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Protective effect of dietary squalene supplementation on mitochondrial function in liver of aged rats
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Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 349–355 www.elsevier.com/locate/plefa
Protective effect of dietary squalene supplementation on mitochondrial function in liver of aged rats S. Buddhana, R. Sivakumara, N. Dhandapania,b, B. Ganesana,c, R. Anandanc, a
Department of Biochemistry, Vinayaka Mission’s Research Foundation (Deemed University), Ariyanoor, Salem-636308, Tamil Nadu, India b Department of Pharmaceutical Chemistry, R.V.S College of Pharmaceutical Sciences, Sulur, Coimbatore-641402, India c Biochemistry and Nutrition Division, Central Institute of Fisheries Technology, Matsyapuri (PO), Cochin-682029, India Received 19 December 2006; received in revised form 1 May 2007; accepted 1 May 2007
Abstract Mitochondria are an important intracellular source and target of reactive oxygen species. The life span of a species is thought to be determined, in part, by the rate of mitochondrial damage inflicted by oxygen free radicals during the course of normal cellular metabolism. In the present study, we have investigated the protective effect of squalene supplementation for 15 days and 30 days on energy status and antioxidant defense system in liver mitochondria of 18 young and 18 aged rats. The dietary supplementation of 2% squalene significantly minimized aging associated alterations in mitochondrial energy status by maintaining the activities of TCA cycle enzymes (isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase) and respiratory marker enzymes (NADH dehydrogenase and cytochrome-c-oxidase) at higher level in the liver mitochondria of aged rats compared with unsupplemented controls. It exerted an antioxidant effect by inhibiting mitochondrial lipid peroxidation (malondialdehyde) in liver of young and aged rats. Supplementation with squalene also maintained the mitochondrial antioxidant defense system at higher rate by increasing the level of reduced glutathione and the activities of glutathione-dependent antioxidant enzymes (glutathione peroxidase and glutathione-S-transferase) and antiperoxidative enzymes (superoxide dismutase and catalase) in the liver of young and aged rats. The results of this study provide evidence that dietary supplementation with squalene can improve liver mitochondrial function during aging and minimize the age-associated disorders in which reactive oxygen species are a major cause. r 2007 Elsevier Ltd. All rights reserved.
1. Introduction Aging is a lifelong biological phenomenon, which is the product of an interaction between genetic, environmental and lifestyle factors. It is generally accompanied by a gradual decline in biochemical and physiological functions of the most organs, ultimately leading to an increase in the susceptibility to age-associated disorders [1]. The free radical theory of aging proposes that aging occurs as a consequence of the deleterious effect of free radicals or reactive oxygen species (ROS) produced during the course of cellular metabolism [2]. The ROS Corresponding author. Tel.: +91 484 2666845; fax: +91 484 2668212. E-mail address: [email protected] (R. Anandan).
0952-3278/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.05.001
exert their oxidative action on many cellular components resulting in lipid peroxidation, protein fragmentation and DNA damage [3]. Free radicals also affect the equilibrium between pro-oxidants and antioxidants in biological systems, leading to modifications in genomes, proteins, carbohydrates, lipids, lipid peroxidation, and in inactivating antioxidant defense [4]. Mitochondria are an important intracellular source and target of reactive oxygen species. Mitochondrial aging is characterized by destruction of structural integrity of membranes, leading to a decline in mitochondrial membrane fluidity and activities of enzymes associated with membrane lipids [5]. As the activities of most enzymes are regulated by the physicochemical state of the lipid environment of the membrane, it seems likely that impaired mitochondrial
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membrane function brought about by aging could be related to free radical reactions such as lipid peroxidation, ROS and their metabolites generated by components of electron transport chain during mitochondrial respiration. This potential for self-destruction renders the mitochondrial membrane more vulnerable to oxidative damage than other cellular membranes [6]. Antioxidant nutrients play a significant role in the body’s defense against excess levels of free radicals and delay the onset of aging and age-associated degenerative diseases [7]. In particular, antioxidants that can be stored in cellular membranes may be potential candidates for prevention or treatment of disorders involving oxidative damage during disease progression. In recent times, there has been an increase in interest in drugs derived from marine sources. Squalene, a remarkable bioactive substance present in deep-sea shark liver oil in high quantities, belongs to a class of antioxidants called isoprenoids, which neutralize the harmful effects of excessive free radicals produced in the body [8]. It is an intermediate metabolite in cholesterol metabolism and is secreted in human sebum, where it protects the skin from ultraviolet (UV) radiation [9]. Squalene has been reported to possess antilipidemic, antioxidant and membrane stabilizing properties [8,10]. It has been used to treat cancer, skin disorders, cardiac ailments and liver diseases [11,12]. Several experimental investigations demonstrated the detoxifying activities of the squalene against diverse chemicals such as hexachlorobiphenyl, hexachlorobenzene, arsenic, theophylline, phenobarbital and strychnine [13–15]. It has also been reported to possess anticarcinogenic activity against several carcinogens, including azoxymethane-induced colon cancer [16] and nicotine-derived nitrosaminoketone-(NMK) induced lung carcinogenisis [17]. Scientific research and clinical trials have shown that squalene is safe as a dietary supplement in food and in capsules and no untoward incidents have been reported following the use of squalene [18]. Though these beneficial properties of squalene are promising and well studied, the protective effects of squalene on mitochondrial function during aging have not yet been explored. In the present study, the protective action of dietary squalene supplementation for 15 days and 30 days on mitochondrial function was investigated in liver of 36 rats by virtue of its antioxidant and membranestabilizing properties.
