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Age-related changes in oxidative damage to lipids and DNA in rat skin
Mechanisms of Ageing and Development 122 (2001) 415– 426 www.elsevier.com/locate/mechagedev
Age-related changes in oxidative damage to lipids and DNA in rat skin Shoichi Tahara a, Mitsuyoshi Matsuo b, Takao Kaneko c,* a
Department of Ultrastructure and Research Facilities, Tokyo Metropolitan Institute of Gerontology, 35 -2 Sakaecho, Itabashi-ku, Tokyo 173 -0015, Japan b Department of Biology, Faculty of Science, Konan Uni6ersity, 8 -9 -1 Okamoto, Higashinada-ku, Kobe, Hyogo 658 -8501, Japan c Department of Biochemistry and Isotopes, Tokyo Metropolitan Institute of Gerontology, 35 -2 Sakaecho, Itabashi-ku, Tokyo 173 -0015, Japan Received 6 October 2000; received in revised form 7 December 2000; accepted 9 December 2000
Abstract Skin is a tissue exposed most frequently to oxidative stress from the environment in daily life. Age-related changes of oxidative damage and antioxidant enzyme activity in the skin were examined in male Fischer 344 rats aged 6 to 30 months. The contents of phosphatidylcholine hydroperoxide (PCOOH) and thiobarbituric acid-reacting substances (TBARS) increased linearly with age. The content of cholesterol hydroperoxide increased until 24 months of age and then decreased. The content of 8-oxo-2%-deoxyguanosine (8-oxodG) increased gradually with age, and was significantly higher at 30 months of age than at 6 months of age. Superoxide dismutase activity tended to decrease with age. The activities of catalase and glutathione peroxidase showed no changes with age. We examined the effect of dietary restriction on the accumulation of oxidative damage in rat skin. The increase in PCOOH content in the skin of dietary-restricted rats was suppressed until 30 months of age. The TBARS and cholesterol hydroperoxide contents in the skin of dietary-restricted rats were significantly lower than in the skin of ad libitum-fed rats, while the 8-oxodG content was somewhat lower in the dietary-restricted rats than the ad libitum-fed rats. These results indicate that oxidative damage to the lipids and DNA in rat skin increases with age and that dietary restriction delays the accumulation of oxidative damage in skin. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: +813-3964-3241, ext.: 3155; fax: + 813-3579-4776. E-mail address: [email protected] (T. Kaneko). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 5 7 - 8
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Keywords: Aging; Oxidative damage; Lipid hydroperoxide; 8-oxo-2%-deoxyguanosine; Dietary restriction; Rat skin
1. Introduction Reactive oxygen species (ROS), such as superoxide anion radicals formed during mitochondrial respiration and phagocytosis, have been shown to cause oxidative damage to biological molecules such as lipids, proteins, and DNA. A variety of antioxidant mechanisms in the body protect tissues against damage caused by ROS (McCord and Fridovich, 1969). However, the antioxidant capacity of tissues decreases during aging (Reiss and Gershon, 1976; Massie et al., 1979; Semsei et al., 1989; Rao et al., 1990a,b). It has been suggested that oxidative damage accumulates in biological molecules during aging and that oxidative stress is relevant to the aging process. In fact, 8-oxo-2%-deoxyguanosine (8-oxodG) residues in DNA, carbonyl groups and advanced glycosylation end products (AGE) in proteins, and hydroperoxides and thiobarbituric acid-reacting substances (TBARS) in lipids have been reported to accumulate in the tissues of aged animals. Further, there is strong evidence showing that oxidative stress is involved in a variety of age-related diseases, such as atherosclerosis, Alzheimer’s disease, etc. (Pryor, 1987). Skin is continuously exposed to many hazardous environmental agents including atmospheric oxygen, ultraviolet, visible light, and prooxidant chemicals such as ozone. Free radicals, including ROS, are thought always to be generated in skin by stimulation from these environmental factors (Epstein, 1977). Furthermore, ROS are known to be involved in many skin disorders such as skin cancer, cutaneous autoimmune diseases, xeroderma pigmentosum, and skin aging (Giacomoni and D’Alessio, 1996). Thus, oxidative damage may accumulate much more in skin than in other organs during aging. In rat skin, the content of 7-hydroperoxycholesterol (ChOOH) has been reported to increase linearly up to 45 weeks of age (Ozawa et al., 1991). There are, however, few reports about age-related changes in oxidative damage to skin throughout the aging process. In order to clarify how oxidative damage in skin accumulates during aging, we examined age-related changes in the contents of oxidatively damaged compounds, such as ChOOH, phosphatidylcholine hydroperoxide (PCOOH), TBARS, and 8-oxodG, and in the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). Dietary restriction is the only reproducible method for extending the life span of rodents (Yu et al., 1985). Although the causal factors in the delay of the aging process observed by dietary restriction have not yet been well characterized, it has been proposed that the antiaging action of dietary restriction involves an antioxidant mechanism (Yu, 1996). In this study, we also examined the effect of dietary restriction on the accumulation of oxidative damage in rat skin.
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2. Materials and methods
2.1. Animals Male Fischer 344 rats were obtained at 4 weeks of age from Charles River Japan Inc. (Kanagawa, Japan) and housed three rats per cage under specific-pathogen-free conditions. Rats were fed ad libitum a commercial laboratory diet, CRF-1 (Oriental Yeast Inc., Tokyo, Japan), and water. The animals were maintained at 20°C in a photoperiod-controlled (12 h/day) room.
2.2. Isolation of lipids and DNA from skin Rats were sacrificed by decapitation under anesthesia. Skin was quickly excised from the dehaired backs, frozen immediately in liquid nitrogen, and stored at −80°C until use. Frozen skin samples were powdered by hammering in a steel vessel cooled with liquid nitrogen. The powdered skin (about 1 g) was homogenized using a Polytron homogenizer (Kinematica GmbH; Luzern, Switzerland) in 30 ml of a mixture of 0.85% sodium chloride, chloroform, and methanol (3:4:2, by volume) containing 0.1% butylated hydroxytoluene and 0.015% 2,5-dimethylfuran. The homogenates were centrifuged at 2000 rpm for 10 min and aqueous layers were removed to separate tubes. The lipid extraction was repeated and the organic layers were combined. The organic solution was evaporated to dryness under reduced pressure. Nuclear DNA was isolated as described previously (Kaneko et al., 1996a). Rat skin (about 150 mg) was put into ice-cold 0.85% sodium chloride and minced with a pair of scissors. The minced skin was homogenized in a glass teflon (Potter-Elvehjem) homogenizer in 2 ml of 0.3 M sucrose. The homogenates were centrifuged to remove the cytosolic fraction containing mitochondria. Pellets were incubated for 2 h at 37°C with proteinase K and 1% SDS/1 mM EDTA (pH 8.0) under an argon atmosphere. Nuclear DNA was extracted with ultrapure phenol using Phase Lock Gel L (5Prime-3Prime Inc., Boulder, CO). The gel allowed the quick separation and ready recovery of the aqueous layer from the phenol layer. Crude DNA was treated with ribonucleases T1 and A (Sigma, St. Louis, MO) to remove contaminating RNA. The purity of the isolated DNA was confirmed spectrophotometrically.
