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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O more...

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10−5, 5 × 10−3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9–10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK. more...

Bisphenol A (BPA) is a ubiquitous industrial chemical that has been identified as an endocrine disrupting compound (EDC). There is growing concern that early life exposures to EDCs, such as BPA, can adversely affect the male reproductive tract and function. This study was conducted as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) to further delineate the toxicities associated with continuous exposure to BPA from early gestation, and to comprehensively examine the elicited effects on testes and sperm. NCTR Sprague Dawley rat dams were gavaged from gestational day (GD) 6 until parturition, and their pups were directly gavaged daily from postnatal day (PND) 1 to 90 with BPA (2.5, 25, 250, 2500, 25,000, 250,000 μg/kg/d) or vehicle control. At PND 90, the testes and sperm were collected for evaluation. The testes were histologically evaluated for altered germ cell apoptosis, sperm production, and altered spermiation. RNA and DNA isolated from sperm were assessed for elicited changes in global mRNA transcript abundance and altered DNA methylation. Effects of BPA were observed in changes in body, testis and epididymis weights only at the highest administered dose of BPA of 250,000 μg/kg/d. Genome-wide transcriptomic and epigenomic analyses failed to detect robust alterations in sperm mRNA and DNA methylation levels. These data indicate that prolonged exposure starting in utero to BPA over a wide range of levels has little, if any, impact on the testes and sperm molecular profiles of 90 day old rats as assessed by the histopathologic, morphometric, and molecular endpoints evaluated. more...

Diet is an important variable in toxicology. There are mixed reports on the impact of soy components on energy utilization, fat deposition, and reproductive parameters. Three generations of CD-1 mice were fed irradiated natural ingredient diets with varying levels of soy (NIH-41, 5K96, or 5008/5001), purified irradiated AIN-93 diet, or the AIN-93 formulation modified with ethanol-washed soy protein concentrate (SPC) or SPC with isoflavones (SPC-IF). NIH-41 was the control for pairwise comparisons. Minimal differences were observed among natural ingredient diet groups. F0 males fed AIN-93, SPC, and SPC-IF diets had elevated glucose levels and lower insulin levels compared with the NIH-41 group. In both sexes of the F1 and F2 generations, the SPC and SPC-IF groups had lower body weight gains than the NIH-41 controls and the AIN-93 group had an increased percent body fat at postnatal day 21. AIN-93 F1 pups had higher baseline glucose than NIH-41 controls, but diet did not significantly affect breeding performance or responses to glucose or uterotrophic challenges. Reduced testes weight and sperm in the AIN-93 group may be related to low thiamine levels. Our observations underline the importance of careful selection, manufacturing procedures, and nutritional characterization of diets used in toxicological studies. more...

Bisphenol A (BPA) is a chemical used in the production of numerous consumer products resulting in potential daily human exposure to this chemical. The FDA previously evaluated the body of BPA toxicology data and determined that BPA is safe at current exposure levels. Although consistent with the assessment of some other regulatory agencies around the world, this determination of BPA safety continues to be debated in scientific and popular publications, resulting in conflicting messages to the public. Thus, the National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), and U.S. Food and Drug Administration (FDA) developed a consortium-based research program to link more effectively a variety of hypothesis-based research investigations and guideline-compliant safety testing with BPA. This collaboration is known as the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). This paper provides a detailed description of the conduct of the study and a midterm update on progress of the CLARITY-BPA research program. more...