2. Materials and methods 2.1. Chemicals Epinephrine, isoprenaline, tetraethoxy propane and cholesterol were obtained from M/s. Sigma Chemical Company, St. Louis. MO, USA. Squalene (Specific
gravity: 0.853; Refractive index: 1.493; Saponification value: 30; Iodine value: 344; Boiling point: 240–245 1C) was prepared from the shark liver oil of Centrophorus sp. caught in the Andaman waters [8]. All the other chemicals used were of analytical grade. 2.2. Animals Male Wistar strain albino rats, weighing 120–150 g [18 young rats of 2–3 months old (mean age: 78.576.42 days)] and 350–400 g [18 aged rats of 20–25 months old (mean age: 697747.3 days)] were selected for the study. The animals were housed individually in polypropylene cages under hygienic and standard environmental conditions (2872 1C, humidity 60–70%, 12 h light/dark cycle). The animals were allowed a standard diet [M/s Hindustan Lever Foods, Bangalore, India] and water ad libitum. The squalene content in the standard diet was 1.42 mg/kg. [Average food consumption by young rats: 12.470.97 g/day and by aged rats: 16.271.21 g/day]. The experiment was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India. 2.3. Experimental protocol Seven days after acclimatization, the animals were divided into two major groups: Group I consisted of 18 normal young rats and Group II consisted of 18 normal aged rats. Each group was further sub-divided into three groups (six rats each): one control group (Group Ia and Group IIa) and two experimental groups based on the duration of supplementation of squalene at 2% level along with feed 15 days (Group Ib and Group IIb) and 30 days (Group Ic and Group IIc). [Average squalene consumption by young rats: 0.2370.018 g/day and by aged rats: 0.3470.023 g/day]. On completion of 15 and 30 days of squalene supplementation, the animals were killed and the liver was excised immediately. The hepatic mitochondria were isolated by the method of Johnson and Lardy [19] and used for the determination of TCA cycle enzymes, respiratory marker enzymes, lipid peroxidation, reduced glutathione and antioxidant enzymes. 2.4. Biochemical assays Isocitrate dehydrogenase (EC 1.1.1.42) activity was assayed by the method of Bell and Baron [20] and aketoglutarate dehydrogenase (EC 1.2.4.2) activity by the method of Reed and Mukherjee [21]. The method described Slater and Bonner [22] was followed for estimation of succinate dehydrogenase (EC 1.3.99.1) activity and the activity of malate dehydrogenase (EC 1.1.1.37) was assayed by the method of Mehler et al.
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[23]. NADH dehydrogenase (EC 1.6.99.3) activity was determined by the method of Minakami et al. [24] and cytochrome-c-oxidase (EC 1.9.3.1) activity by the method of Pearl et al. [25]. Lipid peroxide content was determined by the thiobarbituric acid (TBA) reaction as described by Ohkawa et al. [26] and the non-enzymic antioxidant reduced glutathione was measured by the method of Ellman [27]. The method described by Paglia and Valentine [28] was followed for the estimation of glutathione peroxidase (EC 1.11.1.9) activity and the activity of glutathione-S-transferase (EC 2.5.1.18) was assayed by the method of Habig et al. [29]. Catalase (EC 1.11.1.6) activity was determined by the method of Takahara et al. [30] and superoxide dismutase (EC 1.15.1.1) activity by the method of Misra and Fridovich [31]. The level of ATP content in the liver tissue was determined by the method of Ryder [32] using Shimadzu LC 10 ATvp HPLC. 2.5. Statistical analysis Results are expressed as mean 7 SD. Multiple comparisons of the significant analysis of variance were performed by Duncan’s multiple comparison test. A Pvalue o0.05 was considered as statistically significant. All data were analyzed with the aid of a statistical package program, SPSS 10.0 for Windows.
