Effect of Varying Caloric Restriction Levels on Female Rat Growth and 5-Hydroxymethyl-2'-deoxyuridine in DNA

Zora Djuric*,1, Sherry M. Lewis{dagger}, Ming H. Lu{dagger}, Martha Mayhugh{dagger}, Lilian Naegeli*, Ning Tang{dagger} and Ronald W. Hart{dagger}

* Barbara Ann Karmanos Cancer Institute, Wayne State University, 100 E. Warren, Detroit, Michigan 48118; and {dagger} National Center for Toxicological Research, Jefferson, Arkansas 72079

Received August 22, 2001; accepted November 9, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caloric restriction has previously been shown to decrease levels of oxidative stress in rats. In this study, we examined the effects of 5 different caloric intake levels on one type of oxidative DNA damage in rat mammary gland, blood, and liver. Animals were fed modified AIN-93G diets to accommodate 10, 20, 30, or 40% calorie restriction (CR), relative to ad libitum (AL) consumption. The intakes of fat, protein, vitamins, and minerals thus remained constant, but total carbohydrate intake decreased. Body weights of the animals at 20 weeks reflected the degree of restriction, but in the first 10 weeks, weight gain in the 10% CR group was not reduced relative to animals fed ad libitum. Levels of 5-hydroxymethyl-2'-deoxyuridine increased with time in mammary gland and nucleated blood cells regardless of CR level, indicating an effect of animal age, despite the fact that the animals were only 7 months old after the 20-week dietary study. In liver, however, there was a trend towards decreased DNA damage levels with time. The effect of diet on levels of 5-hydroxymethyl-2'-deoxyuridine was not statistically significant, indicating no protective effect of restricted dietary carbohydrate. This dietary study differed from previous work in that the modified AIN-93G dietary formulation contains relatively higher levels of fat and vitamins K, E, and B12, and it has certain added trace minerals. This data raises the question of whether the previously reported effects of calorie restriction on preventing oxidative stress in mammary gland are dependent on the type of dietary formulation used.

Key Words: caloric restriction; oxidative stress; DNA damage; 5-hydroxymethyl-2'-deoxyuridine; aging; female rats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal studies over the years have shown a clear protective effect of calorie restriction (CR) on mammary gland carcinogenesis, and this literature has been reviewed extensively (Freedman et al., 1990Go; Welsch, 1994Go). In the rat model, "control" diets have typically contained 5% fat by weight, which is about 11–12% of energy from fat (Reeves et al., 1993Go). Restricted feeding of such diets generally results in decreased oxidative stress levels, and this is thought to be one mechanism by which CR protects against tumor growth (Kritchevsky, 1999Go). Our observations in rats over the short term (2 weeks) indicated that levels of 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU), which is one cause of oxidative DNA damage, were decreased in the mammary gland when rats were fed a 40% restricted AIN-76 diet (containing 5% corn oil) relative to rats fed ad libitum (AL) (Djuric et al., 1992Go).

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., 1993Go). 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., 2001aGo). 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, 1986Go). 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., 2001bGo). 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., 2001bGo; LaFrancois et al., 1998Go). 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., 2000Go; Frenkel et al., 1991Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and tissues.
One hundred and fifty Fischer-344 female rats, all 45 days of age, were obtained from the breeding colony of the National Center for Toxicological Research (Jefferson, AR). They were adapted to the AIN-93G diet (Reeves et al., 1993Go) for 12 days before 30 rats per group were randomly placed on one of the 5 experimental diets at week 0 (see Table 1Go). The computerized randomization procedure aimed to equalize mean body weights of the rats on each of the diets. The animals were housed singly to facilitate determination of food intake. Food intake and body weight were determined daily. The lights in the animal room were kept on a schedule of 12 h on and 12 h off.


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TABLE 1 Formulation of Diets
 
After 2, 10, or 20 weeks, 10 rats per group were sacrificed by carbon dioxide inhalation followed by decapitation. Blood was collected into heparinized tubes and then mixed with a high sucrose buffer to lyse the cells. Nuclei from the white blood cells were then isolated by the method of Ciulla et al. (1988). The high sucrose buffer contained 320 mmol/l sucrose, 10 mmol/l Tris pH 7.4, 5 mmol/l MgCl2, 1% (by volume) Triton X-100, and 50 mmol/l mannitol. Nuclei were frozen at –70°C in 50 mmol/l mannitol, 10 mmol/l Tris pH 7.4, 1 mmol/l EDTA, and 1% SDS until DNA could be extracted. The mammary gland was dissected free of blood vessels and lymph nodes and frozen for subsequent isolation of epithelial cells. Liver was snap frozen in liquid nitrogen and then stored at –70°C.

Diets.
The diets were prepared according to the compositions shown in Table 1Go. 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., 1992Go). 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., 1991Go). 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, 1999Go).