Bisphenol A (BPA), an endocrine disrupting chemical (EDC), is a ubiquitous pollutant. As part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), we sought to determine whether exposure of Sprague-Dawley rats to 2,500 μg/kg/day BPA (BPA) or 0.5 μg/kg/day ethinyl estradiol (EE) from gestational day 6 through postnatal day 21 induces behavior-relevant gene expression and DNA methylation changes in hippocampus and hypothalamus at adulthood. RNA and DNA were isolated from both regions. Expression of ten genes (Dnmt1, Dnmt3a, Dnmt3b, Esr1, Esr2, Avp, Ar, Ot, Otr, and Bdnf) presumably altered by early-life BPA/EE-exposure was examined. Three genes (Bdnf, Dnmt3b, and Esr1) were studied for DNA methylation changes in their putative 5ʹ promoter regions. Molecular changes in hippocampus were correlated to prior Barnes maze performance, including sniffing correct holes, distance traveled, and velocity. Exposure to BPA and/or EE disrupted patterns of sexually dimorphic gene expression/promoter DNA methylation observed in the hippocampus and hypothalamus of controls. In the hippocampus of female offspring, BPA-exposure resulted in hypermethylation of the putative 5ʹ promoter region of Bdnf, while EE exposure induced hypomethylation. Bdnf methylation was weakly associated with Bdnf expression in hippocampi of female rats. Hippocampal Bdnf expression in females showed a weak negative association with sniffing correct hole in Barnes maze. Hippocampal expression of Avp, Esr2, Oxt, and Otr was strongly associated with velocity of control rats in Barnes maze. Findings suggest BPA exposure induced non-EE-like gene expression and epigenetic changes in hippocampus of adult rats, a region involved in spatial navigation. more...

We report the data from the guideline-compliant two-year toxicology study conducted as part of the Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA). BPA (0, 2.5, 25, 250, 2,500, and 25,000 μg/kg body weight (bw)/day) was administered daily by gavage in 0.3% carboxymethylcellulose vehicle to NCTR Sprague-Dawley rats from gestation day 6 through the start of parturition and then directly to pups from the day after birth until postnatal day 21 (stop-dose arm) or continuously until termination at one or two years. The stop-dose arm was included to assess the potential for any BPA effects that were due to developmental exposure. No BPA-related effects were evident in the in-life and non-histopathology data. Neoplastic and nonneoplastic lesions diagnosed in both females and males were common age-associated lesions that were variable across control and BPA-treated groups. The lack of consistent responses within the continuous- and stop-dose arms within and across tissues brought into question the plausible relationship of most of these lesions to BPA treatment. There was a possible relationship between the increased incidences of lesions in the female reproductive tract and the male pituitary and exposure to the 25,000 μg BPA/kg bw/day dose level. more...

To assess a drug's toxic or carcinogenic effects on neonatal and adult mice and rats, researchers often carry out oral gavage studies. Whether dosed singly or in various combinations, provided as soluble solutions or as colloidal suspensions, the drug must be delivered in accurate and precise doses. For studies that require newborn mice to receive multiple daily doses, delicately handling neonates to increase their chances of surviving is just as critical as the ability to accurately dose small volumes. To help ensure accurate and precise delivery of drug doses ranging from 5 microl for neonatal mice to 400 microl for adults, the authors adapted an automated pipetting system. By slightly modifying standard gavage needles, the authors delivered, on average, 98-99% of targeted dose volumes to neonatal mice. more...

Usnic acid is a prominent secondary lichen metabolite that has been used for various purposes worldwide. Crude extracts of usnic acid or pure usnic acid have been marketed in the United States as dietary supplements to aid in weight loss. The US Food and Drug Administration (FDA) received 21 reports of liver toxicity related to the ingestion of dietary supplements that contain usnic acid. This prompted the FDA to issue a warning about one such supplement, LipoKinetix, in 2001 (http://www.cfsan.fda.gov/~dms/ds-lipo.html). Subsequently, usnic acid and Usnea barbata lichen were nominated by the National Toxicology Program (NTP) for toxicity evaluations. At present, a toxicological evaluation of usnic acid is being conducted by the NTP. This review focuses on the recent findings of usnic acid-induced toxicities and their underlying mechanisms of action. more...