3. Results and discussion Aging is a complex biological process that leads to gradual loss of ability of an individual to maintain homeostasis. Modern science has made tremendous attempts to explain/understand the aging process. Liver is a crucial organ in the body and the disorders associated with this are many and varied during aging. The focus of the present investigation was to determine
the beneficial effects of the highly lipophilic antioxidant nutrient squalene on hepatic mitochondrial energy status, lipid peroxidation and antioxidant defense system during aging in rats. A significant (po0.05) decline was noticed in the level of ATP (Fig. 1) and in activities of TCA cycle enzymes (isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase) and respiratory marker enzymes (NADH dehydrogenase and cytochrome-c-oxidase) (Table 1) in the liver mitochondria of aged control (Group IIa) compared to that of young control (Group Ia). This suggested that the mitochondrial oxidative phosphorylation was operating at a lower level in aged rats despite the higher energy demand during aging. This observation is in accordance with an earlier reported study [33]. NADH/NAD ratio has been reported to rise in the aged liver mitochondria due to prolonged metabolic overload on cells [34]. This in turn may result in diminution in the activities of TCA enzymes by the mechanism of mass action, as observed in the present study. During aging, decreased NADPH and NADH oxidation accelerates the inactivation of cyt-P450 to cyt-p420 and is associated with destruction of nucleus, mitochondria and endoplasmic reticulum [33,35]. Alterations in the status of respiratory components, phosphorylative activity, cytochrome-c-oxidase activity and hepatic adenylate charge level have been reported in ageassociated disorders [36]. The rate of mitochondrial superoxide anion radical (O2 ) and hydrogen peroxide (H2O2) generation increases in the later part of life [37]. This in turn affects the balance between pro-oxidants and antioxidants in biological systems leading to modifications in vital biomolecules. Accumulation of these free radicals may also lead to increase of membrane damage and impair mitochondrial functions as well as ATP synthesis. In the present study, the 30 days administration of squalene at 2% maintained the level of ATP and the
a
Aged
30d
ATP
b
15d b
Control
c
30d Young
351
d
15d a
Control 0
1
2
3 4 µmol/g wet tissue ATP
5
6
7
Fig. 1. Effect of dietary squalene supplementation on ATP content in the liver tissue of young and aged rats. Results are mean7SD for six rats. Values that have a different superscript letter (a,b,c,d,e) differ significantly with each other (Po0.05; Duncan’s multiple range test).
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Table 1 Effect of dietary squalene supplementation on the activities of TCA cycle enzymes [isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase and respiratory marker enzymes [NADH dehydrogenase and cytochrome-c-oxidase] in the liver mitochondria of young and aged rats Parameters
Young
Aged
Group Ia Control
Group Ib 15d
Group Ic 30d
Group IIa Control
Group IIb 15d
Group IIc 30d
TCA cycle enzymes ICDH a-KDH SDH MDH
832765.1a 75.775.22a 38.671.85a 378724.7a
859768.5a 92.175.74b 46.772.51b 411721.4b
873771.2a 10476.48c 52.972.76c 425726.2b
678752.7b 49.572.76d 21.470.98d 263712.1c
762756.9c 63.873.47e 29.671.21e 305718.0d
818762.3a,c 72.574.83f 35.271.79f 349719.6e
Respiratory marker enzymes NADH dehydrogenase Cytochrome-c-oxidase
38.371.82a 4.15 10 270.29a
51.572.76b 5.03 10 270.36b
57.272.54c 5.42 10 270.34c
24.371.42d 2.98 10 270.12d
30.771.65e 3.49 10 270.22e
34.271.74f 3.98 10 270.27a
Results are mean7SD for six rats. Values expressed: Isocitrate dehydrogenase, nmol of a-ketoglutarate formed/min/mg protein; a-Ketoglutarate dehydrogenase, nmol of ferrocyanide formed/min/mg protein; Succinate dehydrogenase, mmol of succinate oxidized/min/mg protein; Malate dehydrogenase, mmol of NADH oxidized/min/mg protein; NADH-dehydrogenase, mmol of NADH oxidized/min/mg protein; Cytochrome-coxidase, O.D/min/mg protein. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test).
a
Aged
30d
Lipid peroxides b
15d
c
Control d
Young
30d
e
15d
f
Control 0
0.5
1
1.5
2
2.5
nmol malondialdehyde released/mg protein Fig. 2. Effect of dietary squalene supplementation on lipid peroxidation in the liver mitochondria of young and aged rats. Results are mean 7SD for six rats. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test).
