Department of Safety Assessment, Merck Research Laboratories,
* 63963 Clermont-Ferrand Cedex 9, France; and
West Point, Pennsylvania 19486
Received March 6, 2000; accepted July 18, 2000
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ABSTRACT |
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Key Words: Sprague-Dawley rat; dietary restriction; body weight; survival; cause of death; clinical pathology.
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INTRODUCTION |
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While both genetic and environmental factors are involved, rat survival can be improved by simple dietary restriction (DR) (for reviews see Weindruch, 1996). Indeed, DR is the most efficient and convenient means to intervene in aging and disease outcomes (for reviews see Holehan and Merry, 1986; Masoro et al., 1991a,b). In addition, DR appears to be one logical solution, since the decrease in life span of laboratory rats has coincided with a trend to obesity of the affected strains (for reviews see Rao et al., 1990). However, moderate DR is known to prevent or delay both spontaneous and carcinogen-induced tumors (for review see Keenan et al., 1996). Therefore, it seems necessary for each strain and stock of rats to determine an appropriate rate of DR that will increase survival and will not substantially modify other biological parameters.
With this objective in mind, a 2-year study was conducted in SD rats from Charles River France, given either ad libitum (AL) feeding or 2 different dietary-restricted regimens, approximately 75% (25% DR) and 45% (55% DR) of adult AL food consumption. This paper presents data showing the relation between the rate of food intake and the increased survival rate and decreased incidence of causes of death, as well as the effects of food regimens on routine clinical pathology parameters in this stock of SD rats.
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MATERIALS AND METHODS |
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Diet and Dietary Regimens
The experimental groups contained 120 rats/sex/group. All rats were given certified UAR AO4C rodent diet from UAR (Villemoisson sur Orge, France). The mean diet composition over the 106 study weeks (SW) was 15.7% protein, 3.1% fat, and 60.7% carbohydrate, and contained 3.34 kilocalories (kcal)/gram of physiological energy (PE), calculated using Atwater physiologic fuel values. The feeding regimens, summarized in Table 1, were as follows:
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Study Design
This was a 2-year study, with 14-, 29-, and 53-week interim necropsies and a 106-week terminal necropsy. Before study initiation, 20 rats/sex/group were selected to be euthanized at each interim necropsy; all remaining surviving rats were euthanized at terminal necropsy. The protocol was approved by the Institutional Animal Care and Use Committee of Merck Research Laboratories (MRL; West Point, PA, USA). To mimic handling and drug administration throughout carcinogenicity studies done by oral gavage in MRL, rats were given daily, by oral gavage, 5 ml/kg BW of 0.5% aqueous methylcellulose, prepared from methylcellulose 400 cps (Methocel A4C Premium; Colorcon, Bougival, France) in deionized water. The parameters we monitored during the study are presented below.
Antemortem observations.
All animals were observed daily for general appearance, behavior, signs of morbidity, and mortality. They were weighed pretest, once in SW 1, twice a week from SW 2 to SW 13, and once a week from SW 14 to SW 104. In the AL group, food consumption (FC) was generally measured once a week, on 15 rats/sex, and over a 3-day period, up to SW 102. In DR groups, FC was measured generally once a week, on 15 rats/sex/group, and over a 24-h period, up to SW 14; from SW 15, FC was observed twice a week at the time of dosing of each rat.
