Ronald W. Hart

Longevity, Body Weight, and Neoplasia in Ad Libitum-Fed and Diet-Restricted C57BL6 Mice Fed NIH-31 Open Formula Diet

Groups of C57BL6 mice of each sex were assigned to one of 2 dietary regimens, ad libitum (AL) or dietary restriction (DR), to study effects of food restriction on body weight, survival, and neoplasia. The AL and DR groups were subdivided into a scheduled sacrifice group for examination at 6-mo intervals, and a lifetime group to provide longevity data. Necropsies and microscopic examinations were conducted on 911 animals. In the lifetime group food consumption averaged 33.6 and 34.4 g per week by AL males and AL females, respectively; the DR counterparts were given 40% less. The diet contained 4.35 kcal/g. The average lifetime body weights were 34.8, 26.8, 22.6, and 21.6 g for AL males, AL females, DR males, and DR females, respectively, and their age at 50% survival was 27.5, 26.9, 31.7, and 33.5 mo. Maximal lifespan was increased 18% in DR males and females. Lifetime incidence of tumor-bearing mice was 89% and 86% for AL males and females, versus 64% for each sex of DR mice. Dramatic reduction occurred in female DR mice in lymphoma (9% vs 29%), pituitary neoplasms (1% vs 37%), and thyroid neoplasms (0.4% vs 8%). In males, hepatocellular tumors were reduced to 1% from 10% by DR. In contrast, the incidence of histiocytic sarcoma was increased in DR females and unaffected in DR males. Tumor onset was delayed in DR animals; 87% of all neoplasms in males and 95% in females had occurred in the AL mice by 24 mo, whereas the DR animals had only 52% and 39% of their lifetime incidence, respectively, by that age. This study provided comparative AL and DR data from C57BL6 mice examined randomly at 6-mo intervals (cross-sectional group) in parallel with data from animals in similar cohort that was unsampled and allowed to succumb naturally (longevity group). Dietary restriction reduced the lifetime percentage of tumor-bearing animals and the number of tumors per animal, and delayed the age at onset of most neoplasms.

Pesticide induced DNA damage and its repair in cultured human cells

The effects of pesticides on the induction of unscheduled DNA synthesis in SV-40 transformed human cells (VA-4) in culture with and without metabolic activation by liver microsomes was studied. Results showed that ten of the thirteen compounds examined either directly or upon metabolic activation induced unscheduled DNA synthesis in the human cell system used. The DNA repair kinetics and size of the repaired regions resulting from treatment with four of the chemicals (Carbaryl, Chlordane, Dieldrin and 2.4-D Fluid) were studied by 313 nm photolysis of repaired regions containing bromodeoxyuridine (BUdR). The size of the repaired regions differed between compounds but could generally be classified as either of the X-ray (short) or UV-type (long).

Longevity, stability and DNA repair

The functional capacity of a cell, tissue, organ, or organism is dependent upon its ability to maintain the stability of its unit components. The higher the differentiated state of the system, the greater the amount of stability required to maintain that state as a function of time. Stability can be achieved via either redundancy or repair. Redundancy while easily achievable in biological systems is both costly and limited by thermodynamic considerations. Repair, in its general sense, has no such limitations. Repair at the cellular and macromolecular level is multiple in its forms and varies as a function of species, tissue, and stage of the cell cycle. The repair of DNA damage is a dynamic process with many components and subcomponents, each interacting with one another in order to achieve a balance between individual stability and evolutionary diversity. Thus, between internal and external factors which damage DNA and the subsequent expression of alterations in the functional stability of DNA lie the multi-functional pathways which attempt to maintain DNA fidelity. A strong correlation between ulta-violet light induced excision or pre-replication repair, as measured by autoradiogrphy and maximum species lifespan has been reported within different strains of the same species, between related species (e.g. Mus musculus and Peromyscus leucopus), between five orders of mammals, and most recently within members of the primate family. As has been demonstrated by the authors and others, differences in excision repair between species and tissues may relate to the turning off of portions of the repair processes during embryogenesis. Regardless of why such correlations exist or the nature of their mechanisms, it is naive to either assert or deny a causal role for DNA repair in longevity assurance systems. For example, while species-related differences in DNA repair may reflect the turning off of such repair processes during fetal development this does not mean that rates of accumulation of DNA damage are not altered by such changes. Indeed, such a phenomena might well explain the rapid evolution of lifespan within the primates without a concurrent input of new genes.

Survival, body weight, and spontaneous neoplasms in ad Libitum-fed and food-restricted Fischer-344 rats.

