* Barbara Ann Karmanos Cancer Institute, Wayne State University, 100 E. Warren, Detroit, Michigan 48118; and
National Center for Toxicological Research, Jefferson, Arkansas 72079
Received August 22, 2001; accepted November 9, 2001
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ABSTRACT |
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Key Words: caloric restriction; oxidative stress; DNA damage; 5-hydroxymethyl-2'-deoxyuridine; aging; female rats.
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INTRODUCTION |
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The newer dietary formulation for rats that was designed to maximize animal health is AIN-93, containing special formulations for growth (AIN-93G) and maintenance (AIN-93M) (Reeves et al., 1993). These diet formulations contain higher levels of vitamins E, K, and B12 than the AIN-76 formula, and they have added trace minerals. We previously examined levels of oxidative DNA damage in rats fed modified AIN93G diets for 20 weeks with varying levels of corn oil (AIN-93 is usually prepared with 7.5% soy oil). In that study, levels of oxidative DNA damage increased in mammary gland upon increasing fat intake from 3 to 10% (corn oil), but did not increase further, using 15 or 20% corn oil (Djuric et al., 2001a
). We therefore selected 10% corn oil (22% of calories from fat) as the control level of fat intake for further studies on calorie restriction since this would yield a maximal level of DNA damage in the control animals.
In this present study, rats were placed on modified AIN-93G diets containing 10% corn oil. They were then subsequently fed using 10, 20, 30, or 40% CR, and we examined oxidative DNA damage levels in the nucleated blood cells, liver, and mammary gland after 2, 10, and 20 weeks. Although the main goal of the study was to examine oxidative DNA damage levels in mammary gland, a target for the promoting effects of dietary fat and calories, another goal was to gain insight into choices of restriction levels for further studies on the relative influence of energy restriction and dietary fat on mammary gland tumorigenesis.
The marker of oxidative DNA damage that we utilized, 5-OHmdU differs from some of the other altered nucleosides in that it can be formed via hydrogen abstraction, which is thought to be important for initiation of lipid peroxidation, while other hydroxylated nucleosides are formed via hydroxyl radical addition (Jovanovic and Simic, 1986). In comparing 5-OHmdU to the more commonly measured 8-OHdG, we have found that increases in 5-OHmdU are relatively greater than those of 8-OHdG in calf thymus DNA treated with hydrogen peroxide (Djuric et al., 2001b
). Our gas chromatography-mass spectroscopy (GC-MS) method for detection of 5-OHmdU is unique in that we utilize enzymatic hydrolysis while others typically use acid hydrolysis, and acid hydrolysis destroys 5-OHmdU (Djuric et al., 2001b
; LaFrancois et al., 1998
). In most studies that have utilized enzymatic hydrolysis of DNA followed by HPLC or HPLC-MS analysis, levels of 5-OHmdU are relatively greater than that of 8-OHdG (Frelon et al., 2000
; Frenkel et al., 1991
). It is important to note that any methodology for detection of DNA damage is problematic due to the ease with which DNA can be oxidized and due to the instability of the products that are formed. The levels in any given study are thus best interpreted as relative within the study.
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MATERIALS AND METHODS |
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Diets.
The diets were prepared according to the compositions shown in Table 1. The fat, protein, vitamin, and mineral contents of the diets were adjusted such that these nutrient and micronutrient intakes would be equalized across the different diet groups when the diets were fed in restricted amounts. Amaizo Lo-Dex 10, dextrinized starch (American Maize, Hammond, IN) was added to the diet to aid in pelleting. The diets were stored frozen at 40°C until used, and food was changed daily to avoid degradation problems. The AL consumption of the 10% corn oil diet at various stages of rat growth was obtained from our previous results (Djuric et al., 1992
). The restricted amounts fed to the rats were based on that previously determined AL intake. A premeasured amount of food was given to the animals in the restricted groups each day, immediately before the lights were turned off. For the AL-fed animals, food was constantly available.
Analysis of 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU) levels.
For isolation of DNA from mammary gland tissue, epithelial cells were first isolated from mammary gland using the procedure of Moon et al. (1969). Briefly, the procedure involved collagenase digestion of the tissue (collagenase type 3, 35 mg/g tissue, Worthington Biochemical Corp., Lakewood, NJ) followed by centrifugation to remove fat from the epithelial cells. The cells were then homogenized in 1% SDS, 1 mmol/l EDTA, 50 mmol/l mannitol, 10 mmol/l Tris, pH 7.4 (3 ml/g tissue) prior to extraction of DNA. For liver, the whole tissue was homogenized in the same solution, treated with proteinase K (5 mg/g tissue) for 2 h, and extracted with phenol. The DNA was precipitated, redissolved, and treated with heat-inactivated RNases A (300 µg/g tissue) and RNase T1 (150 U/g tissue), followed by phenol and chloroform:isoamyl alcohol (48:2) extraction and precipitation.
DNA was isolated from mammary gland epithelial cells or blood nuclei using a modification of the procedure of Miller et al. (1988). Briefly, the nuclei or mammary cells were treated with heat-inactivated RNases A (200 µg/g tissue) and T1 (100 U/g tissue) for 30 min and proteinase K (2.5 mg/g tissue) for 2 h. The proteins were precipitated by addition of 1/3 volume of 6 mol/l sodium chloride. This mixture was then extracted once with chloroform:isoamyl alcohol (48:2). One final extraction with n-butanol was used to remove residual protein. The DNA was then precipitated and redissolved in 200 µl water and a UV scan was obtained.
