Does Caloric Restriction Induce Hormesis?

Kevin P. Keenan, D.V.M., Ph.D.

Department of Safety Assessment WP45-222, Merck Research Laboratories,

West Point, PA 19486 Tel: 215-652-7714, Fax: 215-652-7758

E-mail: kevin_keenan@merck.com

In this issue Drs. Turturro, Hass and Hart discussed the biological responses of laboratory rodents to caloric restriction (CR) as an example of hormesis, and its implications for toxicological testing. Hormesis is referred to as a beneficial biological effect of lower doses of a factor or agent that is known to be detrimental at higher doses. This common phenomenon has been extensively discussed in past issues of the BELLE Newsletter and the concepts of hormetic dose-response relationships and chemical hormesis were recently reviewed (Calabrese and Baldwin, 1999a, 1999b). Controlling food intake to as much as 50-60% of that eaten by ad libitum (AL) fed rodents improves longevity, prevents or delays age-related diseases and delays the loss of many physiological processes (Weindruch and Walford, 1988, Masoro, 1995, 1996, Yu, 1994). Overnutrition from excessive caloric intake (AL overfeeding) is the primary dietary factor that accelerates aging and disease onset in laboratory rodents (Keenan, et al., 1994a, 1994b, 1996, 1998). This factor is as serious and poorly controlled an adverse event as the opposite extreme of severe food restriction leading to starvation or early death (Masoro, 1998). Moderate degrees of food restriction by CR without malnutrition extend life and prevent disease. Thus, CR meets the criteria of hormesis. The mechanisms of CR's effects on aging appear linked to the sustained reduction of intrinsic damaging agents (such as oxygen radicals) generated during normal food metabolism (oxidation processes and glycation reactions), in addition to sustained periods of protective, daily physiological hyperadrenocorticism induced by CR (Sabatino et al., 1991, Masoro, 1998).

The idea of CR acting as a co-hormetic agent in the presence of another nonnutritive extrinsic damaging agent (physical or chemical) is discussed and may be mechanistically linked through the enhanced protective action of glucocorticoids induced by sustained CR (Sabatino, et al., 1991; Masoro, 1998). Clearly, laboratory rodents maintained by CR are better able to withstand physical stressors such as surgical trauma, heat stress and inflammatory stimuli (Masoro 1996, 1995, 1998; Yu, 1994) as well as withstand increasing doses of xenobiotics (Duffy, et al., 1995, Keenan, et al., 1996). Moreover, the dose response curve showing the characteristics of an hormetic zone for nonessential, nonnutritive chemicals can be applied to CR (Calabrese and Baldwin, 1999b). The positive health effects of CR over the negative effects of either AL overfeeding or starvation (both forms of malnutrition) are hormetic.

The lack of appreciation of CR's hormetic dose-response effect is similar to the oversight of low dose hormetic effects of chemicals. This omission appears to be due to the practical emphasis of most toxicologic scientists on the upper end of the dose response curve in determining the maximum tolerated doses (MTD) and the no observable effect levels (NOEL). The selection of decreased body weight gain relative to untreated controls is used as the common endpoint for both general "toxicity" and MTD determination. However, the adverse effect of a decrease in body weight gain due to the multiple pathological mechanisms induced by high doses of exogenous substances should not be considered the same as the nontoxic, healthful effects of moderate CR in controlling excessive body growth and the numerous adverse effects of AL-overfeeding that include obesity, degenerative disease, early tumors and decreased longevity. The assumption that the controlled growth seen with CR is similar to the loss of body weight secondary to chemical toxicity is not correct. Furthermore, the mechanisms of organ toxicity and carcinogenicity of a specific chemical are not necessarily the same as those associated with age-related spontaneous disease and tumors.

The tendency to overlook dose-response relationships also applies to the consideration of the co-hormetic effects of CR and toxicity. The assumption that the typical CR technique of a 40% reduction in AL food intake is a consistent restriction across studies is not correct. AL food intake shows wide interlaboratory variability, resulting in a wide variation in body weights and survival from study to study, even with the same rodent stock, fed the same diet (Keenan et al., 1994a, l994b, Turturro, et al., 1995). We have observed a strong correlation between very low AL food intakes and improved survival in the same stock of Sprague-Dawley (SD) rat fed the same diet in numerous North American laboratories due to unintentional restricted feeders and other uncontrolled limitations of food intake (Keenan, et al., 1994b). Thus many laboratories have been conducting uncontrolled CR studies under so called "AL" feeding conditions. The variability in AL food intake accounts for much of the poor reproducibility seen in these bioassays as presently conducted (Keenan, et al., 1994b, 1998).

Excessive AL overfeeding results in declining survival which lowers the statistical sensitivity to detect treatment-related tumors, particularly late occurring ones (Keenan, et al., 1994a, 1996). By increasing 2 year survival with moderate CR, statistical sensitivity of the bioassay can be increased. Statistical analyses are simplified and there is a significant increase in the duration of treatment. In our studies of SD rats, 3 to 5 months of additional exposure time is gained by moderate CR in comparison to AL-fed animals in the course of a 2-year study (Keenan, et al., 1994a, 1994b, 1996).

