To.uicology Letters, 63 ( 1992) 9 l-96 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$ 5.00
91
TOXLET 02793
Dietary restriction decreases thiobarbituric acidreactive substances generation in the small intestine and in the liver ofyoung rats
R. Albrecht, M.A. Pelissier, S. Atteba and M. Smaili Conservatoire Nutionaldes Arts et M&iers (C.N.h.M,l,
Laboratoire de Siologie, Paris (France)
(Received 18 May 1992) (Revision received 15 July 1992) (Accepted 16 July 1992) Key wordst Diet restriction; Thiobarbituric Liver; Rat
acid-reactive
substances;
Antioxidant
enzymes; Intestine;
SUMMARY This study investigated the influence of dietary restriction on thiobarbituric acid-reactive substances (TBARS) contents and on the intracellular antioxidant defense system in the small intestine or liver of young rats. Four weeks of diet restriction (-40%) lowered the TBARS level in both organs. No variations were found for the superoxide dismutase and catalase activities; only liver seleno-dependent glutathione peroxidase was enhanced by the restriction. The protection appeared more marked in the intestine than in the liver, and would be dependent on glutathione concentration.
INTRODUCTION
It is recognized that dietary restriction without essential nutrient deficiency extends both the maximum and mean life-span in rodents [l]. Furthermore, underfeeding delays a variety of age-related pathological changes [2]. Harman (31 proposed an hypothesis that a major cause of the aging process is accumulation of free radical: induced damage occurring under normal aerobic conditions. Cutler [4] considered the possibility that active oxygen species could react with the genetic apparatus of cells involved in regulatory functions of the whole organism; this ‘dysdifferentiation’ could act as a primary aging process. Sohal [5] postulated that oxidative stress increases
Correspondence to: R. Albrecht, Conservatoire National des Arts et Metiers (C.N.A.M.), Laboratoire de Biologie, 2 rue Con& 75003 Paris, France.
92
during aging in a programmed fashion and plays a physiological role in the sequential expression of genes associated with development and aging. Evidence substantiating the interactions of dietary restriction with oxidative damage at the levels of genomic integrity was obtained by recent works [6,7]. Free radicals are known to preferentially attack lipids in cell membrane structures [8]. Chilpakatti et al. [9] were the first to show a decrease in thiobarbituric acid-reactive peroxide in liver homonogenates of food-restricted mice. Since then the available data in the literature has been supportive of the notion that dietary restriction is probably the most effective means of modulating the aging processes by lowering oxidative stress [2,10]. However, almost all studies concern changes in the liver. Also of particular interest in this respect are the activities of the gastrointestinal tract, which is a major route for entering xenobiotics into the body, because oxygen-derived free radicals are an important component of injury in ischemia [ 11,121, inflammatory bowel disease [ 131, gastric ulceration [ 141 and necrotizing enterocolitis [ 14,15]. The present study was designed to investigate the influence of short-term dietary restriction on TBARS content and on the intracellular antioxidant defence system in the small intestine or in the liver of young rats. MATERIALS
AND METHODS
Weanling male Sprague-Dawley rats (n = 36) were divided into two groups: controls, fed ad libitum a well-balanced diet [ 161over 4 weeks; restricted animals, fed 60% of the same diet. Rats were killed after overnight fasting. Livers were perfused in situ with ice-cold saline, rapidly excised and weighed. They were homogenized in 5 ~01s. of 10 mM Tris-HCl buffer containing 150 mM KCl, I mM dithioerythritol (DTET), 0.25 mM phenylmethylsulphonyl fluoride (pH 7.4). The small intestine was flushed with saline to remove mucus and food residues, slit open and the mucosae were scraped with the blunt edge of a glass slide then washed three times by resuspending in 10 mM Tris-HCl, 150 mM KCl, 1 mM EDTA, 1 mM DTET (pH 7.4) and recentrifuged for 5 min at 700 x g. The remaining pellet was homogenized in 5 ~01s. of the same buffer as the liver. Protein contents were determined according to the method of Lowry et al. 1171.We used a modified method for measuring TBA-reactive material, initially described by Ohkawa et al. [ 181and Kikugawa et al. [ 191.Placed in a test tube were: liver or intestine homogenates, 0.45 mM BHT in glacial acetic acid, 0.25 mM EDTA, 7.6 mM sodium dodecyl sulphate, 2 mM t-BuOOH in acetic acid. Then 1.8 mM phosphotungstic acid and 6.8 M HC1 (all the values were final concentrations) were added. After waiting at +4”C for 30 min, the mixture was centrifuged at 5000 x g for 15 min. Twelve mM TBA (in 20 mM Tris-HCl buffer, pH 7.0) was added to an aliquot of the supernatant (final pH 3.5) and heated at 100°C for 45 min. After cooling, the mixture was extracted with 3-5 ml of n-butanol. The amount of red pigment produced was determined by absorbance at 532 nm; 1,1,3,3_tetraethoxypropane was used as an external standard. The activity of superoxide dismutases (SOD) was assayed by the method of Oberley and Spitz 1203.One unit of SOD was