2.3. Quantification of lipid peroxides PCOOH and ChOOH were measured by high-performance liquid chromatography (HPLC; Shimadzu LC-6A, Shimadzu Co., Tokyo, Japan) with chemiluminescence detection (CLD-100, Tohoku Electronic Industrial Co., Sendai, Japan). PCOOH was analyzed using a Finepack SIL NH2-5 column (4.6× 250 mm; Japan Spectroscopic Co., Tokyo, Japan) eluted with a mixture of hexane, iso-propanol, methanol and water (5:7:2:1, by vol.) at a flow rate of 0.3 ml/min (Kaneko et al., 1996b). Borate buffer (50 mM, pH 9.5) containing 20 mg/ml of cytochrome c and 5 mg/ml of luminol was used as a luminescent reagent at a flow rate of 0.5 ml/min.
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ChOOH was analyzed using a Chiralcel OD-R column (4.6× 250 mm; Daicel Chemical Industries, Tokyo, Japan) with a mixture of methanol and water (85:15, by vol.) at a flow rate of 1 ml/min (Ozawa et al., 1991). Borate buffer (50 mM, pH 9.5) containing 20 mg/ml microperoxidase and 5 mg/ml isoluminol was used as a luminescent reagent at a flow rate of 1.1 ml/min. Lipid peroxides were identified by cochromatography with authentic samples synthesized as described previously (Teng et al., 1973; Baba et al., 1990). Amounts of phosphatidylcholine were measured with a Phospholipid B-Test WAKO kit (Wako Pure Chemical Industries, Osaka, Japan) based on the choline oxidase-phenol method (Takayama et al., 1977). Free cholesterol was quantified by HPLC with ultraviolet detection (Shimadzu SPD-6A, Shimadzu Co., Tokyo, Japan). Cholesterol esters and their hydroperoxides were hydrolyzed with cholesterol esterase to free cholesterol and free ChOOH, respectively, and quantified as described above. Thus, the term ‘‘total cholesterol’’ means the sum of the free cholesterol and cholesterol esters, and the term ‘‘total ChOOH’’ means the sum of the free ChOOH and cholesterol ester hydroperoxide. TBARS was measured by the method of Kosugi et al. (1991).
2.4. Quantification of DNA damage DNA was hydrolyzed with nuclease P1 and alkaline phosphatase, and 8-oxodG was analyzed by HPLC with an electrochemical detection (ESA Coulochem II 5200 and analytical cell model 5011, Bedford, MA) (Kaneko et al., 1996a).
2.5. Biochemical analysis SOD activity was measured using an SOD-525 assay kit (OXIS International Inc., Portland, OR). Rats skin (about 100 mg) was homogenized in 0.25 M sucrose (500 ml) with a glass – teflon homogenizer. The homogenates obtained were adjusted with 0.25 M sucrose to : 10% w/v, and centrifuged at 11 000 rpm for 10 min and 4°C. An ice-cold mixture of ethanol and chloroform (62.5:37.5, by vol.) was added to the supernatant. The mixture was shaken for at least 30 s and centrifuged at 6400 rpm for 10 min. The aqueous layer was transferred to another tube and kept at 0–4°C. Aliquots (8 ml) of aqueous solution and 33.3 mM 1,4,6-trimethyl-2vinylpyridinium trifluoromethanesulfonate (6 ml) were added to 50 mM 2-amino-2methyl-1,3-propanediol/HCl buffer (pH 8.8, 180 ml) containing 0.11 mM dimethylenetriaminepentaacetic acid, and mixed on a vortex mixer for several seconds. After incubation for 1 min at 37°C, 0.66 mM 5,6,6a,11b-tetrahydro-3,9,10trihydroxybenzo[c]fluorene (6 ml) was added, and the absorbance change was measured at 525 nm for 1 min. Catalase activity was assayed by the method of Aebi (1984). Rat skin (about 100 mg) was minced in ice-cold RIPA buffer (phosphatebuffered saline containing 5 mM EDTA and 0.01% digitonin, 2 ml), homogenized with a glass – teflon homogenizer, and centrifuged at 11 000 rpm for 10 min. An aliquot (20 ml) of supernatant was immediately added to a mixture of 1 M Tris – HCl buffer (pH 8.0, 50 ml) containing 5 mM EDTA, 10 mM hydrogen peroxide (900 ml), and deionized water (30 ml) and the change in absorbance at 240
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nm was measured for 1 min at 37°C. GPx activity was measured by monitoring the decrease in NADPH concentration according to the method of Leopold and Wolfgang (1984). An aliquot (10 ml) of the supernatant prepared for the catalase activity assay was added to a mixture of TE buffer (1 M Tris –HCl, 5 mM EDTA, pH 8.0; 100 ml), 0.1 M GSH in 0.1×TE buffer (20 ml), GSH reductase (10 units/ml) in TE buffer (100 ml), 2 mM NADPH in 0.1× TE buffer (100 ml), and water (570 ml). The mixture was preincubated for 5–10 min at 37°C, and 10 ml of 7 mM tert-butyl hydroperoxide was added to the mixture. The absorbance change at 340 nm was measured for 1 min. Protein content was measured by the method of Bradford (1976).
2.6. Method of dietary restriction Dietary restriction was carried out by the intermittent feeding method on 4-week-old rats. Dietary-restricted rats were fed on Mondays, Wednesdays, and Fridays only, as reported previously (Kaneko et al., 1997).
2.7. Statistical analysis Data are expressed as mean9S.D. Mean values were assessed for significance by a one-way analysis of variance (ANOVA), followed by the Fisher’s PLSD test. Probability values P B 0.05 were considered statistically significant.
