Over the past two decades, rodent bioassays have shown a steady increase in study-to-study variability, decreases in survival, and increases in the incidence, onset time and severity of degenerative diseases and tumors in most of the rodent species, strains and stocks currently used (Hart et al. 1995a and 1995b, Keenan et al. 1992, 1994a and 1995a, Lang 1991, Roe et al. 1995, Weindruch and Walford 1988). These adverse changes have been associated with increases in rodent weights, which seem to be influenced by selection for more rapid growth and greater fecundity but are also greatly influenced by excessive food intake (Masoro 1995, Yu 1995). The uncontrolled variable of allowing rodents to consume excessive energy and the complicating effects of this procedure on the design, results and interpretation of toxicology and carcinogenicity studies continues to be largely neglected by many regulators, toxicologists and pathologists (Hart et al. 1995a and 1995b).

Many toxicology laboratories have observed a steady but variable decline in survival in 2-y rodent carcinogenicity studies over the past 20 y that correlates with increased food consumption and adult body weight. This decline has been observed in the outbred Wistar and Sprague-Dawley rat stocks and in the formally long-lived inbred Fischer-344 rat strain (Hart et al. 1995, Haseman and Rao 1992, Keenan et al. 1992 and 1994a, Rao et al. 1990, Roe et al. 1995). This decreased survival has caused some to question the adequacy of studies that have less than 50% of the animals treated with the test substance for the full 2-y period because of high mortality. Potential statistical problems arise from evaluating treatment-related mortality in studies with low control survival (Keenan et al. 1994a, Keenan and Soper 1995). The simplest solution to the potential loss of statistical power is to increase 2-y survival to 50% or more, because the sensitivity of the bioassay to distinguish a true treatment effect from concurrent controls is greatly increased (Keenan and Soper 1995).

To improve poor rodent survival it is necessary to recognize that the cause of these early deaths is the early development of spontaneous tumors and severe degenerative disease, such as chronic nephropathy and cardiomyopathy, that are secondary to dietary (energy) overfeeding (Keenan and Soper 1995, Keenan et al. 1992, Roe et al. 1995). Although genetic, nutritional and environmental interactions are involved, laboratory rodent survival can be significantly improved by simple energy intake restriction or dietary restriction (DR). This procedure will improve survival and thus increase exposure time to a test compound and improve the statistical sensitivity of these bioassays to detect a true treatment effect during the test period.

MATERIALS AND METHODS

Table 1. Nutrient content of purina certified rodent diets1 [View Table]

In an ongoing study, Sprague-Dawley rats were fed Purina Certified Rodent Chow 5002 with AL access (Group 1), as daily measured amounts at approximately 75-80% of AL intake (Group 2), at approximately 70-75% of AL intake (Group 3), and at 50% of AL intake (Group 4). This latter 50% DR was the most severe tested in our laboratory and represents the range of intakes kJ/d (kcal/d) reported by Klurfield et al. (1989) who fed purified diets at a 40% DR to Charles River Sprague-Dawley rats.

RESULTS AND DISCUSSION

For example, restricting protein intake by 40% without restriction of calories in F-344 rats had minor effects on renal disease and longevity, but few other aging processes were changed (Iwasaki et al. 1988, Maeda et al. 1985, Masoro et al. 1989). However, a 40% caloric restriction without protein restriction was as effective as caloric restriction with protein restriction in increasing F-344 rat survival (Iwasaki et al. 1988, Maeda et al. 1985, Masoro et al. 1989). Similar studies in F-344 rats, Sprague-Dawley rats and Wistar rats with restriction of dietary protein, fat or minerals or increased fiber content without caloric restriction demonstrated no survival benefit (Keenan et al. 1994a, Masoro et al. 1992, Roe et al. 1995, Yu 1995). Thus, numerous studies have shown that dietary modifications alone do not improve survival if animals are allowed ad libitum food consumption. Restriction of fat, protein or carbohydrates without caloric restriction does not increase survival or decrease disease incidence or severity. The restriction of total energy intake per animal is the main factor that improves their longevity (Finch 1990, Hart et al. 1995b, Masoro 1995, Yu 1995, Weindruch and Walford 1988).

F-344 rats, DR-fed or with AL food intake, have similar rates of oxygen consumption per unit of lean body mass because body mass is rapidly reduced in the restricted animals (within 6 wk) in proportion to the reduction of energy intake (Masoro 1995, Masoro and McCarter 1991, Masoro et al. 1992). The DR-fed rats apparently reset their energy utilization mechanisms to be able to use glucose and insulin more efficiently (Masoro 1995, Masoro and McCarter 1991). This anti-aging action of DR does not seem to be a reduction in metabolic rate per unit of "metabolic mass" but is rather a reduction in the total amount of food consumed and thus energy intake per animal (Duffy et al. 1989, Masoro and McCarter 1991, Weindruch and Walford 1988).