Bisphenol A (BPA) is a ubiquitous industrial chemical used in the production of a wide variety of items. Previous studies suggest BPA exposure may result in neuro-disruptive effects; however, data are inconsistent across animal and human studies. As part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), we sought to determine whether female and male rats developmentally exposed to BPA demonstrated later spatial navigational learning and memory deficits. Pregnant NCTR Sprague-Dawley rats were orally dosed from gestational day 6 to parturition, and offspring were directly orally dosed until weaning (postnatal day 21). Treatment groups included a vehicle control, three BPA doses (2.5μg/kg body weight (bw)/day-[2.5], 25μg/kg bw/day-[25], and 2500μg/kg bw/day-[2500]) and a 0.5μg/kg/day ethinyl estradiol (EE)-reference estrogen dose. At adulthood, 1/sex/litter was tested for seven days in the Barnes maze. The 2500 BPA group sniffed more incorrect holes on day 7 than those in the control, 2.5 BPA, and EE groups. The 2500 BPA females were less likely than control females to locate the escape box in the allotted time (p value=0.04). Although 2.5 BPA females exhibited a prolonged latency, the effect did not reach significance (p value=0.06), whereas 2.5 BPA males showed improved latency compared to control males (p value=0.04), although the significance of this result is uncertain. No differences in serum testosterone concentration were detected in any male or female treatment groups. Current findings suggest developmental exposure of rats to BPA may disrupt aspects of spatial navigational learning and memory. more...

Background and aims: The primary purpose of the present study was to investigate the effects of 10, 25, and 40% dietary restriction (DR) on non-neoplastic diseases in rodents at 58 and 110 weeks of age, and to determine whether low-level DR (10 and 25%) can increase the survival rate and decrease variability in chronic bioassay studies. Methods: Male Sprague-Dawley (SD) rats (NCTR colony) were divided into four nutritional groups, consisting of an ad libitum (AL) group with unlimited access to the NIH-31 diet, and three dietary restricted (DR) groups given the NIH-31 diet reduced in amount by 10, 25, and 40%. Results: At 110 weeks of age, the incidence of cardiomyopathy was 95, 75, 45, and 15% for AL and 10, 25, and 40% DR rats, respectively; the incidence of nephropathy was 55, 20, 15, and 0% for AL and 10, 25, and 40% DR rats, respectively. The severity of chronic heart and kidney diseases was significantly reduced in all DR rat groups, with significant DR-dependent linear trends for these diseases. Moreover, DR prevented the progression of skin irritation to foot ulcers, and reduced the age-related degeneration in the adrenal, lacrimal, and thymus glands, and the liver. Conclusions: These results clearly indicate that even low DR levels were effective in preventing or slowing the progression of these non-neoplastic diseases. more...

Background and aims: Daily nutrient intake and growth of male Sprague-Dawley (SD) rats fed compositionally different diets were monitored over 114 weeks to determine whether rats fed ad libitum (AL) or diet restricted (DR) followed normal growth parameters. A second objective was to evaluate the usefulness of the American Institutes of Nutrition’s AIN-93M (maintenance formulation) diet for aging and DR studies. Methods: Rats were fed NIH-31 cereal-based diet AL, or a vitamin-fortified modification of the NIH-31 diet at 10, 25, or 40% DR. Other SD rats were fed AIN-93M diet AL or 31% DR; daily nutrient intake and growth response were reported. Results: At all intervals up to 36 weeks of age, rats fed AL the NIH-31 diet consumed significantly (p more...

Survival, growth and dietary intake (DI) variables were monitored in a chronic 114-wk study in which male Sprague-Dawley rats [n = 120; National Center for Toxicological Research (NCTR) colony] consumed the AIN-93M purified diet ad libitum (AL), or an amount reduced by 31% of total AL intake inclusive of all macro- and micronutrients. The main objectives were to ascertain the survival characteristics of rats fed the AIN-93M diet and to determine whether dietary restriction (DR) increases longevity of rats fed this casein-based diet compared with the use of mixed-protein sources of the NIH-31 cereal-based diet in an earlier study. Body, liver, brain, the brain/body ratio, spleen, thymus and kidney weights, body length and body density were decreased (P < 0.05) by DR, whereas testis weight and skull length were not altered by DR. Significant age effects at 58 and 114 wk were found for body, brain, the brain/body ratio, liver and testis weights, and body density. Survival rates for the AL and 31% DR groups were 43.3 and 57.5%, respectively. Survival curves were not significantly different. The survival rate for AL rats fed the AIN-93M diet was not different from that of AL rats fed the NIH-31 diet (43.3 and 51.7%, respectively). However, the survival rate for 31% DR rats fed the AIN-93M diet was significantly lower than 25% DR rats fed the NIH-31 diet (57.5 and 87.5%, respectively) although both groups had similar body weights and energy intake at various ages. Nutritional components in the NIH-31 diet that are missing and/or reduced in the AIN-93M diet may interact with DR to increase 114-wk survival. Although the survivability, growth and anatomical results of this study suggest that the AIN-93M diet is suitable for chronic rodent studies, additional studies such as comprehensive histopathologic and physiologic investigations must be undertaken to complete the evaluation process. more...

