Kevin P. Keenan

The Effects of Diet, Overfeeding and Moderate Dietary Restriction on Sprague-Dawley Rat Survival, Disease and Toxicology

Overfeeding by ad libitum (AL) food consumption is the most significant, uncontrolled variable affecting the outcome of the current rodent bioassay. The correlation of food consumption, the resultant adult body weight and the 2-y survival in Sprague-Dawley rats is highly significant. Feeding natural ingredient diets that varied in protein, fiber and metabolizable energy content did not improve low 2-y survival if Sprague-Dawley rats were allowed AL food consumption. Moderate dietary restriction (DR) of all diets tested significantly improved survival and delayed the onset of spontaneous degenerative disease (i.e., nephropathy and cardiomyopathy) and diet-related tumors. By 2 y, moderate DR resulted in an incidence of spontaneous tumors similar to that seen with AL consumption; however, the tumors were more likely to be incidental and did not result in early mortality. There was a decreased age-adjusted incidence in pituitary and mammary gland tumors, but tumor volume and growth time were similar in the AL and DR groups, indicating a similar tumor progression with a delay in tumor onset. Moderate DR did not significantly alter drug-metabolizing enzyme activities or the toxicologic response to five pharmaceuticals tested at maximum tolerated doses (MTD). However, moderate DR did require higher doses of compounds to be given before classical MTD were produced with four pharmaceutical drug candidates. Toxicokinetic studies of two of these compounds demonstrated steady-state systemic exposures that were equal or higher in moderate DR-fed rats. These and other data indicate that moderate DR is the most appropriate method of dietary control for rodent bioassays used to assess human safety of candidate pharmaceuticals.

Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague-Dawley rats treated with tumorigenic doses of clofibrate

Clofibrate, a peroxisome proliferator, is hepatocarcinogenic in rats in a dose-dependent fashion. While there is a relationship between peroxisome proliferation and rodent liver carcinogenesis, recent evidence also suggests an association between the tumorigenicity of peroxisome proliferators and sustained cell proliferation. To investigate the role of early cell proliferation in clofibrate-induced carcinogenesis and the predictive potential of this endpoint, in a 3-month study, rats were fed clofibrate doses equivalent to those used in the chronic bioassay, and cell proliferation was determined after 1 week and 3 months, using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique. Adult Sprague-Dawley rats were fed clofibrate at 1500, 4500, or 9000 ppm. Six rats/sex/group were killed after 1 or 13 weeks of treatment. Osmotic minipumps containing BrdU were implanted into rats 7 days prior to necropsy to determine the cumulative 7-day hepatocyte labeling index immunohistochemically. A dose-related increase in hepatocyte labeling index was seen after 1 week of treatment. However, at 13 weeks, sustained increases in hepatocyte proliferation were not seen; but a dose-related decrease in the hepatocyte labeling index was observed. Liver stereology at 13 weeks demonstrated a dose-related increase in liver weight and volume, but a decrease in hepatocyte nuclei per unit volume, a minimal increase or no change in the total number of hepatocyte nuclei per liver, and an absolute decline in the total number of BrdU-labeled hepatocyte nuclei per liver. These data suggest that in rats, clofibrate may influence hepatocarcinogenicity by decreases in normal hepatocyte proliferation over time and this effect may influence the pathogenesis of tumors at time points beyond 13 weeks of treatment.

Need for dietary control by caloric restriction in rodent toxicology and carcinogenicity studies