Because CR can prevent both spontaneous tumors and those induced by a given dose of a chemical carcinogen there is concern over the co-hormetic effects of CR and the potential loss of carcinogenic sensitivity in bioassays (Klurfeld, et al., 1989; Kritchevsky, 1993; Keenan et al., 1996, 1998). However, a factor infrequently considered in studies of induced carcinogenesis in CR rodents is the effect of different degrees of CR on the dose selection of the test compound. Most studies of CR select arbitrary doses or determine doses of the test compound in young growing AL-fed animals and then test them in adult animals under varying levels of CR (Klurfeld, et al., 1989; Kritchevsky, 1993). The differences observed should not be surprising because CR-fed animals are more resistant to long-term metabolic injury and better able to handle the consequences of a xenobiotic load. Moderate CR in the SD rat does not significantly alter phase I and phase II drug metabolizing enzyme activities and has only minimal quantitative, but not qualitative, changes in the toxicological response to many pharmaceuticals given at an MTD (Keenan, et al., 1994a, 1996). When MTD's are determined in a CR fed model, higher MTD's will be obtained (Keenan, et al., 1996, 1998). Examination of 4 pharmaceutical candidates from AL overfed and moderate CR fed SD rats to determine MTD and NOEL doses and pharmacokinetic parameters demonstrated that the moderate CR fed animals were not only healthier but also better able to tolerate dosages of the pharmaceuticals, resulting in the estimated oral MTD's and NOEL's for these compounds being approximately 2-4 fold higher under these conditions. Interestingly, toxicokinetic studies of these compounds demonstrated steady state systemic drug and/or metabolic exposures in moderate CR fed animals that were either equal to or higher than those in their AL fed counterparts (Keenan, et al., 1996, 1998). Therefore, to understand the hormetic effect of a chemical or the co-hormetic effect of CR it is necessary to control the degree of CR employed in the dose selection process.

The effects of CR in the reproductive system illustrate both dose effects and species differences and the response to reduced energy intake. In the example that Turturro, et al., use of C57BL/6J mice placed on an alternative day feeding schedule to achieve CR, it was shown the CR-fed females had a delayed loss of estrous cyclicity and maintained the number of primary ovarian follicles for a longer time (Nelson et al., 1985). But in CD-1 Swiss mice maintained by CR at 90, 80 and 70% of their adult AL body weight for 21 weeks, estrous cyclicity was not affected by the mildest amount of CR, while cyclicity was affected in the more severe CR groups. All of the CR females had dose-related decreases in various fertility parameters while on CR (Chapin et al., 1993a). In contrast, studies in adult SD rats subjected to CR to maintain 90, 80 or 70% of their AL-fed body weight up to 17 weeks were largely resistant to adverse reproductive changes with no effects being seen in fertility and only a transient increase in the length of estrous cyclicity during the initiation of CR in the most severely restricted group (Chapin et al., 1993a, 1993b). We have observed little or no effect of moderate CR on the estrous cyclicity of SD rats when restricted to 75 or 80% of their adult AL-intake and further observed a similar pattern of reproductive senescence in these animals compared to their AL-fed counterparts. In contrast, a 50% CR in this stock extended the time of their estrous cyclicity and appeared to delay their reproductive senescence. Compared to AL feeding, moderate CR fed females did not show a difference in their pattern of reproductive senescence through one year. It was only with a marked CR (50% of AL) that a delay in reproductive senescence was observed. By one year, 45% of the marked CR females still exhibited normal cyclicity compared to only 12% of the AL or moderate DR fed females (Keenan et al., 1996). For this reason, the species as well as the degree of CR must be considered in any evaluation of the hormetic or co-hormetic effects of CR on reproductive parameters and related endocrinological modulations.

The overall case that Turturro, et al. present for CR inducing hormesis is reasonable, but should not be confused with the hormetic effects induced by xenobiotics. Moderate CR, without malnutrition, maintains normal physiological systems in a healthy state and avoids the accelerated aging induced by AL-overfeeding. Moderate CR allows control of dietary intake and permits a more precise measurement of the effects of a test compound with a lower signal to noise ratio from background diseases. The major concern with attempts to link CR's effects and chemical effects is over changes in body weight. The adverse effects of a toxic body weight loss from xenobiotics is not the same as the healthy control of growth seen with moderate CR. The toxic effects of a compound on body weight can best be determined if the controls and treated groups are given the same amount of feed and energy by moderate CR (Keenan, et al., 1998). This simple procedure allows the patterns of xenobiotic toxicity to be clearly distinguished from age-related disease and mortality without the need for additional statistical adjustments for mortality or body weight loss as suggested by others (Gaylor and Kodell, 1999). This method of moderate CR would best control the potential hormetic effects of CR and allow a clear detection of both the high-dose toxic and low-dose hormetic effects of xenobiotics in these bioassays.

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