Hematological and serum biochemical determinations were performed after an overnight fast, in SW 4, 8, and 11, on the 20 rats/sex/group to be euthanized at the 14-week interim necropsy; in SW 27 on the 20 rats/sex/group to be euthanized at the 29-week interim necropsy; in SW 39 and 51 on all surviving rats to be euthanized at the 53-week interim necropsy; and in SW 79 and 103 on 1920 rats/sex/group to be euthanized at final necropsy. Blood samples were drawn from the orbital sinus under ether anesthesia into EDTAtreated tubes (hematology) or serum separator tubes (serum biochemistry). Hematological parameters included red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Hct), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count (PLT), and white blood cell count (WBC) (Coulter Counter Model S Plus IV H.D.; Coultronics France, Margency, France). Leukocyte differential counting was obtained from microscopic examination (x 1000) of blood smears stained using the Hematek staining system (Ames, Puteaux, France), according to a modified Wright's procedure. Serum biochemical parameters included glucose, urea nitrogen, creatinine, total protein, albumin, high density lipoprotein (HDL) cholesterol, total cholesterol, triglycerides, activities for aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase (ALK PHOS), and creatine kinase, and levels of sodium, potassium, chloride, phosphorus, and total calcium (Hitachi 717-6000 0407; Roche Diagnostics, Meylan, France). Urinalyses were performed at the same time points as listed above for hematological and serum biochemical parameters on half of the corresponding animals, placed in their own cages overnight for sampling. Analyses included quantitative determination of urinary volume (urine weight determination) and specific gravity (Atago refractometer; Bioblock, Illkirch, France), semi-quantitative determination of pH, protein, glucose, bilirubin, occult blood, ketones, and urobilinogen (Urotron RL9 with Combur 9 test RL Teststrips; Boehringer, Meylan, France), and microscopic examination (x 100 or 400) of sediments.
Postmortem observations.
Each interim necropsy was conducted on 20 rats/sex/group; all remaining surviving rats were euthanized at terminal necropsy. Prior to scheduled necropsy, rats were fasted overnight; they were euthanized by exsanguination while under carbon dioxide anesthesia and underwent a complete routine necropsy. A complete necropsy was also done on all rats dying spontaneously or sacrificed moribund. Terminal BW, and weights of major organs and mammary masses were recorded from all rats. Samples of most tissues, including all gross changes, were fixed in neutral, 10% formalin. The testes and epididymides were fixed in Bouin's fixative. Representative sections of most tissues were prepared by routine methods and stained with hematoxylin and eosin for microscopic examination, which included salivary glands, stomach, small intestine, large intestine, liver, pancreas, adrenals, pituitary, thyroids and parathyroids, kidneys, urinary bladder, ovaries, uterus, testes and epididymides, prostate, skin, mammary gland, heart, lung, spleen, lymph nodes, thymus, bone and bone marrow, skeletal muscle, brain, spinal cord, sciatic nerve, eyes with optic nerve, Harderian gland, and any gross lesions. The diet-related histopathological changes will be reported in further publications.
Statistics
Statistical analyses were performed on results from determinations of hematological and serum biochemical parameters, and urinary volume and specific gravity. Data were analyzed for homogeneity of variance by Levene test (Levene, 1961), normality by Wilk & Shapiro W statistics (Shapiro and Wilk, 1965
, 1968
; Wilk and Shapiro, 1968
), and statistical significance at p
0.05 was based on an ANOVA, using a trend test. If criteria for homogeneity and/or normality were not met, data were examined after Rankit transformation (Harter, 1961
) followed by an ANOVA.