93
defined as the amount which produced 50% inhibition of the nitrobluetetrazolium absorbance. Catalase activity was measured according to Luck [21]. Selenium-dependent glutathione peroxidase (Se-GPx) was estimated by the method of Burk et al. [22], using hydrogen peroxide as the substrate. Nonprotein sulphydryl groups (mostly reduced glutathione, GSH) were determined by the method of Sedlack and Lindsay 1231.Statistical comparisons were made using analysis of variance (ANOVA). RESULTS AND DISCUSSION
Data on body weight, glutathione and TBARS levels in small intestine and liver are shown in Table I. After 4 weeks, the diet-restricted rats showed a loss in body weight of the magnitude of 29%. Glutathione is the major nonprotein thiol component found in all types of living cells and plays a crucial role in the detoxification of reactive intermediates. Synthesized rapidly in the liver, kidney, and other tissues, including the gastrointestinal tract [24], it has a potent oxidation-reduction capacity and it protects tissues against oxidative stress ]25]. Starvation has been shown to cause a substantial decrease in hepatic GSH level 1261.Possibly, the depletion may be accounted for by an increase in glutathione protein mixed disulphide formation [27]. In contrast, the present study indicates that dietary restriction, without essential deficiency, enhanced GSH content in intestine and liver (42 and 14%, respectively). Moreover, TBA-reactive material, which is a crude measure of malonaldehyde and similar by-products of lipid peroxidation, was reduced in the intestine (-28%) and liver (-16%) of foodrestricted rats. In the liver, the anti-lipoperoxidation action of dietary restriction could not be attributable to the fatty acid composition of mitochondria and microsomes, or vitamin E status: Laganiere and Yu [28] observed no effect on the unsaturated/saturated fatty acids ratio or arachidonic acid content and they noted a decrease in docosapentaenoic acid (22:5) in the membranes of restricted rats (12 months of TABLE I EFFECTS OF DIETARY RESTRICTION AND TBARS CONTENT
Body weight (g)
ON BODY WEIGHT, INTESTINE AND LIVER CSH
Control
Restricted
235
168
t 4
It”
Intestine Glutathione @mol/g) TBARS (nmobg)
1.01 f 0.06 16.8 k 0.7
1.43 rt 0.09” 12.1 + 0.8”
Liver Glutathione @mol/g) TBARS (nmol/g)
6.04 * 0.30 122 f5
6.89 i: 0.28’ 102 + 4’
Results are quoted as mean f SE from 18 rats. 'P < 0.05; '*P< 0.01.
94
age). In contrast to other tissues, intestinal mucosa showed a decrease in TBA-reactive substances between 616 months of age [29]. Balasubramanian et al. [30] found that gastrointestinal mucosa is resistant to lipid peroxidation. They attributed this resistance to oleic and palmitoleic acids [31]. The gastrointestinal mucosa had a lofold higher concentration of free acids than other tissues. In our study, the restrictiondependent decrease in the intestinal TBA-reactive material might, in part, be related to these lipid peroxidation inhibitors. The possibility remains that food restriction can modulate cytosolic antioxidants or scavenger enzymes. Data on SOD, catalase, and Se-GPx activities in two tissues are shown in Table II. In the present experiment, no diet restriction-related variations were found for the enzymatic antioxidant defence (SOD, catalase). In general, the protective system was higher in the liver of mature rats fed the restricted diet and the increase appears to arise from the levels of the mRNAs coding for these enzymes [6]. The animals of our study were younger, and only liver Se-GPx was significantly enhanced (23%). It has been shown that situations which increase oxygen consumption also enhance the rate of active oxygen species [32]. Masoro et al. [33] and Weindruch et al. [34] emphasized that dietary restriction increases the daily and lifetime energy intake per gram of body weight, but if the loss in body weight is taken into account, the total metabolic rate of animals is not modified [35]. These observations suggest that the free radical generation that arises from cellular metabolism would be similar, if not higher, in diet-restricted rats. Thus, at present, there is no direct evidence that food restriction alters the free radical production dependent on mitochondria activity. Nevertheless, we and others have shown a decreased oxidative stress (TBARS or nuclear 8-hydroxyguanosine generation [lo]). One hypothesis is that dietary restriction could lower iron mobilization from the intracellular sites. Iron is required for initiation of lipid peroxidation. Ferritin is a possible source of iron [36,37]. Moreover, TABLE
II
EFFECTS TESTINE
OF DIETARY AND
RESTRICTION
ON ENZYMATIC
Control Intestine SOD (units/mg protein) Catalase @mol/mg protein/min) Se-GPx (nmol/mg
proteinimin)
Liver SOD (units/mg protein) Catalase @mol/mg proteinjmin) Se-GPx Results
(nmol/mg
are quoted
ANTIOXIDANT
DEFENCE
LIVER
proteinimin)
Restricted
0.420 + 0.025 4.30 -t 0.15
0.434 f 0.028 4.29 kO.18
6.34
f 0.78
5.26
2.33 187
5 0.18 +9
2.30 184
kO.12 k8
50.3
k 3.6
61.8
k 3.7’
as mean + SE from 18 rats. ‘P < 0.05.