3. Results
3.1. Lipid peroxides in rat skin during aging The body weight (45.392.6 g) from 4 weeks of age increased rapidly with growth, reaching a maximum (472.39 20.0 g) at around 16 months of age, and then decreased gradually to 371.09 61.1 g at 30 months of age. The 50% survival age of ad libitum-fed rats was 28.2 months (Shumiya and Kuramoto, 1994). Fig. 1 shows the changes in the contents of PCOOH and TBARS in rat skin during aging. The PCOOH content (5.7491.63 mmol/PC mol) at 24 months of age was significantly higher than that (3.469 1.02 mmol/PC mol) at 6 months of age. The PCOOH content (7.1493.26 mmol/PC mol) at 30 months of age was then higher than at 24 months of age. The TBARS content showed changes similar to PCOOH content. The TBARS content (8.3293.12 nmol/mg protein) at 24 months of age was significantly higher than that (4.719 1.53 nmol/mg protein) at 6 months of age, and the content (11.109 2.05 nmol/mg protein) at 30 months of age was higher than at 24 months of age. Contents of free ChOOH and total ChOOH increased up to 24 months of age, and then decreased at 30 months of age as shown in Fig. 2. The free ChOOH content (42.58916.59 mmol/free Ch mol) at 24 months of age was significantly higher than at 6 or 12 months of age, while the total ChOOH content (22.83 93.97 mmol/total Ch mol) at 24 months of age was significantly higher than
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Fig. 1. Age-related changes in the contents of PCOOH (A) and TBARS (B) in the skin of ad libitum-fed Fischer 344 rats. Values represent mean 9 S.D. of 12 – 16 rats. *Significantly different at P B0.05 vs. 6 months of age. **Significantly different at PB0.01 vs. 6 months of age and at P B 0.05 vs. 12 months of age. †Significantly different at PB 0.05 vs. 6 months of age. ††Significantly different at P B0.01 vs. 6 months of age and at PB 0.05 vs. 12 months of age.
at 6, 12, or 30 months. The ratio of free ChOOH to free cholesterol was approximately twice that of total ChOOH to total cholesterol.
3.2. 8 -oxodG content in nuclear DNA of rat skin during aging The 8-oxodG content in nuclear DNA of rat skin increased gradually with age and the level at 30 months of age (2.049 0.27 8-oxodG/105 dG) was significantly higher than at 6 months of age (1.6790.16 8-oxodG/105 dG).
Fig. 2. Age-related changes in the contents of free (A) and total (B) ChOOH in the skin of ad libitum-fed Fischer 344 rats. Values represent mean 9 S.D. of 12 – 16 rats. *Significantly different at P B0.01 vs. 6 months of age and at PB0.05 vs. 12 months of age. †Significantly different at PB0.01 vs. 6, 12 and 30 months of age.
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Fig. 3. Age-related changes in the contents of PCOOH (A) and TBARS (B) in the skin of dietary-restricted Fischer 344 rats. *Significantly different at P B0.01 vs. 6 and 12 months of age and at P B0.05 vs. 24 and 30 months of age. †Significantly different at PB 0.01 vs. 6 and 12 months of age and at P B0.05 vs. 24 months of age.
3.3. Antioxidant enzyme acti6ities in rat skin during aging SOD activity in rat skin decreased gradually with age, and the activity at 30 months of age (0.02969 0.0199 units/mg protein) was 67.6% of that at 6 months of age (0.04389 0.0192 units/mg protein); however, this difference was not significant. Neither catalase nor GPx activities showed significant changes during aging (data not shown).
3.4. Accumulation of oxidati6e damage to lipid and DNA in the skin of dietary-restricted rats The body weights of dietary-restricted rats increased slowly to over 200 g at 16 months of age, and remained relatively stable at about 210 g until 33 months of age. The 50% survival age of the dietary-restricted rats in this study was 35.2 months of age. PCOOH contents showed little change up to 30 months of age (4.09 92.86 mmol/PC mol) in the skin of dietary-restricted rats, after which the content increased significantly at 33 months of age (7.539 2.25 mmol/PC mol) (Fig. 3(A)). The PCOOH contents at 12, 24, and 30 months of age were significantly lower in the skin of dietary-restricted rats than in the skin of ad libitum-fed rats (PB 0.05). Although the TBARS content in the skin of dietary-restricted rats increased similarly to that in ad libitum-fed rats during aging (Fig. 3(B)), the content in dietary-restricted rat skins was significantly lower than that in ad libitum-fed rat skins at the corresponding ages (PB 0.05). Both free and total ChOOH contents in the skin of dietary-restricted rats increased during aging as shown in Fig. 4, although the increase in total ChOOH content was very slow. The free ChOOH content increased significantly at 24 and 30 months of age (31.139
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14.96 mmol/free Ch mol) compared with 6 months of age (11.729 6.90 mmol/free Ch mol), while the total ChOOH content increased significantly at 30 months of age (9.3693.39 mmol/total Ch mol). The free ChOOH content at 24 months of age was significantly lower in the skin of dietary-restricted rats than in the skin of ad libitum-fed rats (P B 0.05) and the total ChOOH contents in the skin of dietary-restricted rats at 12 and 24 months of age were significantly lower than those in the skin of ad libitum-fed rats (P B 0.05 and PB 0.01, respectively). The 8-oxodG content of dietary-restricted rat skin (6 months of age; 1.5390.12 8-oxodG/105 dG) tended to increase during aging, but the increase was not significant, even at 33 months of age (1.7590.13 8-oxodG/105 dG). Although all the 8-oxodG contents were lower than those in the skin of ad libitum-fed rats at the corresponding ages, there were no significant differences between the skin of ad libitum-fed and dietary-restricted rats. Furthermore, antioxidant enzyme activities in the skin of dietary-restricted rats showed no significant differences with the skin of ad libitumfed rats (data not shown).
4. Discussion There have been many reports demonstrating the increase in oxidative damage during aging. Age-dependent increases in the PCOOH contents of rat brain and liver (Miyazawa et al., 1993), gerbil brain (Zhang et al., 1994), human red blood cells (Miyazawa et al., 1996) and cultured human diploid cells (Suzuki et al., 1993) have been reported. Increase in the TBARS contents during aging of rat brain (Santiago et al., 1993; Inanami et al., 1995), rat plasma (Erdincler et al., 1997),
Fig. 4. Age-related changes in the contents of free (A) and total (B) ChOOH in the skin of dietary-restricted Fischer 344 rats. *Significantly different at P B0.05 vs. 6 months of age. **Significantly different at PB 0.01 vs. 6 months of age and at PB0.05 vs. 12 months of age. † Significantly different at PB 0.05 vs. 6 and 12 months of age. ††Significantly different at PB0.01 vs. 6 and 12 months of age and at PB0.05 vs. 24 months of age.
