DE, Walborg EF Jr, and Klaunig JE. Role of oxidative stress in the mechanism of dieldrin's hepatotoxicity. Ann Clin Lab Sci 27: , Berg TF, Breen PJ, Feuers RJ, Oriaku ET, Chen FX, and Hart RW. Acute toxicity of ganciclovir: effect of dietary restriction and chronobiology. Food Chem Toxicol 32:45-50, Cadenas S, Rojas C, Perez-Campo R, Lopez-Torres M, and Barja G. Caloric and carbohydrate restriction in the kidney: effects on free radical metabolism. Exp Gerontol 29:77-88, Chanda S, and Mehendale HM. Role of nutrition in the survival after hepatotoxic injury. Toxicology 111: , Chipalkatti S, De AK, and Aiyar AS. Effect of diet restriction on some biochemical parameters related to aging in mice. J Nutr 113: , Desai VG, Aidoo A, Li J, Lyn-Cook LE, Casciano DA, and Feuers RJ. Effects of bleomycin on liver antioxidant enzymes and the electron transport system from ad libitumfed and dietary-restricted female and male Fischer 344 rats. Nutr Cancer 36:42-51,

13 10. Desai VG, Weindruch R, Hart RW, and Feuers RJ. Influences of age and dietary restriction on gastrocnemius electron transport system activities in mice. Arch Biochem Biophys 333: , Feuers RJ, Weindruch R, and Hart RW. Caloric restriction, aging, and antioxidant enzymes. Mutat Res 295: , Fraga CG, Leibovitz BE, and Tappel AL. Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. Free Radic Biol Med 4: , Fridovich I. The biology of oxygen radicals. Science 201: , Grattagliano I, Vendemiale G, Caraceni P, Domenicali M, Nardo B, Cavallari A, Trevisani F, Bernardi M, and Altomare E. Starvation impairs antioxidant defense in fatty livers of rats fed a choline-deficient diet. J Nutr 130: , Hardmeier R, Hoeger H, Fang-Kircher S, Khoschsorur A, and Lubec G. Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice. Proc Natl Acad Sci USA 94: , Hunt CR, Sim JE, Sullivan SJ, Featherstone T, Golden W, Von Kapp-Herr C, Hock RA, Gomez RA, Parsian AJ, and Spitz DR. Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress. Cancer Res 58: , Janssen YM, Van Houten B, Borm PJ, and Mossman BT. Cell and tissue responses to oxidative damage. Lab Invest 6: ,

14 18. Keenan KP, Ballam GC, Dixit R, Soper KA, Laroque P, Mattson BA, Adams SP, and Coleman JB. The effects of diet, overfeeding and moderate dietary restriction on Sprague- Dawley rat survival, disease and toxicology. J Nutr 127:851S-856S, Koizumi A, Weindruch R, and Walford RL. Influences of dietary restriction and age on liver enzyme activities and lipid peroxidation in mice. J Nutr 117: , Lass A, Sohal BH, Weindruch R, Forster MJ, and Sohal RS. Caloric restriction prevents age-associated accrual of oxidative damage to mo use skeletal muscle mitochondria. Free Radic Biol Med 25: , Maiti S, and Chatterjee AK. Differential response of cellular antioxidant mechanism of liver and kidney to arsenic exposure and its relation to dietary protein deficiency. Environ Toxicol Pharmacol 8: , Marklund S, and Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47: , Martensson J. The effect of fasting on leukocyte and plasma glutathione and sulfur amino acid concentrations. Metabolism 35: , Mates M. Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 153:83-104, Mattson MP. Neuroprotective signaling and the aging brain: take away my food and let me run. Brain Res 886:47-53, Mote PL, Grizzle JM, Walford RL, and Spindler SR. Influence of age and caloric restriction on expression of hepatic genes for xenobiotic and oxygen metabolizing enzymes in the mouse. J Gerontol 46:B95-100,