activities of TCA cycle enzymes and respiratory marker enzymes significantly (po0.05) at near normalcy (in Group IIc rats) compared to control (Group IIa rats), reflecting its ability to maintain the function of the liver mitochondria at near normal status. Continuous oxygen supply is an inevitable requirement for the production of ATP in the mitochondria for the normal function of the liver. Oxidative damage to mitochondria is a major cause of aging. Mitochondria are a potential target of injury by oxygen radicals, and an alteration in mitochondrial membrane function is an important component of oxidative stress in cells. Lipid peroxidation in vivo has been identified as a basic deteriorative reaction in the aging process [3,38]. In the present study, the level of lipid peroxidation was significantly (po0.05) higher in the liver mitochondria of aged control rats (Group IIa) compared to young control rats (Group Ia) (Fig. 2). This was paralleled by a significant (po0.05) decline in
the level of reduced glutathione (Fig. 3) and the activities of glutathione-dependent antioxidant enzymes and antiperoxidative enzymes in the liver mitochondria of aged control rats (Group IIa) compared to young control rats (Group Ia) (Table 2). The reduction noticed in the level of GSH was either due to increased degradation or decreased synthesis of glutathione and the decreased availability of GSH might have resulted in the lowered activities of GPx and GST. Increased generation of reactive oxygen radicals such as superoxide and hydrogen peroxide is associated with the inhibition of SOD and CAT activities [8]. The intracellular calcium concentration has been reported to rise in aging [39]. The intracellular Ca2+ is an inducer of phospholipase A2, which degrades membrane phospholipids. The free radicals produced as a result of this lipid peroxidation may attack the RNA polymerase encoding these antioxidant enzymes.
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GSH a
Aged
30d b
15d c
Control
d
Young
30d e
15d f
Control 0
1
2
3
4
5
6
7
nmol/g wet tissue GSH Fig. 3. Effect of dietary squalene supplementation on reduced glutathione (GSH) in the liver mitochondria of young and aged rats. Results are mean 7SD for six rats. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test). Table 2 Effect of dietary squalene supplementation on the activities of glutathione-dependent antioxidant enzymes (glutathione peroxidase [GPX] and glutathione-S-transferase [GST]) and antiperoxidative enzymes (superoxide dismutase [SOD] and catalase [CAT]) in the liver mitochondria of young and aged rats Parameters
Young
Aged
Group Ia Control
Group Ib 15d
Group Ic 30d
Group IIa Control
Group IIb 15d
Group IIc 30d
Glutathione-dependent antioxidant enzymes GPX GST
21.871.25a 25167168a
26.971.43b 27257181b
32.171.37c 28327173b
12.271.14d 16537123c
16.771.41e 19247118d
19.271.29f 23127152e
Antiperoxidative enzymes SOD CAT
35.272.18a 98.778.32a
42.372.35b 10877.85b
47.872.15c 124710.29c
21.571.76d 68.574.92d
27.771.94e 79.178.12e
33.572.05a 92.477.96a
Results are mean7SD for six animals. Values expressed: GPx, nmol GSH oxidized min 1 mg 1 protein; GST, mmol 1-chloro-2,4-dinitrobenzene conjugate formed min 1 mg 1 protein; CAT, nmol H2O2 decomposed min 1 mg 1 protein; SOD, one unit of the SOD activity is the amount of protein required to give 50% inhibition of epinephrine autoxidation. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test).
In the present study, 30 days administration of squalene [0.3470.023 g/day] was associated with significant (po0.05) decrease in the aging-induced lipid peroxidation and maintained the level of reduced glutathione and the activities of antioxidant enzymes at near normalcy in the liver mitochondria of Group IIc rats. This may be attributed to the bioactivity of squalene to directly react with various ROS as well as its ability to interfere with oxidation processes in the lipid and in the aqueous cellular compartment. The isoprenoid moiety of squalene enables this biomolecule to prevent Fenton-type reactions by chelating free iron. Thus the free radical scavenging action of squalene [9] might have resulted in the preservation of cellular
viability. Highly lipophilic antioxidant molecules such as vitamin E have been reported to trap free radicals by electron donating and radical capture mechanisms, thereby blocking the lipid peroxidation chain reaction [40]. Squalene is more potent than vitamin E as a free radical scavenger [9] and as a stabilizer of cellular and subcellular membranes [41]. It is possible that stabilization of cellular membranes by squalene, particularly the mitochondrial membranes, may prolong the viability of hepatocytes from free radicals mediated peroxidative damage. The results of the present study indicate that dietary supplementation with the antioxidant squalene at 2% level [i.e. average squalene consumption: 0.3470.023 g/
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day] was effective in improving mitochondrial energy status and antioxidant defense system in aged rats, and that 30 days supplementation was more effective than 15 days. It was concluded that dietary squalene could be an effective therapeutic agent in treatment of age-associated disorders where free radicals are a major causative factor.