Ad libitum- fed (AL) and food-restricted (FR) Fischer-344 male and female rats were monitored for survival, body weight, and spontaneous neoplasms. Mean and maximal lifespans for each group were inversely related to mean body weights. AL males were the shortest lived (mean lifespan 101 wk) followed by AL females (118 wk), FR males (125 wk), and FR females (132 wk). Gross and microscopic examinations were performed on 851 rats from cross-sectional and longevity components of the study. In FR groups, the incidence of mammary gland fibroadenomas, testicular interstitial cell tumors, and pituitary neoplasms was decreased while the latency of these neoplasms was increased. In longevity components, most FR groups had a higher incidence of leukemia than AL cohorts, but all FR groups had a higher mean age at death for the rats with leukemia. Higher leukemia rate in the FR groups was thought to be a result of their extended mean lifespan.

Age-Related Neoplasia in a Lifetime Study of Ad Libitum-Fed and Food-Restricted B6C3F1 Mice

Longevity, body weight, and age-specific neoplasia were determined in 1,064 B6C3F1 mice as part of a coordinated study of food restriction (FR). Restricted animals were offered 60% of the diet consumed by the ad libitum (AL) group. Longevity data were derived from a set of 56 animals of each sex from each diet group, which were examined whenever dead or moribund. For cross-sectional data, a parallel set of 210 animals were sacrificed in groups of 12-15 at 6-mo intervals. Lifetime body weight was reduced in the FR mice approximately proportional to restriction (i.e., 40%). Food restriction increased the age at 50% survival (median) by 36% in both sexes and increased the maximal lifespan (mean age of oldest 10%) by 21.5% in males and by 32.5% in females. In 56 males of the longevity groups, there were 89 neoplasms in the AL subgroup versus 53 in FR; 56 AL females had 100, versus 58 in 55 FR females. Increase in lifespan of the restricted animals was achieved primarily by decrease in incidence and delay of onset of fatal tumors, of which lymphoma was the most prominent. This report catalogs all of the neoplasms (1,103) observed in longevity and cross-sectional groups, by diet, sex, and age. These data add to the existing knowledge base needed for future studies of dietary restriction and aging as well to evaluate nutrition of animals used in bioassays.

Role of Glucocorticoids and “Caloric Stress” in Modulating the Effects of Caloric Restriction in Rodentsa

Caloric restriction (i.e., the balanced reduction of the protein, carbohydrate and fat content of the diet without reduction of its micronutrient content) has been shown, in rodents, to be an extremely powerful modulator of a broad spectrum of age-associated degenerative diseases as well as life span.’-5 Caloric restriction has been found to delay the occurrence of many age-associated neoplastic diseases or to slow their progression, often to such an extent that the clinical expression of the diseases is eliminated.* Since reducing individual dietary components such as protein or fat without reducing the overall caloric intake is less effective in increasing longevity or suppressing neoplasia than caloric restriction by itself, it is reasonable to assume that the observed effects of caloric restriction are primarily dependent upon a specific reduction in calories and not specific dietary compon e n t ~ . ~ ~ ’ The multiple actions of caloric restriction suggest that it may influence primary aging processes themselves; however, recent examination of pathological data suggests that caloric restriction delays different age-dependent pathological conditions at different rates.8 This observation would argue against the modulation by caloric restriction of a simple fundamental process of cellular aging, but rather, it implies that caloric restriction modifies the organism’s homeostatic set-point to one that is less susceptible to some but not all pathologies. Unfortunately, such theories on the nature of the caloric restriction effect will remain merely speculatory until the precise molecular and biochemical mechanisms by which caloric restriction influences the processes of aging and disease are fully elucidated.

Rationale for the Use of Dietary Control in Toxicity Studies—B6C3F1 Mouse:

Significant variability in critical study parameters such as tumor incidences and survival, increasing tumor incidence and decreasing survival in common toxicity test models, and agent-induced changes in body weight (BW) and BW distribution all generate concern about the reproducibility, consistency, and equity of chronic toxicity tests used in regulation. These concerns have led to suggestions to control BW in chronic tests by the modulation of dietary intake without inducing malnutrition [dietary control (DC)] thereby minimizing tumor and survival variability both between and within studies. Evaluating the reports of the best controlled set of chronic experiments, the National Toxicology Program bioassay series, from studies initiated from 1981 to 1990, there is an increase in tumor incidence, especially liver tumors, with a consistent increase in BW. The studies are classified as to whether normal or aberrant BW growth curves occur. When the studies with normal growth curves are considered, the variance in the BW at 12 mo on test (BW12) can account for over 50% of the variance in liver tumor incidence. Additional stratification by study type, which alter tumor prevalences, as well as appreciation of housing effects [group housing decreases survival (in male mice) and induces tumors in males and females when compared to individual housing], further increase the strength of the correlations, accounting for up to 90% of the variance seen in tumor incidences. These updated analyses further support the hypothesis that it is the BW variation that is resulting in much of the variability seen in tumor incidences and refine the suggestions for the BW curves used as the desired targets for DC.