15 27. O'Prey J, Ramsay S, Chambers I, and Harrison PR. Transcriptional up-regulation of the mouse cytosolic glutathione peroxidase gene in erythroid cells is due to a tissue-specific 3' enhancer containing functionally important CACC/GT motifs and binding sites for GATA and Ets transcription factors. Mol Cell Biol 13: , Paoletti F, and Mocali A. Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation. Methods Enzymol 186: , Ramaiah SK, Soni MG, Bucci TJ, and Mehendale HM. Diet restriction enhances compensatory liver tissue repair and survival following administration of lethal dose of thioacetamide. Toxicol Appl Pharmacol 150:12-21, Ramsey JJ, Harper ME, and Weindruch R. Restriction of energy intake, energy expenditure, and aging. Free Radic Biol Med 29: , Rao G, Xia E, Nadakavukaren MJ, and Richardson A. Effect of dietary restriction on the age-dependent changes in the expression of antioxidant enzymes in rat liver. J Nutr 120: , Reeves PG, Nielsen FH, and Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123: , Ross MH. Aging, nutrition and hepatic enzyme activity patterns in the rat. J Nutr 97: , Seo SJ, Kang SS, Cho G, Rho HM, and Jung G. C/EBP alpha and C/EBPbeta play similar roles in the transcription of the human Cu/Zn SOD gene. Gene 203:11-15, Seo SJ, Kim HT, Cho G, Rho HM, and Jung G. Sp1 and C/EBP-related factor regulate the transcription of human Cu/Zn SOD gene. Gene 178: ,

16 36. Shaikh ZA, Jordan SA, and Tang W. Protection against chronic cadmium toxicity by caloric restriction. Toxicology 133:93-103, Shimizu M, and Morita S. Effects of feeding and fasting on hepatolobular distribution of glutathione and cadmium-induced hepatotoxicity. Toxicology 75:97-107, Stohs, SJ. Oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Free Radic Biol Med 9:79-90, Tappel AL. Glutathione peroxidase and hydroperoxides. Methods Enzymol 52: , Yu BP. Aging and oxidative stress: modulation by dietary restriction. Free Radic Biol Med 21: , Weindruch R, Walford RL, Fligiel S, and Guthrie D. The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J Nutr 116: , Wendel A. Glutathione peroxidase. Methods Enzymol 77: , Wu A, Wan F, Sun X, and Liu Y. Effects of dietary restriction on growth, neurobehavior, and reproduction in developing Kunmin mice. Toxicol Sci 70: ,

17 Table 1. Composition of diets in each group Restriction level Ingredient 0% (Control) 20% DR 35% DR g/kg Protein Fat Carbohydrate Minerals mix 1, Vitamins mix 1, Fiber H 2 O The contents of indicated ingredients were adjusted so that intake of these nutrients would be constant. 2 Prepared as previously described (32). 17

18 Figure legends Figure 1. Body weight in male and female mice at 12 wk (A) and 24 wk (B) after DR. In each group, n=10. Values represent mean ± SEM. *, P < 0.05; **, P < Figure 2. Effect of DR on liver SOD activity in male and female mice. (A) 12 wk of DR; (B) 24 wk of DR. In each group, n=10. SOD, superoxide dismutase. Values represent mean ± SEM. *, P < 0.05; **, P < Figure 3. Effect of DR on liver CAT activity in male and female mice. (A) 12 wk of DR; (B) 24 wk of DR. In each group, n=10. CAT, catalase. Values represent mean ± SEM. *, P < 0.05; **, P < Figure 4. Effect of DR on liver GPx activity in male and female mice. (A) 12 wk of DR; (B) 24 wk of DR. In each group, n=10. GPx, glutathione peroxidase. Values represent mean ± SEM. *, P < 0.05; **, P < Figure 5. Effect of DR on liver LP level in male and female mice. (A) 12 wk of DR; (B) 24 wk of DR. In each group, n=10. LP, lipid peroxidation; MDA, malondialdehyde. Values represent mean ± SEM. **, P <

19 Fig

20 Fig

21 Fig

22 Fig

23 Fig