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Prostaglandins, Leukotrienes and Essential Fatty Acids 76 (2007) 349–355 www.elsevier.com/locate/plefa
Protective effect of dietary squalene supplementation on mitochondrial function in liver of aged rats S. Buddhana, R. Sivakumara, N. Dhandapania,b, B. Ganesana,c, R. Anandanc, a
Department of Biochemistry, Vinayaka Mission’s Research Foundation (Deemed University), Ariyanoor, Salem-636308, Tamil Nadu, India b Department of Pharmaceutical Chemistry, R.V.S College of Pharmaceutical Sciences, Sulur, Coimbatore-641402, India c Biochemistry and Nutrition Division, Central Institute of Fisheries Technology, Matsyapuri (PO), Cochin-682029, India Received 19 December 2006; received in revised form 1 May 2007; accepted 1 May 2007
Abstract Mitochondria are an important intracellular source and target of reactive oxygen species. The life span of a species is thought to be determined, in part, by the rate of mitochondrial damage inflicted by oxygen free radicals during the course of normal cellular metabolism. In the present study, we have investigated the protective effect of squalene supplementation for 15 days and 30 days on energy status and antioxidant defense system in liver mitochondria of 18 young and 18 aged rats. The dietary supplementation of 2% squalene significantly minimized aging associated alterations in mitochondrial energy status by maintaining the activities of TCA cycle enzymes (isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase) and respiratory marker enzymes (NADH dehydrogenase and cytochrome-c-oxidase) at higher level in the liver mitochondria of aged rats compared with unsupplemented controls. It exerted an antioxidant effect by inhibiting mitochondrial lipid peroxidation (malondialdehyde) in liver of young and aged rats. Supplementation with squalene also maintained the mitochondrial antioxidant defense system at higher rate by increasing the level of reduced glutathione and the activities of glutathione-dependent antioxidant enzymes (glutathione peroxidase and glutathione-S-transferase) and antiperoxidative enzymes (superoxide dismutase and catalase) in the liver of young and aged rats. The results of this study provide evidence that dietary supplementation with squalene can improve liver mitochondrial function during aging and minimize the age-associated disorders in which reactive oxygen species are a major cause. r 2007 Elsevier Ltd. All rights reserved.
1. Introduction Aging is a lifelong biological phenomenon, which is the product of an interaction between genetic, environmental and lifestyle factors. It is generally accompanied by a gradual decline in biochemical and physiological functions of the most organs, ultimately leading to an increase in the susceptibility to age-associated disorders [1]. The free radical theory of aging proposes that aging occurs as a consequence of the deleterious effect of free radicals or reactive oxygen species (ROS) produced during the course of cellular metabolism [2]. The ROS Corresponding author. Tel.: +91 484 2666845; fax: +91 484 2668212. E-mail address: [email protected] (R. Anandan).
0952-3278/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.05.001
exert their oxidative action on many cellular components resulting in lipid peroxidation, protein fragmentation and DNA damage [3]. Free radicals also affect the equilibrium between pro-oxidants and antioxidants in biological systems, leading to modifications in genomes, proteins, carbohydrates, lipids, lipid peroxidation, and in inactivating antioxidant defense [4]. Mitochondria are an important intracellular source and target of reactive oxygen species. Mitochondrial aging is characterized by destruction of structural integrity of membranes, leading to a decline in mitochondrial membrane fluidity and activities of enzymes associated with membrane lipids [5]. As the activities of most enzymes are regulated by the physicochemical state of the lipid environment of the membrane, it seems likely that impaired mitochondrial
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membrane function brought about by aging could be related to free radical reactions such as lipid peroxidation, ROS and their metabolites generated by components of electron transport chain during mitochondrial respiration. This potential for self-destruction renders the mitochondrial membrane more vulnerable to oxidative damage than other cellular membranes [6]. Antioxidant nutrients play a significant role in the body’s defense against excess levels of free radicals and delay the onset of aging and age-associated degenerative diseases [7]. In particular, antioxidants that can be stored in cellular membranes may be potential candidates for prevention or treatment of disorders involving oxidative damage during disease progression. In recent times, there has been an increase in interest in drugs derived from marine sources. Squalene, a remarkable bioactive substance present in deep-sea shark liver oil in high quantities, belongs to a class of antioxidants called isoprenoids, which neutralize the harmful effects of excessive free radicals produced in the body [8]. It is an intermediate metabolite in cholesterol metabolism and is secreted in human sebum, where it protects the skin from ultraviolet (UV) radiation [9]. Squalene has been reported to possess antilipidemic, antioxidant and membrane stabilizing properties [8,10]. It has been used to treat cancer, skin disorders, cardiac ailments and liver diseases [11,12]. Several experimental investigations demonstrated the detoxifying activities of the squalene against diverse chemicals such as hexachlorobiphenyl, hexachlorobenzene, arsenic, theophylline, phenobarbital and strychnine [13–15]. It has also been reported to possess anticarcinogenic activity against several carcinogens, including azoxymethane-induced colon cancer [16] and nicotine-derived nitrosaminoketone-(NMK) induced lung carcinogenisis [17]. Scientific research and clinical trials have shown that squalene is safe as a dietary supplement in food and in capsules and no untoward incidents have been reported following the use of squalene [18]. Though these beneficial properties of squalene are promising and well studied, the protective effects of squalene on mitochondrial function during aging have not yet been explored. In the present study, the protective action of dietary squalene supplementation for 15 days and 30 days on mitochondrial function was investigated in liver of 36 rats by virtue of its antioxidant and membranestabilizing properties.