In our ongoing studies of different levels of DR compared with AL food intake, metabolizable energy (ME) intake of Sprague-Dawley rats was compared with the predicted maintenance energy requirements determined from the equation ME, 470 kJ BW^0.75_kg/day (kcal per day = 112 BW^0.75_kg) (NRC 1995). As shown in Table 2, the actual ME intake for Sprague-Dawley females fed AL or DR was only slightly greater than the predicted maintenance energy requirement calculated for all groups except Group 4 (50% DR). In contrast, the actual ME intake of AL and DR-fed males was slightly less than the predicted maintenance energy requirements, with the greatest differences occurring in Group 4 (50% DR). It should be noted that the energy intake of the 50% DR groups in our study is consistent with the published energy intake using a 30-40% DR with this Sprague-Dawley stock (Klurfeld et al. 1989). These data indicate that AL-fed and moderate DR-fed Sprague-Dawley rats have remarkably similar ME maintenance energy requirements (NRC 1995).

Table 2. One-Year Sprague-Dawley rat average body weight, food consumption, energy intake and requirements1 [View Table]

Our laboratory was surprised by the wide interlaboratory variability in the Sprague-Dawley rats daily food consumption, body weights and 2-y survival (Keenan et al. 1994a). Subsequently, we studied the effects of AL food consumption (overfeeding) and moderate DR that was within the range of AL food intake in other laboratories (Keenan et al. 1994a). Feeding Sprague-Dawley rats diets varying in protein, fiber and energy content did not improve 2-y Sprague-Dawley rat survival above 50% if AL access to food was allowed (Keenan et al. 1994a). However, moderate DR did improve survival by delaying the onset and progression of spontaneous degenerative disease and tumors in Sprague-Dawley rats (Keenan et al. 1995a and 1995b). Nephropathy, a common diet-related disease in Sprague-Dawley, Wistar and F-344 rats that can be responsible for early non-tumor mortality, was greatly improved by moderate DR (Gumprecht et al. 1993, Keenan et al. 1995a). The greatest benefit gained from moderate DR in preventing the progression of chronic nephropathy in Sprague-Dawley rats seemed to be a result of caloric restriction and not protein restriction (Gumprecht et al. 1993, Keenan et al. 1995a). Our results are similar to those reported with Wistar rats (Roe et al. 1995) and F-344 rats (Masoro et al. 1989). Masoro et al. (1989) convincingly demonstrated that AL-fed F-344 rats developed more severe nephropathy than DR-fed rats given 1.7 times the protein intake per unit body mass.

The most common neoplastic cause of death in Sprague-Dawley rats are pituitary tumors in both sexes. Because pituitary tumors are prolactin secreting, mammary gland tumors in females are the second most common cause of death in Sprague-Dawley rats (Keenan et al. 1992, 1994a, 1994b and 1995b). Ad libitum-fed Sprague-Dawley rats of both sexes have an early development of hyperplastic, large pituitaries, higher DNA labeling indices and a high incidence of focal pituitary cell hyperplasias and adenomas compared with moderate DR groups when examined at 1-y intervals (Keenan et al. 1994a). These lesions lead to fatal tumors in the second year of life. This mortality lowers the statistical sensitivity of the bioassay to detect treatment-related tumors, particularly late-occurring ones (Keenan and Soper 1995). By increasing 2-y survival, statistical sensitivity of the bioassay increases, statistical analysis is simplified, and there is a significant increase in exposure time to treatment (Keenan and Soper 1995). In our Sprague-Dawley rat studies, 3-5 mo of additional time on study was gained during the 2-y period (Keenan et al. 1994a).

Many have expressed concern over the potential loss of sensitivity in carcinogenicity studies conducted with DR, because many studies do show that marked to severe DR prevents both spontaneous tumors and those induced by a given dose of carcinogen (Albanes 1987, Fishbein 1991, Hart et al. 1995b, Klurfeld et al. 1989, Kritchevsky 1993, Pollard and Luckert 1985). However, these studies used more severe energy restriction than we have proposed, usually with shorter (5-12 mo) endpoints that do not account for the delayed tumor onset under severe DR or for the confounding effects of tissue damage and early mortality induced by AL overfeeding on the potential development of induced tumors.

The spontaneous tumor incidence in moderately DR-fed Sprague-Dawley rats was in the range for AL-fed Sprague-Dawley rats, but most tumors were found incidentally at study termination, rather than at early necropsy (Keenan et al. 1995b). In our studies of mammary gland tumors, AL-fed females were the only rats that had tumors before 1 y, and they also had the highest incidence of proliferative lesions in the mammary gland at that time (Keenan et al. 1994a). By 2-y, the final overall incidences of benign and malignant mammary gland tumors were similar in all groups except for the 5002-9 DR females. A clear delay in the time of onset was evident in the DR groups, but the mean growth time and doubling time were not different (Keenan et al. 1995b). These results show that moderate DR by energy restriction of either diet caused a delay in the time of onset of mammary tumors but did not affect mammary tumor growth after initial palpation (Keenan et al. 1995b). Moderate DR resulted in only a decreased 2-y age-adjusted incidence of pituitary, mammary gland and islet cell tumors in these Sprague-Dawley rats fed either diet (Keenan et al. 1995b).