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RESULTS |
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DISCUSSION |
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Increases in BW in most of the laboratory rodents have been shown to be associated with increases in the onset and severity of spontaneous degenerative diseases and tumors (Hart et al., 1995; Haseman and Rao, 1992
; Keenan et al., 1992
; Lang, 1991
; Rao et al., 1990
; Turturro et al., 1995
, 1996
) resulting in decreased survival (Allaben et al., 1991
, 1996
; Duffy et al., 1989
; Hart et al., 1995
; Haseman and Rao, 1992
; Keenan et al., 1992
, 1994a
, b
, 1995a
, b
, 1996
, 1997
; Lang, 1991
; Newberne and Sotnikov, 1996
; Rao et al., 1990
; Roe, 1994
; Turturro et al., 1995
). Many of these adverse effects are the results of increased normal and neoplastic tissue growth by AL overfeeding of calories (for review see Keenan et al., 1998). DR in rodents has long been known to extend the life span by retarding the aging processes (for review see Masoro et al., 1991a). The main concept, which has emerged from different studies, is that DR enables rodents to utilize nutritional fuel for metabolism in less damaging ways than in AL conditions (for review see K. P. Keenan et al., 1998). At study termination, the incidence of deaths caused by both neoplasms and non neoplastic lesions was increased in the AL group, compared to the DR groups, but, pituitary adenoma, which is the most common cause of death in SD rats (Chandra et al., 1992
; Chvedoff et al., 1982
; Keenan et al., 1995a
, 1996
; Nohynek et al., 1993
; Pettersen et al., 1996
), remained the main cause of death among the different groups. Keenan et al. (1995a) suggested that moderate DR acts on pituitary tumors by delaying the time of onset, but not the growth rate or progression once these tumors develop. Among the degenerative lesions as causes of spontaneous deaths, chronic renal disease (CRD) was noted only in AL-fed males. It has been shown that AL-fed SD males, compared to SD rats on DR, have the earliest onset, highest incidence, and most severe CRD and that this is a common cause of death in this population (Keenan et al., 1995b
). One hypothesis for CRD pathogenesis is that AL feeding induces hemodynamic changes and hyperfiltration at the level of the glomerulus, which results in glomerular hypertrophy leading to CRD; DR is thought to delay the progression of the glomerular diseases and, subsequently, the CRD onset (Keenan et al., 1995b
).
In AL-fed males, BW mean values observed throughout the study were similar to those obtained by Nohynek et al. (1993) in SD rats from the same stock, given the same diet, housed individually, and maintained under similar experimental conditions. The survival rate obtained in this group after exclusion of the accidental deaths was 27% and corresponds to the worst rate given by these authors in studies starting in 1988 and 1989. Since mean BW curves, as functions of time, were similar among AL and 25 and 55% DR male groups, both DR rates can be considered to increase survival without impairing physiological growth in this gender. In AL-fed females, mean values for BW, compared to data from Nohynek et al. (1993), were very slightly higher and, subsequently, the survival rate was very slightly lower. As noted in males, survival was improved by both rates of DR. However, BW loss and BW retardation occurred in females given 55% DR during the first 3 months of the study and it is known that in female SD rats, there is a disproportionate loss of body fat with DR (Turturro et al., 1993). Therefore, such BW alterations during early adult life may impair sensitivity of toxicity studies and the rate of 55% DR is considered undesirable in studies conducted with female SD rats. The highest rate of BW gain was observed in AL-fed rats and was associated with the highest absolute daily FC. However, when FC was calculated as grams of ingested food (and subsequently of kilocalories) per gram of BW, there was no significant difference among groups. Therefore, rats fed AL consumed the greatest number of grams and kilocalories per rat but approximately the same number of grams and kilocalories per gram of BW as rats on DR. This has been previously noted in SD rats by Keenan et al. (1994b, 1995b). These results rule out hypotheses that DR acts by reducing the intake of calories or other nutrients per unit of body mass. BW apparently adjusts to the reduced food intake in such a way that there is no decrease in the intake of nutrients or calories per gram of BW. This adjustment suggests that the conversion of calories to body mass is similar in rats fed under both AL and DR conditions, and that metabolic rate, oxygen consumption, and food utilization may be similar. The reduction in nutrient intake per rat rather than per unit of body mass is the maneuver that prevents excessive growth, early BW gain, the early onset of spontaneous degenerative diseases and tumors, and poor survival (Keenan et al., 1994a
). The antiaging action of DR seems to be a reduction in the total amount of food consumed and thus energy intake per animal (Duffy et al., 1989
; Masoro and McCarter, 1991b
; Masoro et al., 1982
; Weindruch and Walford, 1988
).