k 0.68
IN IN-
95
a small pool of microsome-bound nonheme iron, which was increased in the liver of aging rats, has been recently identified as a possible catalyst of peroxidation [38]. In conclusion, the present experiment suggested that: (i) anti-lipoperoxidation of diet restriction (4 weeks in a young rat) was more pronounced in the intestine than in the liver and (ii) in both organs the protection appeared linked to GSH content (and Se-GPx activity in the liver only). ACKNOWLEDGEMENTS
We thank Nicole Darmon, Martine Heyman, Pr. J.-F. Desjeux, INSERM U 290, Hopital St. Lazare, Paris, for helpful discussions; C. Desfontaines for maintenance and care of the animals; John Lambert for having corrected this text. REFERENCES I McCay, C.M., Crowell, M.F. and Maynard, L.A. (1935) The effect of retarded growth upon the length of the life span and upon the ultimate body size. J. Nutr. 10, 63-79. 2 Masoro, E.J. (1985) Nutrition and aging - a current assessment. J. Nutr. 115, 842-848. 3 Harman,
D. (1956) Aging.
A theory
based
on free radical
and radiation
chemistry.
J. Gerontol.
1 I,
298-300. 4 Cutler,
R.G. (1991) Antioxidants
5 Sohal, R.S. (1989) Oxidative
and aging. Am. J. Clin. Nutr.
stress hypothesis
6 Rao, G., Xia, E., Nakavukaren, M.J. and Richardson, age-dependent changes in the expression of antioxidant 7 Weraarchakul,
N., Strong,
R., Wood,
53, 373553795.
of aging. Adv. Myochem.
G. and Richardson,
A. (1989) The effect of aging and dietary
restriction on DNA repair. Exp. Cell. Res. 18 I, 1977204. 8 Kappus, H. (1985) Lipid peroxidation: mechanisms, analysis,
enzymology
H. Sies (Ed.), Oxidative Stress, Academic Press, London, pp. 273-309. 9 Chilpakatti, S., De, A.K. and Aiyar, AS. (1983) Effect of diet restriction ters related to aging in mice. J. Nutr. 113, 9444950. 10 Yu. B.P. (1991) Free radicals and modulation by dietary 1 I Granger, derived
D.N.,
Hollwarth,
free radicals.
M.E. and Park,
Acta Physiol.
Stand.
2, 21-34.
A. (1990) Effect of dietary restriction on the enzymes in rat liver. J. Nutr. 120,602-609.
restriction,
and biological
relevance.
on some biochemical
In:
parame-
Age Nutr. 2, 8489.
D.A. (1986) Ischemia-reperfusion
injury:
role of oxygen-
548 (Suppl.) 47-63.
12 Perry, M.A., Wadhwa, S., Parks, D.A., Pickard, W. and Granger, in ischemia-induced lesions in the cat stomach. Gastroenterology
D.N. (1986) Role of oxygen 90, 3622367.
13 Craven, P.A.. Pfanstiez, J. and DeRubertis, F.R. (1986) Role of reactive of colonic epithelial proliferation. J. Clin. Invest. 77, 850-859.
oxygen
radicals
in bile salt stimulation
14 Korthuis, R.J. and Granger, D.N. (1986) Ischemia-reperfusion injury: role of oxygen-derived free radicals. In: A.E. Taylor, S. Malaton and P. Ward (Eds.), Physiology of Oxygen Radicals, American Physiological
Society,
Bethesda,
pp. 2177249.
15 Miller, J.S., McNeil], P., Mullane, K.M., Caravella, S.J. and Clark, and attenuates release in a rabbit model of necrotizing enterocolitis.