DNA REPAIR AND MUTAGENESIS IN MAMMALIAN CELLS

Abstract— Mutations may result from imperfect DNA replication, unrepaired DNA damage, and errorprone DNA repair. DNA damage may induce any or all of these effects. Numerous physical and chemical agents damage DNA, and the repair of such damage may be either incomplete, error‐free, or error‐prone. It is assumed that correctly repaired DNA damage has no deleterious biological consequence. Unrepaired or misrepaired DNA damage has been related to such physiological changes as cell death, division suppression, gene repression and derepression, altered transcription, elevated cAMP levels, predisposition of the cell to viral transformation, decreased cellular respiration, and mutation. Defective DNA repair has been linked in man to cancer, birth defects, arteriosclerosis, high blood pressure, aging, and neurological dysfunction. Various clinical syndromes including xeroderma pigmentosum (XP), ataxia telangiectasia (AT), Fanconis anemia (FA), progeria, actinic keratosis (AK), and possibly Cockayne syndrome (CS) have been described as being defective in at least one of the various forms of DNA repair.

FDA Points-to-Consider Documents: The Need for Dietary Control for the Reduction of Experimental Variability within Animal Assays and the Use of Dietary Restriction to Achieve Dietary Control

Standard protocols for conducting chronic toxicity and carcinogenicity studies have been refined over the years to carefully control for many variables. Nevertheless, over the last 2 decades, there has been a steady increase in variability, a decrease in survival, an increase in tumor incidence rates, and an increase in the average body weight of control animals among the various rodent species and strains used for toxicity testing. These observations have prompted an evaluation of chronic study designs to determine what factor(s) may be responsible for such confounding changes. Ad libitum feeding and the selection of successful breeders with rapid offspring growth is believed to be at least partially responsible for the heavier, obese rodents with which many laboratories are coping today. As a result of these changes, some studies used for the evaluation of safety have been deemed inconclusive or inadequate for regulatory purposes and either additional supportive studies have been requested and/or studies per se have been repeated. Research on the molecular mechanisms of caloric restriction and agent-induced toxicity at the Food and Drug Administration (FDA) National Center for Toxicological Research stimulated the first international conference on the biological effects of dietary restriction in 1989; this was followed in 1993 by an FDA workshop exploring the utility of dietary restriction in controlling reduced survival in chronic tests and an international conference in 1994 exploring the implications for the regulatory community of using dietary restriction in toxicity and carcinogenicity studies used in support of a sponsors submission or in risk assessments. The outcome of that conference was the FDAs commitment to develop Points-to-Consider documents that address the issue of dietary control in chronic toxicity and carcinogenicity studies.

Effects of Cell Density on Lipids of Human Glioma and Fetal Neural Cells

Abstract: Gangliosides, phospholipids, and cholesterol of human glioma (12‐18) and fetal neural cells (CH) were analyzed at specified cell densities, from sparse to confluent. Total ganglioside sialic acid, phospholipid phosphorus, and cholesterol increased in the glioma cells on a per cell, mg protein, or mg total lipid basis two‐ to threefold as cell density increased 25‐fold. These same three constituents in the fetal cells increased with cell density on a per cell and mg protein basis but not on a per mg total lipid basis. In glioma cells, the di‐ and trisialogangliosides (GD2+ GDlb+ GT1) increased from 1–2% of total ganglioside sialic acid at sparse densities to 7–8% at intermediate (logarithmic phase) densities to 10–13% at confluent densities. The set of simpler gangliosides (GM4+ GM3+ GM2) decreased from 50% of total ganglioside sialic acid at sparse glioma cell densities, to 36% at intermediate and 30% at confluent densities. In the fetal neural cells, the set of gangliosides (GM4+ GM3+ GM2) had about 48% of total ganglioside sialic acid in both sparse and confluent preparations. The fetal cells were twofold higher in GM3 (32.4 ± 2.1%) than the glioma cells (16.8 ± 1.6%), but lower in GMt (9.1 ± 0.9% versus 18.2 ± 1.8%), cell densities notwithstanding. Confluent cell preparations of both cell lines were consistently higher in ethanolamine plasmalogen than sparse cells. We conclude that in these two neural cell lines quantitative changes in ganglioside and phospholipid species occurred correlatively as cell densities increased. Higher glioma cell densities were associated with greater proportions of complex ganglioside species. These changes in cell membrane constituents during growth may result from cell contact and may indicate a role for them in cell growth regulation and/or differentiation.