Most of the changes observed in the clinical pathology parameters were similar to those seen in Charles River SD rats in a companion study with similar design and experimental conditions (Coleman et al., 1997). In the literature, DR has been shown to induce decreases in WBC, neutrophils, and lymphocytes in rodents (Coleman et al., 1997
; C. Keenan et al., 1998; Keenan et al., 1994a
; Kim and Gilman-Sachs, 1989
; Kubo et al., 1984
; Prescott-Mathews et al., 1998
; Roe et al., 1995
; Weindruch and Walford, 1988
) and in primates (Walford et al., 1992
). These changes are among the hallmarks of the alterations induced in rodents by antiaging low-calorie regimens (Weindruch and Walford, 1988
). However, it is important to point out that these decreases were only observed in our study with a 55% rate of DR. Less data are available regarding a possible DR-related effect on PLT. In the Biosure Study (Roe et al., 1995
) conducted with Wistar rats, PLT were significantly higher in males on 20% standard maintenance DR at 6, 12, and 18 months, whereas, throughout our study, PLT were lower in both sexes on 55% DR and similar among AL and 25% DR groups. Therefore, this apparent DR effect on PLT remains questionable. No noteworthy changes in RBC parameters were observed in any of the two MRL studies conducted in SD rats, whereas increases in RBC counts, Hb concentration, and Hct have been reported in the literature in Wistar rats (Pickering and Pickering, 1984
; Roe et al., 1995
). Again, a possible DR effect on RBC parameters remains questionable. The decreases in serum lipids are among the most commonly reported effects of DR in rodents (Coleman et al., 1997
; C. Keenan et al., 1998; Keenan et al., 1994a
; Liepa et al., 1980
; Masoro et al., 1983
; Prescott-Mathews et al., 1998
; Reaven and Reaven, 1981
; Sachan and Das, 1982
; Snyder and Towne, 1989
; Turturro et al., 1993
; Yu et al., 1984
). Indeed, DR acts as a modulator of age-related increases in serum lipids (Liepa et al., 1980
; Masoro et al., 1983
; Reaven and Reaven, 1981
; Snyder and Towne, 1989
). Sachan and Das (1982) showed that lower levels of serum lipids do not appear to be indicative of essential fatty acid deficiency since, in their 50% DR-fed SD rats, the levels of linolenic acid were elevated in both neutral and polar lipids, and activities of drug-metabolizing enzymes were not decreased, as observed with deficiency of essential fatty acids. In the present study, decreases in total and HDL cholesterol were observed only in the 55% DR females, whereas decreases in triglycerides more clearly corresponded to the level of DR and to the percentage of decreases in BW gain, with both sexes affected in the 55% DR group and the females in the 25% DR group. This better concordance between triglyceride levels (as opposed to cholesterol levels) and the level of DR has already been mentioned by Turturro et al. (1993), and lowered triglyceride levels with DR have been consistently found by these authors across a number of genotypes of rodents. This suggests that organismic fat metabolism is significantly modified by DR (Turturro et al., 1993
). Reaven and Reaven (1981) have suggested that DR enhances insulin sensitivity and therefore prevents the age-related rise in hepatic triglyceride secretion. DR-related decreases in plasma total protein and albumin have been observed in SD rats by Coleman et al. (1997) and in Wistar rats by Roe et al. (1995), whereas Snyder and Towne (1989) mentioned decreases in serum total protein and globulin without noteworthy changes in serum albumin. Birchenall-Sparks et al. (1985) reported that DR slows the rate of protein synthesis in rats and Snyder and Towne (1989) suggested that the reduction in serum globulin might be due to a decreased antigen stimulus. Therefore, the decreases in serum total protein we observed in 55% DR females may be due to the combination of both effects. Increases in serum ALK PHOS activities have been described in DR SD rats by Coleman et al. (1997), and in DR Wistar rats by Roe et al. (1995) and Snyder and Towne (1989). Ross (1969) found a close correlation between levels of hepatic enzyme (including ALK PHOS) activities in rats and life expectancy, and that long-term caloric restriction led to the persistence into middle and old ages of enzyme profiles seen in young rats. Similarly, the higher serum ALK PHOS activities observed in 55% DR females throughout our study may reflect DR-induced retardation of the age-related decrease in serum ALK PHOS activities (C. Keenan et al., 1998) and, therefore, the persistence of younger profiles of these enzymes in the blood. DR-related decreases in serum total calcium have been described in male Fischer 344 rats (Kalu et al., 1988a