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human serum (Miquel et al., 1998), and cultured human vein endothelial cells (Carrera-Rotllan and Estrada-Garcia, 1998) have also been reported. On the other hand, the ChOOH content in male Fischer 344 rat skin has been found to increase linearly with age up to 45 weeks (Ozawa et al., 1991). In this study, we observed that the contents of PCOOH and TBARS increased linearly in rat skin during aging. The ChOOH contents also increased until 24 months of age. However, the ChOOH content at 30 months of age was unexpectedly lower than that at 24 months of age. The ChOOH isolated from rat skin in this study was found to be a mixture of 7a- and 7b-hydroperoxides by HPLC with chemiluminescence detection. This indicates that ChOOH in rat skin is produced by autoxidation, not by oxygenation by singlet oxygen. Cholesterols are present in the free and esterized forms in skin. Because cholesterol esters are composed of cholesterol and unsaturated fatty acids, ChOOH is also produced at the cholesterol in esters. When cholesterol esters are hydrolyzed by cholesterolesterase to free cholesterol, it becomes possible to measure the total amounts of ChOOH. Thus, the ChOOH obtained from the hydrolysis is called ‘‘total’’ ChOOH. Although the age-related changes in the total ChOOH content are similar to those of free ChOOH, the ratio of total ChOOH/total cholesterol was approximately half that of free ChOOH/free cholesterol. The low concentration of ChOOH in total cholesterol may be due to the high sensitivity of the unsaturated fatty acid moieties contained in cholesterol esters to autoxidation. Although it is unclear why the ChOOH content decreases in 30 month-old rat skin, the structural changes in the skin during aging (Shuster et al., 1975; Jung et al., 1997) may be responsible for the change. The skin of aged rats is characterized by thinning of the epidermis (Lavker et al., 1987), molecular modifications of the connective tissue (Pease and Grimmer, 1972; Lapie´re, 1988), changes in mechanical properties (Daly and Odland, 1979; Leveque et al., 1984), etc. The skin of 30 month-old rats was thinner than that of 24 months old rats in this study. The cholesterol peroxidation that occurs during aging may depend not only on oxidative stress, but also on the structural changes that take place in rat skin during aging. We previously showed that the 8-oxodG content in the DNA of rat tissues, including heart, liver, kidney, and brain, is elevated in rats at over 24 months of age (Kaneko et al., 1996a). In rat skin, the 8-oxodG content increased gradually with age, with the content at 30 months of age significantly higher than that at 6 months of age. The slow accumulation of 8-oxodG in rat skins may be due to the capacity of the antioxidant systems and/or repair systems or their relative short turnover. Although no data concerning the age-related changes in the antioxidant enzyme activities in rat skins have been reported, there are many reports about changes in these activities in other rat organs. For example, it has been observed that the activities of SOD, catalase, and GPx decrease with age in brain, hepatocytes, and kidneys from male rats (Rao et al., 1990a,b). As for the effect of age on antioxidant enzyme activities in the skin, GPx activity has been found to decrease with age in mouse skin (Lopez-Torres et al., 1994). In this study, the activities of SOD, catalase and GPx in rat skin underwent little change during aging. Since no decreases in the antioxidant enzyme activities were observed, the repair system that protects against
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oxidative damage to biological molecules may become weak in aged rats. It has been reported that the activities of the phospholipase A2 and the repair enzyme for DNA damage decrease during aging (Petkova et al., 1986; Edwards et al., 1998). Further studies are required to understand the contribution of repair enzymes to the oxidative damage that accumulates in skin during aging. Dietary restriction is thought to produce a delay in the aging process and/or a reduction in the deleterious effects of overfeeding in ad libitum-fed animals. There is evidence showing that changes in susceptibility to oxidative stress are related to the delay in the aging process brought about by dietary restriction. For example, we have previously shown the retarding effect of dietary restriction on the accumulation of 8-oxodG in rat kidney, heart, liver and brain (Kaneko et al., 1997). In this study, the age-related accumulation of PCOOH and ChOOH in rat skin was delayed by dietary restriction. The TBARS content in the skin of dietary-restricted rats also decreased significantly compared with ad libitum-fed rats. The 8-oxodG content was lower than in the skin of ad libitum-fed rats, but there were no significant differences between ad libitum-fed and dietary-restricted rats. These results confirm that dietary restriction suppresses the accumulation of oxidative damage to biological molecules, and suggest that oxidative stress participate in the aging process.
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Kaneko, T., Tahara, S., Matsuo, M., 1996a. Non-linear accumulation of 8-hydroxy-2’-deoxyguanosine, a marker of oxidized DNA damage, during aging. Mutat. Res. 316, 277– 285. Kaneko, T., Baba, N., Matsuo, M., 1996b. Cytotoxicity of phosphatidylcholine hydroperoxides is exerted through decomposition of fatty acid hydroperoxide moiety. Free. Radic. Biol. Med. 21, 173–179. Kaneko, T., Tahara, S., Matsuo, M., 1997. Retarding effect of dietary restriction on the accumulation of 8-hydroxy-2’-deoxyguanosine in organs of Fischer 344 rats during aging. Free. Radic. Biol. Med. 23, 76–81. Kosugi, H., Kojima, T., Kikugawa, K., 1991. Characteristics of the thiobarbituric acid reactivity of oxidized fats and oils. J. Am. Oil. Chem. Soc. 68, 51 – 55. Lapie´re, C.M., 1988. Aging of fibroblasts, collagen and the dermis. In: Lavker, R.M., Zheng, P., Dong, G. (Eds.), 1987. Aged skin: a study by light, transmission electron, and scanning electron microscopy, J. Invest. Dermatol. 88, 44s– 51s. Lavker, R.M., Zheng, P.S., Dong, G., 1987. Aged skin: a study by light, transmission electron, and scanning electron microscopy. J. Invest. Dermatol. 88, 44s– 51s. Leopold, F., Wolfgang, A.G., 1984. Assays of glutathione peroxidase. Meth. Enzymol. 105, 114– 121. Leveque, J.L., Corcuff, P., De Rigal, J., Agache, P., 1984. In vivo studies of the evolution of physical properties of the human skin with age. Int. J. Dermatol. 23, 322– 329. Lopez-Torres, M., Shindo, Y., Packer, L., 1994. Effect of age on antioxidants and molecular markers of oxidative damage in murine epidermis and dermis. J. Invest. Dermatol. 102, 476– 480. Massie, H.R., Aiello, V.R., Iodice, A.A., 1979. Changes with age in copper and superoxide dismutase levels in brains of C57BL/6J mice. Mech. Ageing. Dev. 10, 93 – 99. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049– 6055. Miquel, J., Ramirez-Bosca, A., Soler, A., Diez, A., Carrion-Gutierrez, M.A., Diaz-Alperi, J., Quintanilla-Ripoll, E., Bernd, A., Quintanilla-Almagro, E., 1998. Increase with age of serum lipid peroxides: implications for the prevention of atherosclerosis. Mech. Ageing. Dev. 100, 17 – 24. Miyazawa, T., Suzuki, T., Fujimoto, K., 1993. Age-dependent accumulation of phosphatidylcholine hydroperoxide in the brain and liver of rat. Lipids 28, 789– 793. Miyazawa, T., Suzuki, T., Fujimoto, K., Kinoshita, M., 1996. Age-related change of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide levels in normal human red blood cells. Mech. Ageing. Dev. 86, 145– 150. Ozawa, N., Yamazaki, S., Chiba, K., Aoyama, H., Tomisawa, H., Tateishi, M., Watabe, T., 1991. Occurrence of cholesterol 7a- and 7b-hydroperoxides in rat skin as aging markers. Biochem. Biophys. Res. Commun. 178, 242–247. Pease, R.H., Grimmer, B.A., 1972. Age and the chemical constitution of normal human skin. J. Invest. Dermatol. 58, 347–361. Petkova, D.H., Momchilova, A.B., Koumanov, K.S., 1986. Age-related changes in rat liver plasma membrane phospholipase A2 activity. Exp. Gerontol. 21, 187– 193. Pryor, W.A., 1987. The free-radical theory of aging revised. In: Warner, H.R., Butler, R.N., Sprott, R.L., Schneider, E.L. (Eds.), A Critique and a Suggested Disease-Specific Theory, in Modern Biological Theories of Aging. Raven Press, New York, pp. 89 – 92. Rao, G., Xia, E., Richardson, A., 1990a. Effect of age on the expression of antioxidant enzymes in male Fischer F344 rats. Mech. Ageing. Dev. 53, 49 – 60. Rao, G., Xia, E., Nadakavukaren, M.J., Richardson, A., 1990b. Effect of dietary restriction on the age-dependent changes in the expression of antioxidant enzymes in rat liver. J. Nutr. 120, 602– 609. Reiss, U., Gershon, D., 1976. Comparison of cytoplasmic superoxide dismutase in liver, heart and brain of aging rats and mice. Biochem. Biophys. Res. Commun. 73, 255– 261. Santiago, L.A., Osato, J.A., Liu, J., Mori, A., 1993. Age-related increases in superoxide dismutase activity and thiobarbituric acid-reactive substances: effect of bio-catalyzer in aged rat brain. Neurochem. Res. 18, 711–717. Semsei, I., Rao, G., Richardson, A., 1989. Changes in the expression of superoxide dismutase and catalase as a function of age and dietary restriction. Biochem. Biophys. Res. Commun. 164, 620– 625.