Aliquots of the isolated DNA, 100 µg, were hydrolyzed enzymatically, derivatized and 5-OHmdU levels determined by GC-MS using isotopically-labeled internal standards as described previously (Djuric et al., 1991). Derivatization was with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane and acetonitrile (2:1) by heating at 120°C for 20 min. GC separations were performed with a 25 m Hewlett-Packard SE54 Ultra 2 column using helium as the carrier gas. In this laboratory we have obtained similar results for 5-OHmdU levels in DNA when derivatization is conducted with and without heating (Yu and Djuric, 1999
).
Statistical analyses.
Descriptive statistics were used to calculate least-square means and standard errors. Statistical comparisons of diet groups were accomplished by two-way ANOVA employing the General Linear Models least-square means comparison procedure to adjust for unbalanced sample sizes across age groups (SAS 1994). Post hoc comparisons of age-group means were made by Duncan's means separation. Response differences were considered significant when p < 0.05.
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RESULTS |
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DISCUSSION |
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The chief difference between this present study and previously reported studies is the diet used. The AIN-93 formulation has higher levels of vitamins and minerals than was used previously in the AIN-76 semipurified diet in order to maximize animal health (Reeves et al., 1993). This may function to improve resistance of AL-fed animals to DNA damage such that caloric restriction then has no additional benefit. The increases of about 50% in vitamin E and selenium contents of the diet may be especially important, since both of these dietary components have been shown to contribute to antioxidant defenses (Clarkson and Thompson, 2000
). It appears that the life-prolonging effects of CR are also attenuated in rats fed AIN-93 diets. In a recent survival study with Sprague-Dawley rats, the effect of restricting AIN-93M food intake on survival was small (odds ratio of 1.7 for survival to 114 weeks of age for 31% CR animals relative to AL-fed animals) versus the effect of restricting a chow-type diet (NIH-31 diet, odds ratio of 17.8 for survival to 114 weeks of age for 40% CR animals relative to AL fed animals) (Duffy et al., 2001). The reduction of tumor incidence in rats fed CR diets is well documented using both purified and nonpurified diets, including AIN-76 (Weindruch and Walford, 1988
). Similar experiments have not been reported with the AIN-93 diet.
Another factor that can affect the metabolic results of CR is the method of restriction that is employed. In caloric restriction studies, vitamin content of the restricted diet is often increased such that intake of vitamins is similar in AL and CR fed animals (Hart et al., 1995). In this study, fat and protein contents of the diets fed in restricted amounts were increased so that absolute intake of those two macronutrients also would remain constant. Restriction of either dietary fat or protein is known to be protective of mammary gland tumorigenesis (Freedman et al., 1990
; Youngman, 1993
). In order to delineate the relative effects of restricting fat or protein versus calories, it therefore was important to hold fat and protein intake constant to single out the effects of carbohydrate restriction. This resulted in a decreased content of carbohydrate in the restricted diets (Table 1
). We found that in the absence of fat or protein restriction, carbohydrate restriction had very little effect on levels of 5-OHmdU in any of the organs examined. If this finding can be replicated with other markers of oxidative stress, it has important implications for the relative influence of these nutrient intakes on cancer prevention.
The effect of animal age on increasing levels of 5-OHmdU in blood and mammary gland was evident in this study (Tables 4 and 5). The animals were placed on the test diets at 57 days of age. The 2- and 20-week time points therefore corresponded to 71 and 197 days of age for the rats (approximately 2.5 and 7.5 months of age). Mammary gland tumorigenesis protocols typically initiate with carcinogen at 50 days of age, and dietary manipulations are subsequently instituted to examine the role of diet in tumor promotion. During this time, the animal mammary gland is maturing and the rats attain their adult weight. Most studies concerned with the effects of aging on levels of oxidative stress have used older animals (Holmes et al., 1992
), but even at 15 weeks of age (105 days), oxidative stress levels were shown to be increased relative to 9 weeks of age in rats fed 20% casein diets (Youngman et al., 1992
). The inverse effect with time in liver was unexpected and contrasts with other literature. Differences in response of various organs to diet have, however, been noted to occur. In mice, 40 days of CR resulted in increased antioxidant capacity of heart, kidney and muscle while antioxidant capacity of liver and small intestine declined (Dubnov et al., 2000
). In the same animals used here, malondialdehyde levels were measured and previously reported to increase with age in kidney but to decrease with age in cerebellum and liver (Danam et al., 1999
).
In summary, this study indicates that the previously reported effectiveness of CR in reducing levels of oxidative DNA damage in mammary gland may be attenuated, depending on the type of dietary formulation used. The AIN-93 diet formulation contains higher levels of vitamins and minerals than the AIN-76 formulation used previously. Many of these vitamins and minerals are known to contribute to the antioxidant status of cells. Caloric restriction of the AIN-93 diet resulted in no detectable protection from one type of oxidative DNA damage, that of 5-OHmdU. The failure of CR to decrease endogenous oxidative damage when micronutrient intakes are high points to possible interactions between CR and micronutrient intakes in regulating oxidant/antioxidant levels.
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ACKNOWLEDGMENTS |
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NOTES |
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