D.A. (1988) SOD prevents damage Am. J. Physiol. 255, G556- 565.
16 Potier de Courcy, G., Durand, G., Abraham, J. and Gueguen, L. (1989) Recommandations sur les conditions d’alimentation des animaux de laboratoire (rats et souris). Sci. Aliments 9, 2099217. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, Folin phenol reagent. J. Biol. Chem. 193, 2655275.
R.J. (1951) Protein
18 Ohkawa, H., Ohnishi, N. and Yagi, K. (1979) Assay for lipid peroxides bituric acid reaction, Anal. Biochem. 95, 351-358.
measurement
in animal
with the
tissues by thiobar-
96
19 Kikugawa, K., Kojima, T. and Kosugi, H. (1990) Major thiobarbituric homogenates are alkadienals. Free Rad. Communs. 8, 107-l 13.
acid-reactive
20 Oberley, L.W. and Spitz, D.R. (1984) Methods Enzymol. 105, 457464. 21 Luck, H. (1965) Catalase. In: H. Bergmeyer (Ed.), Methods of Enzymatic New York,
substances
Analysis,
of liver
Academic
Press,
pp. 885-894.
22 Burk, R.F., Lawrence, the result of paraquat 10241031.
R.A. and Lane, J.M. (1980) Liver necrosis and diquat
administration.
and lipid peroxidation
Effect of selenium
deficiency.
23 Sedlack, J. and Lindsay, R.H. (1968) Estimation of total protein-bound groups in tissue with Ellman’s reagent. Anal. Biochem. 2, 1922205. 24 Manohar, M. and Balasubramanian, K.A. Indian J. Biochem. Biophys. 23,274278. 25 Reed, D.J. (1990) Glutathione:
toxicological
(1986) Antioxidant implications.
and nonprotein
enzymes
Annu.
in the rat as
J. Clin. Invest.
sulfhydryl
in rat gastrointestinal
Rev. Pharmacol.
65,
Toxicol.
tract. 30, 603-
631. 26 Strubelt, O., Dost-Kempf, E., Siegers, C.P., Younes, M., Volpel, M. and Preuss, U. (1981) The influence of fasting on the susceptibility of mice to hepatotoxic injury. Toxicol. Appl. Pharmacol. 60, 6677. 27 Isaacs, J.T. and Binkley, F. (1977) Glutathione dependent control of protein-sulfhydryl subcellular fractions of hepatic tissue. Biochim. Biophys. Acta 497, 192-204. 28 Laganiere, S. and Yu, B.P. (1987) Anti-lipoperoxidation Res. Commun. 145, 118551191.
action
of food restriction.
29 Rao, G., Xia, E. and Richardson, A. (1990) Effect of age on the expression male Fischer F441 rats. Mech. Ageing Dev. 53, 49-60. 30 Balasubramanian, K.A., peroxidation in intestinal
content
Biochem.
of antioxidant
Manohar, M. and Mathan, V.I. (1988) An unidentified mucosa. Biochim. Biophys. Acta 962,51-58.
by
Biophys. enzymes
inhibitor
in
of lipid
31 Diplock, A.T., Balasubramanian, K.A., Manohar, M. and Mathan, V.I. (1988) Purification and chemical characterization of the inhibitor of lipid peroxidation from intestinal mucosa. Biochim. Biophys. Acta 962, 42-50. 32 Davies, K.J.A., produced 33 Masoro, process.
Quintanilha,
A.T., Brooks,
G.A. and Packer,
L. (1982) Free radicals
by exercise. Biochem. Biophys. Res. Commun. 107, 119881205. E.J., Yu, B.P. and Bertrand, H.A. (1982) Action of food restriction Proc. Nat]. Acad.
and tissue damage
in delaying
the aging
Sci. USA 79,42394241_
34 Weindruch, R., Walford, R.L., Fhgiel, S. and Guthrie, D. (1986) The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J. Nutr. 116, 641-654. 35 McCarter,
R., Masoro,
E.J. and Yu, B.P. (1985) Does food restriction
metabolic rate? Am. J. Physiol. 248, E488-E490. 36 Thomas, C.E., Morehouse, L.A. and Aust, S.D. (1985) Ferritin dation. J. Biol. Chem. 260, 3275-3280. 37 Koster, J.F. and Slee, R.G. (1986) Ferritin, tion. FEBS Lett. 199, 85-88. 38 Minotti, G., DiGennaro, M., D’Ugo, peroxidation.