2. Materials and methods 2.1. Chemicals Epinephrine, isoprenaline, tetraethoxy propane and cholesterol were obtained from M/s. Sigma Chemical Company, St. Louis. MO, USA. Squalene (Specific
gravity: 0.853; Refractive index: 1.493; Saponification value: 30; Iodine value: 344; Boiling point: 240–245 1C) was prepared from the shark liver oil of Centrophorus sp. caught in the Andaman waters [8]. All the other chemicals used were of analytical grade. 2.2. Animals Male Wistar strain albino rats, weighing 120–150 g [18 young rats of 2–3 months old (mean age: 78.576.42 days)] and 350–400 g [18 aged rats of 20–25 months old (mean age: 697747.3 days)] were selected for the study. The animals were housed individually in polypropylene cages under hygienic and standard environmental conditions (2872 1C, humidity 60–70%, 12 h light/dark cycle). The animals were allowed a standard diet [M/s Hindustan Lever Foods, Bangalore, India] and water ad libitum. The squalene content in the standard diet was 1.42 mg/kg. [Average food consumption by young rats: 12.470.97 g/day and by aged rats: 16.271.21 g/day]. The experiment was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India. 2.3. Experimental protocol Seven days after acclimatization, the animals were divided into two major groups: Group I consisted of 18 normal young rats and Group II consisted of 18 normal aged rats. Each group was further sub-divided into three groups (six rats each): one control group (Group Ia and Group IIa) and two experimental groups based on the duration of supplementation of squalene at 2% level along with feed 15 days (Group Ib and Group IIb) and 30 days (Group Ic and Group IIc). [Average squalene consumption by young rats: 0.2370.018 g/day and by aged rats: 0.3470.023 g/day]. On completion of 15 and 30 days of squalene supplementation, the animals were killed and the liver was excised immediately. The hepatic mitochondria were isolated by the method of Johnson and Lardy [19] and used for the determination of TCA cycle enzymes, respiratory marker enzymes, lipid peroxidation, reduced glutathione and antioxidant enzymes. 2.4. Biochemical assays Isocitrate dehydrogenase (EC 1.1.1.42) activity was assayed by the method of Bell and Baron [20] and aketoglutarate dehydrogenase (EC 1.2.4.2) activity by the method of Reed and Mukherjee [21]. The method described Slater and Bonner [22] was followed for estimation of succinate dehydrogenase (EC 1.3.99.1) activity and the activity of malate dehydrogenase (EC 1.1.1.37) was assayed by the method of Mehler et al.
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[23]. NADH dehydrogenase (EC 1.6.99.3) activity was determined by the method of Minakami et al. [24] and cytochrome-c-oxidase (EC 1.9.3.1) activity by the method of Pearl et al. [25]. Lipid peroxide content was determined by the thiobarbituric acid (TBA) reaction as described by Ohkawa et al. [26] and the non-enzymic antioxidant reduced glutathione was measured by the method of Ellman [27]. The method described by Paglia and Valentine [28] was followed for the estimation of glutathione peroxidase (EC 1.11.1.9) activity and the activity of glutathione-S-transferase (EC 2.5.1.18) was assayed by the method of Habig et al. [29]. Catalase (EC 1.11.1.6) activity was determined by the method of Takahara et al. [30] and superoxide dismutase (EC 1.15.1.1) activity by the method of Misra and Fridovich [31]. The level of ATP content in the liver tissue was determined by the method of Ryder [32] using Shimadzu LC 10 ATvp HPLC. 2.5. Statistical analysis Results are expressed as mean 7 SD. Multiple comparisons of the significant analysis of variance were performed by Duncan’s multiple comparison test. A Pvalue o0.05 was considered as statistically significant. All data were analyzed with the aid of a statistical package program, SPSS 10.0 for Windows.