The conditions under which laboratory animals are maintained can powerfully influence the results of toxicological studies utilized for risk assessment. Nutrition is of importance in toxicological bioassays and research, because diet composition and the conditions under which it is fed can affect the metabolism and activity of xenobiotic test substances and alter the results and reproducibility of long-term studies. It is known that ad libitum (AL) overfed sedentary laboratory rodents suffer from an early onset of degenerative disease and diet-related tumors that lead to poor survival in chronic bioassays. AL-fed animals are not well-controlled subjects for any experimental studies. Examination of study-to-study variability in food consumption, body weight, and survival in carcinogenicity studies for the same strain or stock of rodents shows tremendous laboratory-to-laboratory variability. However, a significant correlation between average food (calorie) consumption, adult body weight, and survival has been clearly established. The use of moderate dietary restriction (DR) results in a better controlled rodent model with a lower incidence or delayed onset of spontaneous diseases and tumors. Operationally simple, moderate DR significantly improves survival, controls adult body weight and obesity, reduces age-related renal, endocrine, and cardiac diseases, increases exposure time, and increases the statistical sensitivity of these expensive, chronic bioassays to detect a true treatment effect. A moderate DR regimen of 70-75% of the maximum unrestricted AL food intake is recommended as a nutritionally intelligent, well-established method in conducting well-controlled toxicology and carcinogenicity studies.

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay.

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed an increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequence of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.

HPLC-MS/MS determination of a peptide conjugate prodrug of doxorubicin, and its active metabolites, leucine-doxorubicin and doxorubicin, in dog and rat plasma

A HPLC-MS/MS Electrospray (ESI) method was developed and validated to quantify a peptide conjugate prodrug of doxorubicin (Dox-Con) and its active metabolites leucine-doxorubicin (Leu-Dox) and doxorubicin (Dox) in dog and rat plasma. The analytes were extracted from plasma by solid-phase extraction on a Bond Elut C8 cartridge and eluted with chloroform-methanol (2:1). Eluates were evaporated and reconstituted in acetonitrile-5 microM sodium trifluoroacetate in 0.1% aqueous formic acid (20:80) and injected onto a Waters Oasis HLB column. Analytes were eluted from the column with a solvent gradient into the mass analyzer. The ions were quantified in the selected reaction-monitoring mode (SRM), using positive ions, on a triple quadrupole mass spectrometer. The lower limits of quantification for Dox-Con, Leu-Dox, and Dox in plasma, were approximately 5, 1 (dog)/6 (rat), and 0.5 ng/ml, respectively. Intra- and inter-assay accuracy (% of nominal concentration) and precision (%CV) for all analytes were within 15 and 16%, respectively.

Effect of early body weight and moderate dietary restriction on the survival of the sprague-dawley rat

The effects of ad libitum (AL) feeding, moderate dietary restriction (DR), and initial (6-week) and one-year body weights on the two-year survival of the Sprague-Dawley (SD) rat were evaluated. DR-fed rats were given approximately 75 percent of the adult AL food intake. At two years, body weights of DR-fed males and females were approximately 69 and 58 percent of the AL-fed male and female body weights, respectively. The 2-year survival rate was 80 and 74 percent in DR-fed males and females, respectively, and 28 and 38 percent in AL-fed males and females, respectively. This increase in longevity indicates that DR-fed males and females in carcinogenicity studies would have 14.8 and 9.1 additional weeks of exposure in a 2-year period to test compounds, respectively, compared to AL-fed animals. There was no correlation between initial body weight and 2-year survival in DR or AL-fed rats. There was no association between 1-year body weight and 2-year survival among DR-fed rats. However, AL-fed rats with the greatest 1-year body weight had a lower 2-year average survival compared with the lightest AL-fed rats; this trend was statistically significant only in males. Body weights between the first and second years were statistically significantly correlated for both genders and feeding regimens but no correlation was observed between pretest and 2-year body weights. These findings demonstrate that initial body weight is not the determining factor of 2-year survival, but that the total adult food (caloric) intake is important. In conclusion, moderate dietary restriction prevented excessive body weight gain and greatly increased the 2-year survival of the SD rat. Initial body weights did not correlate to 2-year body weight gain and were not a predictive biomarker of 2-year SD rat survival.