Free Rad. Communs.
a physiological
D. and Granone,
12-13, 99-106.
retard
aging by reducing
and superoxide-dependent
iron donor
for microsomal
P. (1991) Possible
sources
the
lipid peroxilipid peroxidaof iron for lipid
91
TOXLET 02793
Dietary restriction decreases thiobarbituric acidreactive substances generation in the small intestine and in the liver ofyoung rats
R. Albrecht, M.A. Pelissier, S. Atteba and M. Smaili Conservatoire Nutionaldes Arts et M&iers (C.N.h.M,l,
Laboratoire de Siologie, Paris (France)
(Received 18 May 1992) (Revision received 15 July 1992) (Accepted 16 July 1992) Key wordst Diet restriction; Thiobarbituric Liver; Rat
acid-reactive
substances;
Antioxidant
enzymes; Intestine;
SUMMARY This study investigated the influence of dietary restriction on thiobarbituric acid-reactive substances (TBARS) contents and on the intracellular antioxidant defense system in the small intestine or liver of young rats. Four weeks of diet restriction (-40%) lowered the TBARS level in both organs. No variations were found for the superoxide dismutase and catalase activities; only liver seleno-dependent glutathione peroxidase was enhanced by the restriction. The protection appeared more marked in the intestine than in the liver, and would be dependent on glutathione concentration.
INTRODUCTION
It is recognized that dietary restriction without essential nutrient deficiency extends both the maximum and mean life-span in rodents [l]. Furthermore, underfeeding delays a variety of age-related pathological changes [2]. Harman (31 proposed an hypothesis that a major cause of the aging process is accumulation of free radical: induced damage occurring under normal aerobic conditions. Cutler [4] considered the possibility that active oxygen species could react with the genetic apparatus of cells involved in regulatory functions of the whole organism; this ‘dysdifferentiation’ could act as a primary aging process. Sohal [5] postulated that oxidative stress increases
Correspondence to: R. Albrecht, Conservatoire National des Arts et Metiers (C.N.A.M.), Laboratoire de Biologie, 2 rue Con& 75003 Paris, France.
92
during aging in a programmed fashion and plays a physiological role in the sequential expression of genes associated with development and aging. Evidence substantiating the interactions of dietary restriction with oxidative damage at the levels of genomic integrity was obtained by recent works [6,7]. Free radicals are known to preferentially attack lipids in cell membrane structures [8]. Chilpakatti et al. [9] were the first to show a decrease in thiobarbituric acid-reactive peroxide in liver homonogenates of food-restricted mice. Since then the available data in the literature has been supportive of the notion that dietary restriction is probably the most effective means of modulating the aging processes by lowering oxidative stress [2,10]. However, almost all studies concern changes in the liver. Also of particular interest in this respect are the activities of the gastrointestinal tract, which is a major route for entering xenobiotics into the body, because oxygen-derived free radicals are an important component of injury in ischemia [ 11,121, inflammatory bowel disease [ 131, gastric ulceration [ 141 and necrotizing enterocolitis [ 14,15]. The present study was designed to investigate the influence of short-term dietary restriction on TBARS content and on the intracellular antioxidant defence system in the small intestine or in the liver of young rats. MATERIALS
AND METHODS
Weanling male Sprague-Dawley rats (n = 36) were divided into two groups: controls, fed ad libitum a well-balanced diet [ 161over 4 weeks; restricted animals, fed 60% of the same diet. Rats were killed after overnight fasting. Livers were perfused in situ with ice-cold saline, rapidly excised and weighed. They were homogenized in 5 ~01s. of 10 mM Tris-HCl buffer containing 150 mM KCl, I mM dithioerythritol (DTET), 0.25 mM phenylmethylsulphonyl fluoride (pH 7.4). The small intestine was flushed with saline to remove mucus and food residues, slit open and the mucosae were scraped with the blunt edge of a glass slide then washed three times by resuspending in 10 mM Tris-HCl, 150 mM KCl, 1 mM EDTA, 1 mM DTET (pH 7.4) and recentrifuged for 5 min at 700 x g. The remaining pellet was homogenized in 5 ~01s. of the same buffer as the liver. Protein contents were determined according to the method of Lowry et al. 1171.We used a modified method for measuring TBA-reactive material, initially described by Ohkawa et al. [ 181and Kikugawa et al. [ 191.Placed in a test tube were: liver or intestine homogenates, 0.45 mM BHT in glacial acetic acid, 0.25 mM EDTA, 7.6 mM sodium dodecyl sulphate, 2 mM t-BuOOH in acetic acid. Then 1.8 mM phosphotungstic acid and 6.8 M HC1 (all the values were final concentrations) were added. After waiting at +4”C for 30 min, the mixture was centrifuged at 5000 x g for 15 min. Twelve mM TBA (in 20 mM Tris-HCl buffer, pH 7.0) was added to an aliquot of the supernatant (final pH 3.5) and heated at 100°C for 45 min. After cooling, the mixture was extracted with 3-5 ml of n-butanol. The amount of red pigment produced was determined by absorbance at 532 nm; 1,1,3,3_tetraethoxypropane was used as an external standard. The activity of superoxide dismutases (SOD) was assayed by the method of Oberley and Spitz 1203.One unit of SOD was