To explore the question of assay sensitivity, standard 14-wk toxicity studies with five pharmaceutical agents were conducted under AL feeding and moderate DR (Purina 5002 diet at 65% of AL food intake). The compounds were given orally by gavage, at the maximum tolerated doses (MTD) as follows: cyclosporine A [25 mg/(kg·d)], phenobarbital [100 mg/(kg·d)], clofibrate [500 mg/(kg·d)], L-647,318, a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor [150 mg/(kg·d)], and MK-458, a dopamine agonist [4 mg/(kg·d)] (Keenan and Soper 1995, Keenan et al. 1994b).

Although minor differences were observed between the AL and DR rats treated for 3 mo with the above agents, all of the expected treatment-related effects (clinical, biochemical, hematological and histopathological) were clearly observed under both feeding regimens (Keenan and Soper 1995, Keenan et al. 1994b). These results indicate that moderate DR did not alter the assay sensitivity to detect treatment-related toxicity or markedly alter drug metabolism of the above pharmaceutical agents given at MTD doses.

To determine whether dose selection under AL feeding conditions is appropriate for moderate DR conditions, we evaluated the dose toxicity responses of four additional investigational compounds under both AL and moderate DR conditions. Additionally, we determined the plasma toxicokinetics of the parent compound and the major metabolites of two of the evaluated drugs (Dixit et al. 1995). Although treatment-related, toxicological and pathological effects were clearly evident under both feeding regimens, there was also a shift in the dose response for all four compounds (Dixit et al. 1995). The moderate DR-fed Sprague-Dawley rats better tolerated larger doses of the drugs, and the estimated MTD and no observable effect levels (NOEL) were at least two- to fourfold greater than those of their AL counterparts. The plasma toxicokinetic profiles indicated that moderate DR animals had either similar or greater systemic exposure to the parent drug and its metabolites compared with their AL-fed counterparts (Dixit et al. 1995). These data indicated it is not appropriate to select doses in AL-fed rodents and then use these doses in DR-fed rodents in chronic carcinogenesis bioassays. However, we have found no adverse effects on drug absorption, metabolism or excretion using moderate DR as a controlled model (Dixit et al. 1995 and 1996, Keenan and Soper 1995).

To further determine whether the shift in the dose-response curve under moderate DR conditions was due to a healthier status of the animals, we evaluated numerous biochemical markers of oxidative stress and P450 isoenzymes, in Sprague-Dawley rats AL-fed and under moderate (70-80% AL) and marked (50%) DR feeding conditions. Although the P450 isozyme profiles were generally not altered under either DR regimen (Dixit et al. 1996), the biochemical markers of oxidative stress, including lipid peroxidation products (malondialdehyde, 4-hydroxy-2-E-nonenal), protein oxidation products (protein carbonyls) and superoxide dismutase were decreased and cytoprotective hepatic glutathione levels were increased in DR-fed animals compared with their AL-fed counterparts (Adams et al. 1995 and 1996, Dixit et al. 1996). A relatively small restriction in the amount of food given moderately DR-fed rats compared with AL-overfed rats significantly improves their health status (reduced oxidative stress) and makes them a more appropriate rodent model to evaluate subchronic and chronic toxicity of test agents.

It has been known since the 1930s that DR profoundly increases the survival of rodents and that DR produces this effect in all animals ranging from invertebrates to higher mammals (Finch 1990, Masoro 1995, McCay et al. 1935, Weindruch and Walford 1988, Yu 1995). The positive consequences of controlling energy intake have led some evolutionary biologists to consider AL-fed rodents to be examples of accelerated pathological senescence, whereas the supposedly postponed aging induced in DR-fed rodents really represents a more normal aging pattern as seen in other species (Finch 1990, Rose 1991). These and other observations have led several very prominent gerontologists to question the suitability of AL feeding as a standard method of husbandry in aging research (Finch 1990, Masoro 1995, Weindruch and Walford 1988). This issue extends beyond the fields of nutrition and gerontology and is pertinent to all fields of experimental biology, including toxicologic pathology and medicine. Increased energy intake resulting from overeating and decreased activity patterns has been identified as the major non-genetic extrinsic factor that contributes to human death in the United States after tobacco use (Manson et al. 1995, McGinnis and Foege 1993). Dietary factors associated with excessive energy intake in humans are also associated with the development of cardiovascular disease, diabetes mellitus and cancers of the colon, uterus, ovaries, breast and prostate (McGinnis and Foege 1993).

It is now clear that AL overfeeding of otherwise nutritious food is one of the most insidious, underestimated and significant factors increasing degenerative disease and tumors and decreasing survival in both humans and laboratory animals. Uncontrolled AL overfeeding of excessive energy needs to be recognized as one of the most important determinate errors in the current rodent bioassays that can compromise the usefulness of these risk-assessment studies. We do not advocate marked to severe DR, (e.g., 40-50% of AL energy intake) as an appropriate dietary control method for toxicologic or carcinogenesis studies; however, a moderate DR regimen (70-80% of the maximum unrestricted AL energy intake) will improve the long-term health of laboratory rodents. This simple and effective method makes a great deal of scientific sense for conducting well-controlled toxicology and pathology studies for human safety assessment.