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Shumiya, S., Kuramoto, K., 1994. Longevity and food restriction. In: Matsuo, M. (Ed.), Aging and Environment Factors. Japan Sci. Soc. Press, Tokyo, pp. 9 – 36. Shuster, S., Black, M.M., McVitie, E., 1975. The influence of age and sex on skin thickness, skin collagen and density. Br. J. Dermatol. 93, 639– 643. Suzuki, T., Miyazawa, T., Fujimoto, K., Otsuka, M., Tsutsumi, M., 1993. Age-related accumulation of phosphatidylcholine hydroperoxide in cultured human diploid cells and its prevention by alpha-tocopherol. Lipids 28, 775–778. Takayama, M., Itoh, S., Nagasaki, T., Tanimizu, I., 1977. A new enzymatic method for determination of serum choline-containing phospholipids. Clin. Chim. Acta. 79, 93 – 98. Teng, J.I., Kulig, M.J., Smith, L.L., Kan, G., Van Lier, J.E., 1973. Sterol metabolism. XX. Cholesterol 7b-hydroperoxide. J. Org. Chem. 38, 119– 123. Yu, B.P., Masoro, E.J., McMahan, C., 1985. Nutritional influences on aging of Fischer 344 rats: I. Physical, metabolic, and longevity characteristics. J. Gerontol. 40, 657– 670. Yu, B.P., 1996. Aging and oxidative stress: modulation by dietary restriction. Free. Radic. Biol. Med. 21, 651–668. Zhang, J.R., Andrus, P.K., Hall, E.D., 1994. Age-related phospholipid hydroperoxide levels in gerbil brain measured by HPLC-chemiluminescence and their relation to hydroxyl radical stress. Brain. Res. 14, 275–282.
Age-related changes in oxidative damage to lipids and DNA in rat skin Shoichi Tahara a, Mitsuyoshi Matsuo b, Takao Kaneko c,* a
Department of Ultrastructure and Research Facilities, Tokyo Metropolitan Institute of Gerontology, 35 -2 Sakaecho, Itabashi-ku, Tokyo 173 -0015, Japan b Department of Biology, Faculty of Science, Konan Uni6ersity, 8 -9 -1 Okamoto, Higashinada-ku, Kobe, Hyogo 658 -8501, Japan c Department of Biochemistry and Isotopes, Tokyo Metropolitan Institute of Gerontology, 35 -2 Sakaecho, Itabashi-ku, Tokyo 173 -0015, Japan Received 6 October 2000; received in revised form 7 December 2000; accepted 9 December 2000
Abstract Skin is a tissue exposed most frequently to oxidative stress from the environment in daily life. Age-related changes of oxidative damage and antioxidant enzyme activity in the skin were examined in male Fischer 344 rats aged 6 to 30 months. The contents of phosphatidylcholine hydroperoxide (PCOOH) and thiobarbituric acid-reacting substances (TBARS) increased linearly with age. The content of cholesterol hydroperoxide increased until 24 months of age and then decreased. The content of 8-oxo-2%-deoxyguanosine (8-oxodG) increased gradually with age, and was significantly higher at 30 months of age than at 6 months of age. Superoxide dismutase activity tended to decrease with age. The activities of catalase and glutathione peroxidase showed no changes with age. We examined the effect of dietary restriction on the accumulation of oxidative damage in rat skin. The increase in PCOOH content in the skin of dietary-restricted rats was suppressed until 30 months of age. The TBARS and cholesterol hydroperoxide contents in the skin of dietary-restricted rats were significantly lower than in the skin of ad libitum-fed rats, while the 8-oxodG content was somewhat lower in the dietary-restricted rats than the ad libitum-fed rats. These results indicate that oxidative damage to the lipids and DNA in rat skin increases with age and that dietary restriction delays the accumulation of oxidative damage in skin. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: +813-3964-3241, ext.: 3155; fax: + 813-3579-4776. E-mail address: [email protected] (T. Kaneko). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 5 7 - 8
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Keywords: Aging; Oxidative damage; Lipid hydroperoxide; 8-oxo-2%-deoxyguanosine; Dietary restriction; Rat skin
1. Introduction Reactive oxygen species (ROS), such as superoxide anion radicals formed during mitochondrial respiration and phagocytosis, have been shown to cause oxidative damage to biological molecules such as lipids, proteins, and DNA. A variety of antioxidant mechanisms in the body protect tissues against damage caused by ROS (McCord and Fridovich, 1969). However, the antioxidant capacity of tissues decreases during aging (Reiss and Gershon, 1976; Massie et al., 1979; Semsei et al., 1989; Rao et al., 1990a,b). It has been suggested that oxidative damage accumulates in biological molecules during aging and that oxidative stress is relevant to the aging process. In fact, 8-oxo-2%-deoxyguanosine (8-oxodG) residues in DNA, carbonyl groups and advanced glycosylation end products (AGE) in proteins, and hydroperoxides and thiobarbituric acid-reacting substances (TBARS) in lipids have been reported to accumulate in the tissues of aged animals. Further, there is strong evidence showing that oxidative stress is involved in a variety of age-related diseases, such as atherosclerosis, Alzheimer’s disease, etc. (Pryor, 1987). Skin is continuously exposed to many hazardous environmental agents including atmospheric oxygen, ultraviolet, visible light, and prooxidant chemicals such as ozone. Free radicals, including ROS, are thought always to be generated in skin by stimulation from these environmental factors (Epstein, 1977). Furthermore, ROS are known to be involved in many skin disorders such as skin cancer, cutaneous autoimmune diseases, xeroderma pigmentosum, and skin aging (Giacomoni and D’Alessio, 1996). Thus, oxidative damage may accumulate much more in skin than in other organs during aging. In rat skin, the content of 7-hydroperoxycholesterol (ChOOH) has been reported to increase linearly up to 45 weeks of age (Ozawa et al., 1991). There are, however, few reports about age-related changes in oxidative damage to skin throughout the aging process. In order to clarify how oxidative damage in skin accumulates during aging, we examined age-related changes in the contents of oxidatively damaged compounds, such as ChOOH, phosphatidylcholine hydroperoxide (PCOOH), TBARS, and 8-oxodG, and in the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). Dietary restriction is the only reproducible method for extending the life span of rodents (Yu et al., 1985). Although the causal factors in the delay of the aging process observed by dietary restriction have not yet been well characterized, it has been proposed that the antiaging action of dietary restriction involves an antioxidant mechanism (Yu, 1996). In this study, we also examined the effect of dietary restriction on the accumulation of oxidative damage in rat skin.