3. Results and discussion Aging is a complex biological process that leads to gradual loss of ability of an individual to maintain homeostasis. Modern science has made tremendous attempts to explain/understand the aging process. Liver is a crucial organ in the body and the disorders associated with this are many and varied during aging. The focus of the present investigation was to determine
the beneficial effects of the highly lipophilic antioxidant nutrient squalene on hepatic mitochondrial energy status, lipid peroxidation and antioxidant defense system during aging in rats. A significant (po0.05) decline was noticed in the level of ATP (Fig. 1) and in activities of TCA cycle enzymes (isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase) and respiratory marker enzymes (NADH dehydrogenase and cytochrome-c-oxidase) (Table 1) in the liver mitochondria of aged control (Group IIa) compared to that of young control (Group Ia). This suggested that the mitochondrial oxidative phosphorylation was operating at a lower level in aged rats despite the higher energy demand during aging. This observation is in accordance with an earlier reported study [33]. NADH/NAD ratio has been reported to rise in the aged liver mitochondria due to prolonged metabolic overload on cells [34]. This in turn may result in diminution in the activities of TCA enzymes by the mechanism of mass action, as observed in the present study. During aging, decreased NADPH and NADH oxidation accelerates the inactivation of cyt-P450 to cyt-p420 and is associated with destruction of nucleus, mitochondria and endoplasmic reticulum [33,35]. Alterations in the status of respiratory components, phosphorylative activity, cytochrome-c-oxidase activity and hepatic adenylate charge level have been reported in ageassociated disorders [36]. The rate of mitochondrial superoxide anion radical (O2 ) and hydrogen peroxide (H2O2) generation increases in the later part of life [37]. This in turn affects the balance between pro-oxidants and antioxidants in biological systems leading to modifications in vital biomolecules. Accumulation of these free radicals may also lead to increase of membrane damage and impair mitochondrial functions as well as ATP synthesis. In the present study, the 30 days administration of squalene at 2% maintained the level of ATP and the
a
Aged
30d
ATP
b
15d b
Control
c
30d Young
351
d
15d a
Control 0
1
2
3 4 µmol/g wet tissue ATP
5
6
7
Fig. 1. Effect of dietary squalene supplementation on ATP content in the liver tissue of young and aged rats. Results are mean7SD for six rats. Values that have a different superscript letter (a,b,c,d,e) differ significantly with each other (Po0.05; Duncan’s multiple range test).
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Table 1 Effect of dietary squalene supplementation on the activities of TCA cycle enzymes [isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase and respiratory marker enzymes [NADH dehydrogenase and cytochrome-c-oxidase] in the liver mitochondria of young and aged rats Parameters
Young
Aged
Group Ia Control
Group Ib 15d
Group Ic 30d
Group IIa Control
Group IIb 15d
Group IIc 30d
TCA cycle enzymes ICDH a-KDH SDH MDH
832765.1a 75.775.22a 38.671.85a 378724.7a
859768.5a 92.175.74b 46.772.51b 411721.4b
873771.2a 10476.48c 52.972.76c 425726.2b
678752.7b 49.572.76d 21.470.98d 263712.1c
762756.9c 63.873.47e 29.671.21e 305718.0d
818762.3a,c 72.574.83f 35.271.79f 349719.6e
Respiratory marker enzymes NADH dehydrogenase Cytochrome-c-oxidase
38.371.82a 4.15 10 270.29a
51.572.76b 5.03 10 270.36b
57.272.54c 5.42 10 270.34c
24.371.42d 2.98 10 270.12d
30.771.65e 3.49 10 270.22e
34.271.74f 3.98 10 270.27a
Results are mean7SD for six rats. Values expressed: Isocitrate dehydrogenase, nmol of a-ketoglutarate formed/min/mg protein; a-Ketoglutarate dehydrogenase, nmol of ferrocyanide formed/min/mg protein; Succinate dehydrogenase, mmol of succinate oxidized/min/mg protein; Malate dehydrogenase, mmol of NADH oxidized/min/mg protein; NADH-dehydrogenase, mmol of NADH oxidized/min/mg protein; Cytochrome-coxidase, O.D/min/mg protein. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test).
a
Aged
30d
Lipid peroxides b
15d
c
Control d
Young
30d
e
15d
f
Control 0
0.5
1
1.5
2
2.5
nmol malondialdehyde released/mg protein Fig. 2. Effect of dietary squalene supplementation on lipid peroxidation in the liver mitochondria of young and aged rats. Results are mean 7SD for six rats. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test).