93
defined as the amount which produced 50% inhibition of the nitrobluetetrazolium absorbance. Catalase activity was measured according to Luck [21]. Selenium-dependent glutathione peroxidase (Se-GPx) was estimated by the method of Burk et al. [22], using hydrogen peroxide as the substrate. Nonprotein sulphydryl groups (mostly reduced glutathione, GSH) were determined by the method of Sedlack and Lindsay 1231.Statistical comparisons were made using analysis of variance (ANOVA). RESULTS AND DISCUSSION
Data on body weight, glutathione and TBARS levels in small intestine and liver are shown in Table I. After 4 weeks, the diet-restricted rats showed a loss in body weight of the magnitude of 29%. Glutathione is the major nonprotein thiol component found in all types of living cells and plays a crucial role in the detoxification of reactive intermediates. Synthesized rapidly in the liver, kidney, and other tissues, including the gastrointestinal tract [24], it has a potent oxidation-reduction capacity and it protects tissues against oxidative stress ]25]. Starvation has been shown to cause a substantial decrease in hepatic GSH level 1261.Possibly, the depletion may be accounted for by an increase in glutathione protein mixed disulphide formation [27]. In contrast, the present study indicates that dietary restriction, without essential deficiency, enhanced GSH content in intestine and liver (42 and 14%, respectively). Moreover, TBA-reactive material, which is a crude measure of malonaldehyde and similar by-products of lipid peroxidation, was reduced in the intestine (-28%) and liver (-16%) of foodrestricted rats. In the liver, the anti-lipoperoxidation action of dietary restriction could not be attributable to the fatty acid composition of mitochondria and microsomes, or vitamin E status: Laganiere and Yu [28] observed no effect on the unsaturated/saturated fatty acids ratio or arachidonic acid content and they noted a decrease in docosapentaenoic acid (22:5) in the membranes of restricted rats (12 months of TABLE I EFFECTS OF DIETARY RESTRICTION AND TBARS CONTENT
Body weight (g)
ON BODY WEIGHT, INTESTINE AND LIVER CSH
Control
Restricted
235
168
t 4
It”
Intestine Glutathione @mol/g) TBARS (nmobg)
1.01 f 0.06 16.8 k 0.7
1.43 rt 0.09” 12.1 + 0.8”
Liver Glutathione @mol/g) TBARS (nmol/g)
6.04 * 0.30 122 f5
6.89 i: 0.28’ 102 + 4’
Results are quoted as mean f SE from 18 rats. 'P < 0.05; '*P< 0.01.
94
age). In contrast to other tissues, intestinal mucosa showed a decrease in TBA-reactive substances between 616 months of age [29]. Balasubramanian et al. [30] found that gastrointestinal mucosa is resistant to lipid peroxidation. They attributed this resistance to oleic and palmitoleic acids [31]. The gastrointestinal mucosa had a lofold higher concentration of free acids than other tissues. In our study, the restrictiondependent decrease in the intestinal TBA-reactive material might, in part, be related to these lipid peroxidation inhibitors. The possibility remains that food restriction can modulate cytosolic antioxidants or scavenger enzymes. Data on SOD, catalase, and Se-GPx activities in two tissues are shown in Table II. In the present experiment, no diet restriction-related variations were found for the enzymatic antioxidant defence (SOD, catalase). In general, the protective system was higher in the liver of mature rats fed the restricted diet and the increase appears to arise from the levels of the mRNAs coding for these enzymes [6]. The animals of our study were younger, and only liver Se-GPx was significantly enhanced (23%). It has been shown that situations which increase oxygen consumption also enhance the rate of active oxygen species [32]. Masoro et al. [33] and Weindruch et al. [34] emphasized that dietary restriction increases the daily and lifetime energy intake per gram of body weight, but if the loss in body weight is taken into account, the total metabolic rate of animals is not modified [35]. These observations suggest that the free radical generation that arises from cellular metabolism would be similar, if not higher, in diet-restricted rats. Thus, at present, there is no direct evidence that food restriction alters the free radical production dependent on mitochondria activity. Nevertheless, we and others have shown a decreased oxidative stress (TBARS or nuclear 8-hydroxyguanosine generation [lo]). One hypothesis is that dietary restriction could lower iron mobilization from the intracellular sites. Iron is required for initiation of lipid peroxidation. Ferritin is a possible source of iron [36,37]. Moreover, TABLE
II
EFFECTS TESTINE
OF DIETARY AND
RESTRICTION
ON ENZYMATIC
Control Intestine SOD (units/mg protein) Catalase @mol/mg protein/min) Se-GPx (nmol/mg
proteinimin)
Liver SOD (units/mg protein) Catalase @mol/mg proteinjmin) Se-GPx Results
(nmol/mg
are quoted
ANTIOXIDANT
DEFENCE
LIVER
proteinimin)
Restricted
0.420 + 0.025 4.30 -t 0.15
0.434 f 0.028 4.29 kO.18
6.34
f 0.78
5.26
2.33 187
5 0.18 +9
2.30 184
kO.12 k8
50.3
k 3.6
61.8
k 3.7’
as mean + SE from 18 rats. ‘P < 0.05.