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2. Materials and methods
2.1. Animals Male Fischer 344 rats were obtained at 4 weeks of age from Charles River Japan Inc. (Kanagawa, Japan) and housed three rats per cage under specific-pathogen-free conditions. Rats were fed ad libitum a commercial laboratory diet, CRF-1 (Oriental Yeast Inc., Tokyo, Japan), and water. The animals were maintained at 20°C in a photoperiod-controlled (12 h/day) room.
2.2. Isolation of lipids and DNA from skin Rats were sacrificed by decapitation under anesthesia. Skin was quickly excised from the dehaired backs, frozen immediately in liquid nitrogen, and stored at −80°C until use. Frozen skin samples were powdered by hammering in a steel vessel cooled with liquid nitrogen. The powdered skin (about 1 g) was homogenized using a Polytron homogenizer (Kinematica GmbH; Luzern, Switzerland) in 30 ml of a mixture of 0.85% sodium chloride, chloroform, and methanol (3:4:2, by volume) containing 0.1% butylated hydroxytoluene and 0.015% 2,5-dimethylfuran. The homogenates were centrifuged at 2000 rpm for 10 min and aqueous layers were removed to separate tubes. The lipid extraction was repeated and the organic layers were combined. The organic solution was evaporated to dryness under reduced pressure. Nuclear DNA was isolated as described previously (Kaneko et al., 1996a). Rat skin (about 150 mg) was put into ice-cold 0.85% sodium chloride and minced with a pair of scissors. The minced skin was homogenized in a glass teflon (Potter-Elvehjem) homogenizer in 2 ml of 0.3 M sucrose. The homogenates were centrifuged to remove the cytosolic fraction containing mitochondria. Pellets were incubated for 2 h at 37°C with proteinase K and 1% SDS/1 mM EDTA (pH 8.0) under an argon atmosphere. Nuclear DNA was extracted with ultrapure phenol using Phase Lock Gel L (5Prime-3Prime Inc., Boulder, CO). The gel allowed the quick separation and ready recovery of the aqueous layer from the phenol layer. Crude DNA was treated with ribonucleases T1 and A (Sigma, St. Louis, MO) to remove contaminating RNA. The purity of the isolated DNA was confirmed spectrophotometrically.
2.3. Quantification of lipid peroxides PCOOH and ChOOH were measured by high-performance liquid chromatography (HPLC; Shimadzu LC-6A, Shimadzu Co., Tokyo, Japan) with chemiluminescence detection (CLD-100, Tohoku Electronic Industrial Co., Sendai, Japan). PCOOH was analyzed using a Finepack SIL NH2-5 column (4.6× 250 mm; Japan Spectroscopic Co., Tokyo, Japan) eluted with a mixture of hexane, iso-propanol, methanol and water (5:7:2:1, by vol.) at a flow rate of 0.3 ml/min (Kaneko et al., 1996b). Borate buffer (50 mM, pH 9.5) containing 20 mg/ml of cytochrome c and 5 mg/ml of luminol was used as a luminescent reagent at a flow rate of 0.5 ml/min.
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ChOOH was analyzed using a Chiralcel OD-R column (4.6× 250 mm; Daicel Chemical Industries, Tokyo, Japan) with a mixture of methanol and water (85:15, by vol.) at a flow rate of 1 ml/min (Ozawa et al., 1991). Borate buffer (50 mM, pH 9.5) containing 20 mg/ml microperoxidase and 5 mg/ml isoluminol was used as a luminescent reagent at a flow rate of 1.1 ml/min. Lipid peroxides were identified by cochromatography with authentic samples synthesized as described previously (Teng et al., 1973; Baba et al., 1990). Amounts of phosphatidylcholine were measured with a Phospholipid B-Test WAKO kit (Wako Pure Chemical Industries, Osaka, Japan) based on the choline oxidase-phenol method (Takayama et al., 1977). Free cholesterol was quantified by HPLC with ultraviolet detection (Shimadzu SPD-6A, Shimadzu Co., Tokyo, Japan). Cholesterol esters and their hydroperoxides were hydrolyzed with cholesterol esterase to free cholesterol and free ChOOH, respectively, and quantified as described above. Thus, the term ‘‘total cholesterol’’ means the sum of the free cholesterol and cholesterol esters, and the term ‘‘total ChOOH’’ means the sum of the free ChOOH and cholesterol ester hydroperoxide. TBARS was measured by the method of Kosugi et al. (1991).
2.4. Quantification of DNA damage DNA was hydrolyzed with nuclease P1 and alkaline phosphatase, and 8-oxodG was analyzed by HPLC with an electrochemical detection (ESA Coulochem II 5200 and analytical cell model 5011, Bedford, MA) (Kaneko et al., 1996a).