activities of TCA cycle enzymes and respiratory marker enzymes significantly (po0.05) at near normalcy (in Group IIc rats) compared to control (Group IIa rats), reflecting its ability to maintain the function of the liver mitochondria at near normal status. Continuous oxygen supply is an inevitable requirement for the production of ATP in the mitochondria for the normal function of the liver. Oxidative damage to mitochondria is a major cause of aging. Mitochondria are a potential target of injury by oxygen radicals, and an alteration in mitochondrial membrane function is an important component of oxidative stress in cells. Lipid peroxidation in vivo has been identified as a basic deteriorative reaction in the aging process [3,38]. In the present study, the level of lipid peroxidation was significantly (po0.05) higher in the liver mitochondria of aged control rats (Group IIa) compared to young control rats (Group Ia) (Fig. 2). This was paralleled by a significant (po0.05) decline in
the level of reduced glutathione (Fig. 3) and the activities of glutathione-dependent antioxidant enzymes and antiperoxidative enzymes in the liver mitochondria of aged control rats (Group IIa) compared to young control rats (Group Ia) (Table 2). The reduction noticed in the level of GSH was either due to increased degradation or decreased synthesis of glutathione and the decreased availability of GSH might have resulted in the lowered activities of GPx and GST. Increased generation of reactive oxygen radicals such as superoxide and hydrogen peroxide is associated with the inhibition of SOD and CAT activities [8]. The intracellular calcium concentration has been reported to rise in aging [39]. The intracellular Ca2+ is an inducer of phospholipase A2, which degrades membrane phospholipids. The free radicals produced as a result of this lipid peroxidation may attack the RNA polymerase encoding these antioxidant enzymes.
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353
GSH a
Aged
30d b
15d c
Control
d
Young
30d e
15d f
Control 0
1
2
3
4
5
6
7
nmol/g wet tissue GSH Fig. 3. Effect of dietary squalene supplementation on reduced glutathione (GSH) in the liver mitochondria of young and aged rats. Results are mean 7SD for six rats. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test). Table 2 Effect of dietary squalene supplementation on the activities of glutathione-dependent antioxidant enzymes (glutathione peroxidase [GPX] and glutathione-S-transferase [GST]) and antiperoxidative enzymes (superoxide dismutase [SOD] and catalase [CAT]) in the liver mitochondria of young and aged rats Parameters
Young
Aged
Group Ia Control
Group Ib 15d
Group Ic 30d
Group IIa Control
Group IIb 15d
Group IIc 30d
Glutathione-dependent antioxidant enzymes GPX GST
21.871.25a 25167168a
26.971.43b 27257181b
32.171.37c 28327173b
12.271.14d 16537123c
16.771.41e 19247118d
19.271.29f 23127152e
Antiperoxidative enzymes SOD CAT
35.272.18a 98.778.32a
42.372.35b 10877.85b
47.872.15c 124710.29c
21.571.76d 68.574.92d
27.771.94e 79.178.12e
33.572.05a 92.477.96a
Results are mean7SD for six animals. Values expressed: GPx, nmol GSH oxidized min 1 mg 1 protein; GST, mmol 1-chloro-2,4-dinitrobenzene conjugate formed min 1 mg 1 protein; CAT, nmol H2O2 decomposed min 1 mg 1 protein; SOD, one unit of the SOD activity is the amount of protein required to give 50% inhibition of epinephrine autoxidation. Values that have a different superscript letter (a,b,c,d,e,f) differ significantly with each other (Po0.05; Duncan’s multiple range test).
In the present study, 30 days administration of squalene [0.3470.023 g/day] was associated with significant (po0.05) decrease in the aging-induced lipid peroxidation and maintained the level of reduced glutathione and the activities of antioxidant enzymes at near normalcy in the liver mitochondria of Group IIc rats. This may be attributed to the bioactivity of squalene to directly react with various ROS as well as its ability to interfere with oxidation processes in the lipid and in the aqueous cellular compartment. The isoprenoid moiety of squalene enables this biomolecule to prevent Fenton-type reactions by chelating free iron. Thus the free radical scavenging action of squalene [9] might have resulted in the preservation of cellular
viability. Highly lipophilic antioxidant molecules such as vitamin E have been reported to trap free radicals by electron donating and radical capture mechanisms, thereby blocking the lipid peroxidation chain reaction [40]. Squalene is more potent than vitamin E as a free radical scavenger [9] and as a stabilizer of cellular and subcellular membranes [41]. It is possible that stabilization of cellular membranes by squalene, particularly the mitochondrial membranes, may prolong the viability of hepatocytes from free radicals mediated peroxidative damage. The results of the present study indicate that dietary supplementation with the antioxidant squalene at 2% level [i.e. average squalene consumption: 0.3470.023 g/
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day] was effective in improving mitochondrial energy status and antioxidant defense system in aged rats, and that 30 days supplementation was more effective than 15 days. It was concluded that dietary squalene could be an effective therapeutic agent in treatment of age-associated disorders where free radicals are a major causative factor.
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