k 0.68
IN IN-
95
a small pool of microsome-bound nonheme iron, which was increased in the liver of aging rats, has been recently identified as a possible catalyst of peroxidation [38]. In conclusion, the present experiment suggested that: (i) anti-lipoperoxidation of diet restriction (4 weeks in a young rat) was more pronounced in the intestine than in the liver and (ii) in both organs the protection appeared linked to GSH content (and Se-GPx activity in the liver only). ACKNOWLEDGEMENTS
We thank Nicole Darmon, Martine Heyman, Pr. J.-F. Desjeux, INSERM U 290, Hopital St. Lazare, Paris, for helpful discussions; C. Desfontaines for maintenance and care of the animals; John Lambert for having corrected this text. REFERENCES I McCay, C.M., Crowell, M.F. and Maynard, L.A. (1935) The effect of retarded growth upon the length of the life span and upon the ultimate body size. J. Nutr. 10, 63-79. 2 Masoro, E.J. (1985) Nutrition and aging - a current assessment. J. Nutr. 115, 842-848. 3 Harman,
D. (1956) Aging.
A theory
based
on free radical
and radiation
chemistry.
J. Gerontol.
1 I,
298-300. 4 Cutler,
R.G. (1991) Antioxidants
5 Sohal, R.S. (1989) Oxidative
and aging. Am. J. Clin. Nutr.
stress hypothesis
6 Rao, G., Xia, E., Nakavukaren, M.J. and Richardson, age-dependent changes in the expression of antioxidant 7 Weraarchakul,
N., Strong,
R., Wood,
53, 373553795.
of aging. Adv. Myochem.
G. and Richardson,
A. (1989) The effect of aging and dietary
restriction on DNA repair. Exp. Cell. Res. 18 I, 1977204. 8 Kappus, H. (1985) Lipid peroxidation: mechanisms, analysis,
enzymology
H. Sies (Ed.), Oxidative Stress, Academic Press, London, pp. 273-309. 9 Chilpakatti, S., De, A.K. and Aiyar, AS. (1983) Effect of diet restriction ters related to aging in mice. J. Nutr. 113, 9444950. 10 Yu. B.P. (1991) Free radicals and modulation by dietary 1 I Granger, derived
D.N.,
Hollwarth,
free radicals.
M.E. and Park,
Acta Physiol.
Stand.
2, 21-34.
A. (1990) Effect of dietary restriction on the enzymes in rat liver. J. Nutr. 120,602-609.
restriction,
and biological
relevance.
on some biochemical
In:
parame-
Age Nutr. 2, 8489.
D.A. (1986) Ischemia-reperfusion
injury:
role of oxygen-
548 (Suppl.) 47-63.
12 Perry, M.A., Wadhwa, S., Parks, D.A., Pickard, W. and Granger, in ischemia-induced lesions in the cat stomach. Gastroenterology
D.N. (1986) Role of oxygen 90, 3622367.
13 Craven, P.A.. Pfanstiez, J. and DeRubertis, F.R. (1986) Role of reactive of colonic epithelial proliferation. J. Clin. Invest. 77, 850-859.
oxygen
radicals
in bile salt stimulation
14 Korthuis, R.J. and Granger, D.N. (1986) Ischemia-reperfusion injury: role of oxygen-derived free radicals. In: A.E. Taylor, S. Malaton and P. Ward (Eds.), Physiology of Oxygen Radicals, American Physiological
Society,
Bethesda,
pp. 2177249.
15 Miller, J.S., McNeil], P., Mullane, K.M., Caravella, S.J. and Clark, and attenuates release in a rabbit model of necrotizing enterocolitis.
D.A. (1988) SOD prevents damage Am. J. Physiol. 255, G556- 565.