2.5. Biochemical analysis SOD activity was measured using an SOD-525 assay kit (OXIS International Inc., Portland, OR). Rats skin (about 100 mg) was homogenized in 0.25 M sucrose (500 ml) with a glass – teflon homogenizer. The homogenates obtained were adjusted with 0.25 M sucrose to : 10% w/v, and centrifuged at 11 000 rpm for 10 min and 4°C. An ice-cold mixture of ethanol and chloroform (62.5:37.5, by vol.) was added to the supernatant. The mixture was shaken for at least 30 s and centrifuged at 6400 rpm for 10 min. The aqueous layer was transferred to another tube and kept at 0–4°C. Aliquots (8 ml) of aqueous solution and 33.3 mM 1,4,6-trimethyl-2vinylpyridinium trifluoromethanesulfonate (6 ml) were added to 50 mM 2-amino-2methyl-1,3-propanediol/HCl buffer (pH 8.8, 180 ml) containing 0.11 mM dimethylenetriaminepentaacetic acid, and mixed on a vortex mixer for several seconds. After incubation for 1 min at 37°C, 0.66 mM 5,6,6a,11b-tetrahydro-3,9,10trihydroxybenzo[c]fluorene (6 ml) was added, and the absorbance change was measured at 525 nm for 1 min. Catalase activity was assayed by the method of Aebi (1984). Rat skin (about 100 mg) was minced in ice-cold RIPA buffer (phosphatebuffered saline containing 5 mM EDTA and 0.01% digitonin, 2 ml), homogenized with a glass – teflon homogenizer, and centrifuged at 11 000 rpm for 10 min. An aliquot (20 ml) of supernatant was immediately added to a mixture of 1 M Tris – HCl buffer (pH 8.0, 50 ml) containing 5 mM EDTA, 10 mM hydrogen peroxide (900 ml), and deionized water (30 ml) and the change in absorbance at 240
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nm was measured for 1 min at 37°C. GPx activity was measured by monitoring the decrease in NADPH concentration according to the method of Leopold and Wolfgang (1984). An aliquot (10 ml) of the supernatant prepared for the catalase activity assay was added to a mixture of TE buffer (1 M Tris –HCl, 5 mM EDTA, pH 8.0; 100 ml), 0.1 M GSH in 0.1×TE buffer (20 ml), GSH reductase (10 units/ml) in TE buffer (100 ml), 2 mM NADPH in 0.1× TE buffer (100 ml), and water (570 ml). The mixture was preincubated for 5–10 min at 37°C, and 10 ml of 7 mM tert-butyl hydroperoxide was added to the mixture. The absorbance change at 340 nm was measured for 1 min. Protein content was measured by the method of Bradford (1976).
2.6. Method of dietary restriction Dietary restriction was carried out by the intermittent feeding method on 4-week-old rats. Dietary-restricted rats were fed on Mondays, Wednesdays, and Fridays only, as reported previously (Kaneko et al., 1997).
2.7. Statistical analysis Data are expressed as mean9S.D. Mean values were assessed for significance by a one-way analysis of variance (ANOVA), followed by the Fisher’s PLSD test. Probability values P B 0.05 were considered statistically significant.
3. Results
3.1. Lipid peroxides in rat skin during aging The body weight (45.392.6 g) from 4 weeks of age increased rapidly with growth, reaching a maximum (472.39 20.0 g) at around 16 months of age, and then decreased gradually to 371.09 61.1 g at 30 months of age. The 50% survival age of ad libitum-fed rats was 28.2 months (Shumiya and Kuramoto, 1994). Fig. 1 shows the changes in the contents of PCOOH and TBARS in rat skin during aging. The PCOOH content (5.7491.63 mmol/PC mol) at 24 months of age was significantly higher than that (3.469 1.02 mmol/PC mol) at 6 months of age. The PCOOH content (7.1493.26 mmol/PC mol) at 30 months of age was then higher than at 24 months of age. The TBARS content showed changes similar to PCOOH content. The TBARS content (8.3293.12 nmol/mg protein) at 24 months of age was significantly higher than that (4.719 1.53 nmol/mg protein) at 6 months of age, and the content (11.109 2.05 nmol/mg protein) at 30 months of age was higher than at 24 months of age. Contents of free ChOOH and total ChOOH increased up to 24 months of age, and then decreased at 30 months of age as shown in Fig. 2. The free ChOOH content (42.58916.59 mmol/free Ch mol) at 24 months of age was significantly higher than at 6 or 12 months of age, while the total ChOOH content (22.83 93.97 mmol/total Ch mol) at 24 months of age was significantly higher than
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Fig. 1. Age-related changes in the contents of PCOOH (A) and TBARS (B) in the skin of ad libitum-fed Fischer 344 rats. Values represent mean 9 S.D. of 12 – 16 rats. *Significantly different at P B0.05 vs. 6 months of age. **Significantly different at PB0.01 vs. 6 months of age and at P B 0.05 vs. 12 months of age. †Significantly different at PB 0.05 vs. 6 months of age. ††Significantly different at P B0.01 vs. 6 months of age and at PB 0.05 vs. 12 months of age.
at 6, 12, or 30 months. The ratio of free ChOOH to free cholesterol was approximately twice that of total ChOOH to total cholesterol.
3.2. 8 -oxodG content in nuclear DNA of rat skin during aging The 8-oxodG content in nuclear DNA of rat skin increased gradually with age and the level at 30 months of age (2.049 0.27 8-oxodG/105 dG) was significantly higher than at 6 months of age (1.6790.16 8-oxodG/105 dG).
Fig. 2. Age-related changes in the contents of free (A) and total (B) ChOOH in the skin of ad libitum-fed Fischer 344 rats. Values represent mean 9 S.D. of 12 – 16 rats. *Significantly different at P B0.01 vs. 6 months of age and at PB0.05 vs. 12 months of age. †Significantly different at PB0.01 vs. 6, 12 and 30 months of age.
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Fig. 3. Age-related changes in the contents of PCOOH (A) and TBARS (B) in the skin of dietary-restricted Fischer 344 rats. *Significantly different at P B0.01 vs. 6 and 12 months of age and at P B0.05 vs. 24 and 30 months of age. †Significantly different at PB 0.01 vs. 6 and 12 months of age and at P B0.05 vs. 24 months of age.
3.3. Antioxidant enzyme acti6ities in rat skin during aging SOD activity in rat skin decreased gradually with age, and the activity at 30 months of age (0.02969 0.0199 units/mg protein) was 67.6% of that at 6 months of age (0.04389 0.0192 units/mg protein); however, this difference was not significant. Neither catalase nor GPx activities showed significant changes during aging (data not shown).