The effects of overfeeding and moderate dietary restriction on Sprague-Dawley rat survival, pathology, carcinogenicity, and the toxicity of pharmaceutical agents

Ad libitum (AL) overfeeding is the most significant uncontrolled variable effecting the rodent bioassay. There is a highly significant correlation between food consumption, the resultant body weight, and two-year survival in laboratory rats. We have studied the effects of AL overfeeding, moderate dietary restriction (DR) and several modified diets on Sprague-Dawley (SD) rat longevity, spontaneous disease, carcinogenesis and the toxicity of pharmaceuticals. AL feeding of diets varying in protein, fiber and metabolizable energy content did not significantly alter two-year rat survival. Moderate DR (within the range of reported AL food intake) of all diets tested significantly improved survival and delayed the onset of spontaneous degenerative disease and diet-related tumors compared to AL-fed rats. Moderate DR resulted in a similar incidence of spontaneous tumors by 2 years, however, the tumors were more likely to be incidental and not result in early mortality. There was a decreased, age-adjusted incidence of pituitary and mammary gland tumors, but tumor volume and growth time was similar between AL and DR groups indicating similar tumor progression with a delay in tumor onset. Moderate DR did not change Phase I and Phase II drug metabolizing enzyme levels and did not significantly alter the toxicological response to 5 pharmaceuticals tested at maximum tolerated doses (MTDs). Additional studies with 4 pharmaceutical candidates did demonstrate that moderate DR allowed higher doses of compounds to be given before classical MTDs were observed. However, toxicokinetic studies of two of these compounds demonstrated steady state systemic exposures that were either equal of higher in the moderate DR fed rats. These and other data indicate that the moderate DR fed SD rat is a more appropriately controlled rodent model for toxicity and carcinogenicity studies to assess human safety of candidate pharmaceuticals.

Chapter 5 – Nutrition

Publisher Summary nThe environmental and nutritional conditions under which laboratory animals are maintained can powerfully influence the experimental results measured. Nutrition is of major importance in toxicological bioassays and research, because diet composition and the conditions under which it is fed can affect the metabolism and activity of xenobiotic test substances and alter the results and reproducibility of long-term studies. It is known that ad libitum (AL)-overfed sedentary laboratory rodents suffer from an early onset of degenerative disease and diet-related tumors that lead to poor survival in chronic bioassays. AL fed animals are not well-controlled subjects for any experimental studies. However, a significant correlation among average food (calorie) consumption, adult body weight, and survival has been clearly established. The use of moderate dietary restriction (DR) of a nutritionally balanced diet results in a better controlled rodent model with a lower incidence or delayed onset of spontaneous diseases and tumors. Operationally simple, moderate DR of balanced diets significantly improves survival, controls adult body weight and obesity, reduces age-related renal, endocrine and cardiac diseases, and reduces study-to-study variability, increases treatment exposure time, and increases the statistical sensitivity of these expensive, chronic bioassays to detect a true treatment effect.

Effects of Diet and Overfeeding On Body Weight and Survival in the Rodent Bioassay: the Impact On Pharmaceutical Safety Assessment

Uncontrolled body weight due to ad libitum (AL) overfeeding is the most significant variable affecting the rodent bioassay. The correlation between AL food consumption, the resultant excessive adult body weight, and the low 2-yr survival in Sprague-Dawley (SD) rats was highly significant. However, initial body weight does not correlate with adult body weight or survival. AL feeding of diets that varied in protein, fiber, and energy content did not improve low 2-yr survival. Only moderate dietary restriction (DR) of all diets tested significantly improved survival and delayed the onset or severity of spontaneous degenerative diseases and tumors. Moderate DR-fed rats had a similar incidence of tumors as AL-fed rats by 2 yr; however, the tumors were more likely to be incidental and not fatal. There was a decreased age-adjusted incidence of pituitary and mammary-gland tumors, but tumor volume and growth time were similar between AL and moderate DR groups. These data indicate a similar tumor progression, but only a delay in tumor onset. Moderate DR did not significantly alter drug-metabolizing enzyme activities nor the toxicologic response to five pharmaceuticals tested at maximum tolerated doses (MTDs). However, moderate DR did require higher doses of compounds to be given before classical MTDs were produced with four drug candidates. Toxicokinetic studies of two of these compounds demonstrated steady-state systemic exposures that were equal or higher in moderate DR-fed rats. Biochemical markers of oxidative stress (lipid peroxidation, protein oxidation) were decreased and cytoprotective markers were increased by moderate DR. These and other data indicate that moderate DR is the most appropriate method of body weight control when used to assess pharmaceuticals for human safety.