16 Potier de Courcy, G., Durand, G., Abraham, J. and Gueguen, L. (1989) Recommandations sur les conditions d’alimentation des animaux de laboratoire (rats et souris). Sci. Aliments 9, 2099217. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, Folin phenol reagent. J. Biol. Chem. 193, 2655275.
R.J. (1951) Protein
18 Ohkawa, H., Ohnishi, N. and Yagi, K. (1979) Assay for lipid peroxides bituric acid reaction, Anal. Biochem. 95, 351-358.
measurement
in animal
with the
tissues by thiobar-
96
19 Kikugawa, K., Kojima, T. and Kosugi, H. (1990) Major thiobarbituric homogenates are alkadienals. Free Rad. Communs. 8, 107-l 13.
acid-reactive
20 Oberley, L.W. and Spitz, D.R. (1984) Methods Enzymol. 105, 457464. 21 Luck, H. (1965) Catalase. In: H. Bergmeyer (Ed.), Methods of Enzymatic New York,
substances
Analysis,
of liver
Academic
Press,
pp. 885-894.
22 Burk, R.F., Lawrence, the result of paraquat 10241031.
R.A. and Lane, J.M. (1980) Liver necrosis and diquat
administration.
and lipid peroxidation
Effect of selenium
deficiency.
23 Sedlack, J. and Lindsay, R.H. (1968) Estimation of total protein-bound groups in tissue with Ellman’s reagent. Anal. Biochem. 2, 1922205. 24 Manohar, M. and Balasubramanian, K.A. Indian J. Biochem. Biophys. 23,274278. 25 Reed, D.J. (1990) Glutathione:
toxicological
(1986) Antioxidant implications.
and nonprotein
enzymes
Annu.
in the rat as
J. Clin. Invest.
sulfhydryl
in rat gastrointestinal
Rev. Pharmacol.
65,
Toxicol.
tract. 30, 603-
631. 26 Strubelt, O., Dost-Kempf, E., Siegers, C.P., Younes, M., Volpel, M. and Preuss, U. (1981) The influence of fasting on the susceptibility of mice to hepatotoxic injury. Toxicol. Appl. Pharmacol. 60, 6677. 27 Isaacs, J.T. and Binkley, F. (1977) Glutathione dependent control of protein-sulfhydryl subcellular fractions of hepatic tissue. Biochim. Biophys. Acta 497, 192-204. 28 Laganiere, S. and Yu, B.P. (1987) Anti-lipoperoxidation Res. Commun. 145, 118551191.
action
of food restriction.
29 Rao, G., Xia, E. and Richardson, A. (1990) Effect of age on the expression male Fischer F441 rats. Mech. Ageing Dev. 53, 49-60. 30 Balasubramanian, K.A., peroxidation in intestinal
content
Biochem.
of antioxidant
Manohar, M. and Mathan, V.I. (1988) An unidentified mucosa. Biochim. Biophys. Acta 962,51-58.
by
Biophys. enzymes
inhibitor
in
of lipid
31 Diplock, A.T., Balasubramanian, K.A., Manohar, M. and Mathan, V.I. (1988) Purification and chemical characterization of the inhibitor of lipid peroxidation from intestinal mucosa. Biochim. Biophys. Acta 962, 42-50. 32 Davies, K.J.A., produced 33 Masoro, process.
Quintanilha,
A.T., Brooks,
G.A. and Packer,
L. (1982) Free radicals
by exercise. Biochem. Biophys. Res. Commun. 107, 119881205. E.J., Yu, B.P. and Bertrand, H.A. (1982) Action of food restriction Proc. Nat]. Acad.
and tissue damage
in delaying
the aging
Sci. USA 79,42394241_
34 Weindruch, R., Walford, R.L., Fhgiel, S. and Guthrie, D. (1986) The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. J. Nutr. 116, 641-654. 35 McCarter,
R., Masoro,
E.J. and Yu, B.P. (1985) Does food restriction
metabolic rate? Am. J. Physiol. 248, E488-E490. 36 Thomas, C.E., Morehouse, L.A. and Aust, S.D. (1985) Ferritin dation. J. Biol. Chem. 260, 3275-3280. 37 Koster, J.F. and Slee, R.G. (1986) Ferritin, tion. FEBS Lett. 199, 85-88. 38 Minotti, G., DiGennaro, M., D’Ugo, peroxidation.
Free Rad. Communs.
a physiological
D. and Granone,
12-13, 99-106.
retard
aging by reducing
and superoxide-dependent
iron donor
for microsomal
P. (1991) Possible
sources
the
lipid peroxilipid peroxidaof iron for lipid