3.4. Accumulation of oxidati6e damage to lipid and DNA in the skin of dietary-restricted rats The body weights of dietary-restricted rats increased slowly to over 200 g at 16 months of age, and remained relatively stable at about 210 g until 33 months of age. The 50% survival age of the dietary-restricted rats in this study was 35.2 months of age. PCOOH contents showed little change up to 30 months of age (4.09 92.86 mmol/PC mol) in the skin of dietary-restricted rats, after which the content increased significantly at 33 months of age (7.539 2.25 mmol/PC mol) (Fig. 3(A)). The PCOOH contents at 12, 24, and 30 months of age were significantly lower in the skin of dietary-restricted rats than in the skin of ad libitum-fed rats (PB 0.05). Although the TBARS content in the skin of dietary-restricted rats increased similarly to that in ad libitum-fed rats during aging (Fig. 3(B)), the content in dietary-restricted rat skins was significantly lower than that in ad libitum-fed rat skins at the corresponding ages (PB 0.05). Both free and total ChOOH contents in the skin of dietary-restricted rats increased during aging as shown in Fig. 4, although the increase in total ChOOH content was very slow. The free ChOOH content increased significantly at 24 and 30 months of age (31.139
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14.96 mmol/free Ch mol) compared with 6 months of age (11.729 6.90 mmol/free Ch mol), while the total ChOOH content increased significantly at 30 months of age (9.3693.39 mmol/total Ch mol). The free ChOOH content at 24 months of age was significantly lower in the skin of dietary-restricted rats than in the skin of ad libitum-fed rats (P B 0.05) and the total ChOOH contents in the skin of dietary-restricted rats at 12 and 24 months of age were significantly lower than those in the skin of ad libitum-fed rats (P B 0.05 and PB 0.01, respectively). The 8-oxodG content of dietary-restricted rat skin (6 months of age; 1.5390.12 8-oxodG/105 dG) tended to increase during aging, but the increase was not significant, even at 33 months of age (1.7590.13 8-oxodG/105 dG). Although all the 8-oxodG contents were lower than those in the skin of ad libitum-fed rats at the corresponding ages, there were no significant differences between the skin of ad libitum-fed and dietary-restricted rats. Furthermore, antioxidant enzyme activities in the skin of dietary-restricted rats showed no significant differences with the skin of ad libitumfed rats (data not shown).
4. Discussion There have been many reports demonstrating the increase in oxidative damage during aging. Age-dependent increases in the PCOOH contents of rat brain and liver (Miyazawa et al., 1993), gerbil brain (Zhang et al., 1994), human red blood cells (Miyazawa et al., 1996) and cultured human diploid cells (Suzuki et al., 1993) have been reported. Increase in the TBARS contents during aging of rat brain (Santiago et al., 1993; Inanami et al., 1995), rat plasma (Erdincler et al., 1997),
Fig. 4. Age-related changes in the contents of free (A) and total (B) ChOOH in the skin of dietary-restricted Fischer 344 rats. *Significantly different at P B0.05 vs. 6 months of age. **Significantly different at PB 0.01 vs. 6 months of age and at PB0.05 vs. 12 months of age. † Significantly different at PB 0.05 vs. 6 and 12 months of age. ††Significantly different at PB0.01 vs. 6 and 12 months of age and at PB0.05 vs. 24 months of age.
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human serum (Miquel et al., 1998), and cultured human vein endothelial cells (Carrera-Rotllan and Estrada-Garcia, 1998) have also been reported. On the other hand, the ChOOH content in male Fischer 344 rat skin has been found to increase linearly with age up to 45 weeks (Ozawa et al., 1991). In this study, we observed that the contents of PCOOH and TBARS increased linearly in rat skin during aging. The ChOOH contents also increased until 24 months of age. However, the ChOOH content at 30 months of age was unexpectedly lower than that at 24 months of age. The ChOOH isolated from rat skin in this study was found to be a mixture of 7a- and 7b-hydroperoxides by HPLC with chemiluminescence detection. This indicates that ChOOH in rat skin is produced by autoxidation, not by oxygenation by singlet oxygen. Cholesterols are present in the free and esterized forms in skin. Because cholesterol esters are composed of cholesterol and unsaturated fatty acids, ChOOH is also produced at the cholesterol in esters. When cholesterol esters are hydrolyzed by cholesterolesterase to free cholesterol, it becomes possible to measure the total amounts of ChOOH. Thus, the ChOOH obtained from the hydrolysis is called ‘‘total’’ ChOOH. Although the age-related changes in the total ChOOH content are similar to those of free ChOOH, the ratio of total ChOOH/total cholesterol was approximately half that of free ChOOH/free cholesterol. The low concentration of ChOOH in total cholesterol may be due to the high sensitivity of the unsaturated fatty acid moieties contained in cholesterol esters to autoxidation. Although it is unclear why the ChOOH content decreases in 30 month-old rat skin, the structural changes in the skin during aging (Shuster et al., 1975; Jung et al., 1997) may be responsible for the change. The skin of aged rats is characterized by thinning of the epidermis (Lavker et al., 1987), molecular modifications of the connective tissue (Pease and Grimmer, 1972; Lapie´re, 1988), changes in mechanical properties (Daly and Odland, 1979; Leveque et al., 1984), etc. The skin of 30 month-old rats was thinner than that of 24 months old rats in this study. The cholesterol peroxidation that occurs during aging may depend not only on oxidative stress, but also on the structural changes that take place in rat skin during aging. We previously showed that the 8-oxodG content in the DNA of rat tissues, including heart, liver, kidney, and brain, is elevated in rats at over 24 months of age (Kaneko et al., 1996a). In rat skin, the 8-oxodG content increased gradually with age, with the content at 30 months of age significantly higher than that at 6 months of age. The slow accumulation of 8-oxodG in rat skins may be due to the capacity of the antioxidant systems and/or repair systems or their relative short turnover. Although no data concerning the age-related changes in the antioxidant enzyme activities in rat skins have been reported, there are many reports about changes in these activities in other rat organs. For example, it has been observed that the activities of SOD, catalase, and GPx decrease with age in brain, hepatocytes, and kidneys from male rats (Rao et al., 1990a,b). As for the effect of age on antioxidant enzyme activities in the skin, GPx activity has been found to decrease with age in mouse skin (Lopez-Torres et al., 1994). In this study, the activities of SOD, catalase and GPx in rat skin underwent little change during aging. Since no decreases in the antioxidant enzyme activities were observed, the repair system that protects against
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oxidative damage to biological molecules may become weak in aged rats. It has been reported that the activities of the phospholipase A2 and the repair enzyme for DNA damage decrease during aging (Petkova et al., 1986; Edwards et al., 1998). Further studies are required to understand the contribution of repair enzymes to the oxidative damage that accumulates in skin during aging. Dietary restriction is thought to produce a delay in the aging process and/or a reduction in the deleterious effects of overfeeding in ad libitum-fed animals. There is evidence showing that changes in susceptibility to oxidative stress are related to the delay in the aging process brought about by dietary restriction. For example, we have previously shown the retarding effect of dietary restriction on the accumulation of 8-oxodG in rat kidney, heart, liver and brain (Kaneko et al., 1997). In this study, the age-related accumulation of PCOOH and ChOOH in rat skin was delayed by dietary restriction. The TBARS content in the skin of dietary-restricted rats also decreased significantly compared with ad libitum-fed rats. The 8-oxodG content was lower than in the skin of ad libitum-fed rats, but there were no significant differences between ad libitum-fed and dietary-restricted rats. These results confirm that dietary restriction suppresses the accumulation of oxidative damage to biological molecules, and suggest that oxidative stress participate in the aging process.
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