,b
) and, in the present and companion (Coleman et al., 1997
) studies, only in 55% DR female SD rats. The prevention by DR of age-related increases in serum parathyroid hormone and calcitonin, and decreases in 25-hydroxyvitamin D, and, therefore, the inhibition of age-related hyperparathyroidism and senile bone loss could explain these decreases in serum total calcium (Kalu et al., 1984
, 1988a
,b
). DR increases in plasma chloride levels have been reported only in Wistar rats together with increases in RBC parameters, and may have reflected some hemoconcentration (Pickering and Pickering, 1984
). Since the increases in chloride in 55% DR females were not observed in the companion study (Coleman et al., 1997
) and in the absence of DR-related increases in RBC parameters, this DR change remains questionable. DR-related decreases in glycemia are commonly reported in rats (Coleman et al., 1997
; Keenan et al., 1994a
,b
; Masoro et al., 1989
; Prescott-Mathews et al., 1998
; Roe et al., 1995
; Sachan and Das, 1982
; Snyder and Towne, 1989
) and support the widely held theory that DR acts by preventing long-term damage of fuel use from the glycation reaction (for review see Keenan et al., 1994b). Surprisingly, there was no DR-induced hypoglycemia in the present study. The increased urinary pH in DR rats has been described in various strains (Coleman et al., 1997
; Pickering and Pickering, 1984
; Prescott-Mathews et al., 1998
; Roe et al., 1995
) as has been the decrease in proteinuria (Prescott-Mathews et al., 1998
; Roe et al., 1995
; Tapp et al., 1989
; Wu et al., 1989
). Both DR regimens induced these changes in the present study. According to Roe et al. (1995), DR-related reduced proteinuria seen in rats correlates with the delay in the onset of CRD, and urinary pH proves to be one of the best early indicators of survival, with high urinary pH associated with high survival. In opposition to the present data, increases in urinary volume have been described in DR rats (Pickering and Pickering, 1984
; Roe et al., 1995
). However, on at least one occasion, the increase was almost certainly related to the times when the animals were fed and when the urine samples were collected (Roe et al., 1995
). Therefore, DR-related changes in absolute and relative (to BW) urinary volume should be further investigated in order to reach a conclusion.
The results of the present study confirm the beneficial effects of DR in rodents. AL-fed rats, with their early and high incidence of tumors and endocrine disturbances, cannot be regarded as appropriate models for detecting carcinogenicity relevant to humans (Abelson, 1992; Roe, 1981
, 1988
, 1993
; Roe and Lee, 1991
; Roe et al., 1995
). In addition, CRD being generally a common cause of death in AL-fed rats (Keenan et al., 1994b
; Roe et al., 1995
; Short and Goldstein, 1992
), the chronic damage and progressive loss of renal function may either mask a subtle compound-related nephrotoxicity or enhance renal injury to the remaining hypertrophied nephrons (Hard and Alden, 1992
; Keenan et al., 1994a
; Short and Goldstein, 1992
) and thus, compromise toxicity studies. Under moderate DR, BW gain is reduced, but growth remains regular, survival is improved and reaches the regulatory agencies' expectations in carcinogenicity studies, the incidence of tumors and diseases, including CRD, is reduced, and routine clinical pathology parameters are not significantly modified. Therefore, a moderate rate of DR of approximately 25% is recommended in carcinogenicity and toxicity assays conducted with SD rats. The adjustment of the DR rate to the different SD stocks should be made under the experimental conditions described in the present study. The condition of note is the determination of the adult AL food consumption mean values that should be made in single-housed rats given daily AL regular rodent diet.
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NOTES |
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