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Food Consumption
Food intake, in grams of food consumed, was 17, 20, 26, and 34% restricted relative to AL at week 2 in the 10, 20, 30, and 40% CR groups. The corresponding numbers were 15, 22, 31, and 40% restricted at 10 weeks and 10, 19, 28, and 38% restricted at 20 weeks (the 20-week data is shown in Table 2Go). Caloric intake was 16,18, 24, and 30% less than that of AL at week 2, it was 14, 20, 29, and 37% less at week 10, and it was 9, 18, 26, and 35% less at 20 weeks in the 10, 20, 30, and 40% CR groups, respectively (Table 2Go). The intended calorie restriction levels thus were achieved within about 5%.


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TABLE 2 Total Food Intake over 20 Weeks of Study
 
Body Weight
The initial body weights of the animals ranged from 89 to 135 g, and they were allocated into the 5 diet groups randomly. The randomization program sought to equalize mean body weights. The initial body weight means obtained for each group ranged from 101 to 112 g (n = 30 per group). Average group allocation weights (108 g) were similar for the AL, 20%, and 40% CR groups. Despite the randomization procedure, the mean weights of the 10% and 30% CR groups differed with means of 105 vs. 111 g, respectively (p < 0.05). Body weight gain of the animals in the group kept a full 20 weeks is shown in Figure 1Go. At 20 weeks, the body weight gains were 86, 73, 60, and 50% of AL in the 10, 20, 30, and 40% CR groups (Table 3Go), indicating that the decrease in body weight gain is greater than the decrease in food consumption. Interestingly, the rate of body weight gain in the animals restricted 10% was not decreased relative to AL during weeks 2 through 10, and in fact was slightly higher, although this was not significant (Table 3Go). Later, at weeks 10 to 20, the rate of body weight gain was similar in all diet groups and reflects a slowing of growth in mature animals.



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FIG. 1. Mean body weight gain for female F344 rats fed diets restricted at varying levels for 20 weeks. Statistical analyses of the data are shown in Table 3Go.

 

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TABLE 3 Growth Parameters of Female F344 Rats Fed Varying Levels of Fat
 
DNA Damage
Levels of 5-OHmdU in nucleated blood cells and mammary glands both increased with time (Tables 4 and 5GoGo). In nucleated blood cells this increase was the greatest, with roughly a doubling of levels from 2 to 20 weeks (Table 4Go). The increases with time in nucleated blood cells were significant at 10 weeks in the AL and 10% CR groups. In the 20, 30, and 40% CR groups, the increases relative to the 2-week time point were significant only after 20 weeks, indicating a delay in the time-dependent increase in oxidative damage with increasing CR levels. In mammary gland, levels of oxidative DNA damage were the same or decreased after 10 weeks of feeding but were significantly increased by 20 weeks in all diet groups (Table 5Go). In liver, however, levels of 5-OHmdU somewhat decreased with time (Table 6Go). There were no significant effects of diet on levels of 5-OHmdU in the two-way ANOVA analyses in nucleated blood cells, mammary gland epithelium, or liver.


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TABLE 4 DNA Damage in Nucleated Blood Cells of Female F344 Rats on ad Libitum or Restricted Diets
 

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TABLE 5 DNA Damage Levels in Mammary Gland of Female F344 Rats on ad Libitum or Restricted Diets
 

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TABLE 6 DNA Damage Levels in Livers of Female F344 Rats Receiving ad Libitum or Restricted Diets
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study examined both weight gain and levels of one type of oxidative DNA damage in animals fed diets varying in the extent of caloric restriction. The animals selected for study were 45 days old at the start of the feeding study, which is the age used in many tumor protocols (Freedman et al., 1990Go). Body weight gain of the animals in the group kept a full 20 weeks is shown in Figure 1Go and indicates that the CR was effective in limiting body weight gain overall. Interestingly, the rate of body weight gain in the animals restricted 10% was not decreased relative to AL during weeks 2–10 (Table 3Go). This indicates an increased metabolic "efficiency" in the animals restricted by this small amount or perhaps decreased digestion and absorption of the diet quantity consumed by the animals in the AL group (McDonald et al., 1978Go). With regard to levels of 5-OHmdU, time did have a statistically significant effect, but the effects of CR were largely not significant. This was surprising since caloric restriction has been shown in many laboratories, including our own, to decrease levels of oxidative stress and increase antioxidant levels (Djuric et al., 1992Go; Feuers et al., 1989Go).

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., 1993Go). 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, 2000Go). 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, 1988Go). 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., 1995Go). 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., 1990Go; Youngman, 1993Go). 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 1Go). 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 5GoGo). 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., 1992Go), 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., 1992Go). 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., 2000Go). 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., 1999Go).

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.


    ACKNOWLEDGMENTS
 
This work was funded in part by the American Institute of Cancer Research-National Center for Toxicological Research Collaborative Research Program grant # NC93B61.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (313) 966-7368. E-mail: djuricz{at}karmanos.org. Back


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 DISCUSSION
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