Bisphenol A (BPA), an industrial chemical used in the manufacture of polycarbonate and epoxy resins, binds to the nuclear estrogen receptor with an affinity 4–5 orders of magnitude lower than that of estradiol. We reported previously that “high BPA” [100,000 and 300,000 µg/kg body weight (bw)/day], but not “low BPA” (2.5–2700 µg/kg bw/day), induced clear adverse effects in NCTR Sprague-Dawley rats gavaged daily from gestation day 6 through postnatal day (PND) 90. The “high BPA” effects partially overlapped those of ethinyl estradiol (EE 2 , 0.5 and 5.0 µg/kg bw/day). To evaluate further the potential of “low BPA” to induce biological effects, here we assessed the global genomic DNA methylation and gene expression in the prostate and female mammary glands, tissues identified previously as potential targets of BPA, and uterus, a sensitive estrogen-responsive tissue. Both doses of EE 2 modulated gene expression, including of known estrogen-responsive genes, and PND 4 global gene expression data showed a partial overlap of the “high BPA” effects with those of EE 2 . The “low BPA” doses modulated the expression of several genes; however, the absence of a dose response reduces the likelihood that these changes were causally linked to the treatment. These results are consistent with the toxicity outcomes. more...

Concerns have been raised regarding the long-term impacts of early life exposure to the ubiquitous environmental contaminant bisphenol A (BPA) on brain organization. Because BPA has been reported to affect estrogen signaling, and steroid hormones play a critical role in brain sexual differentiation, there is also concern that BPA exposure could alter neural sex differences. Here, we examine the impact of subchronic exposure from gestation to adulthood to oral doses of BPA below the current no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day on estrogen receptor (ESR) expression in sexually dimorphic brain regions of prepubertal and adult female rats. The dams were gavaged daily with vehicle (0.3% carboxymethylcellulose), 2.5, 25, 260, or 2700 μg BPA/kg bw/day, or 0.5 or 5.0 μg ethinyl estradiol (EE)/kg bw/day from gestational day 6 until labor began. Offspring were then gavaged directly from the day after birth until the day before scheduled sacrifice on postnatal days 21 or 90. Using in situ hybridization, one or more BPA doses produced significant decreases in Esr1 expression in the juvenile female rat anteroventral periventricular nucleus (AVPV) of the hypothalamus and significant decreases in Esr2 expression in the adult female rat AVPV and medial preoptic area (MPOA), relative to vehicle controls. BPA did not simply reproduce EE effects, indicating that BPA is not acting solely as an estrogen mimic. The possible consequences of long-term changes in hypothalamic ESR expression resulting from subchronic low dose BPA exposure on neuroendocrine effects are discussed and being addressed in ongoing, related work. more...

A study was undertaken to determine the effects of incremental levels of dietary restriction (DR) in rats. Survival, growth, reproductive, and dietary intake (DI) variables were monitored in a chronic study in which male Sprague Dawley (SD) rats (NCTR colony) were fed their ration ad libitum (AL), or DR. The main objectives were to determine if low levels of DR could be used to increase the survival rate of SD rats in the chronic bioassay, and to identify the survival characteristics of a long-lived SD rat strain (NCTR colony). The average life span of AL rats was 115 months. At 104 weeks on study (110 weeks of age), the survival rate for the AL and 10%, 25%, and 40% DR groups was 63.4, 87.5, 87.5, and 97.5%, respectively. The largest increase in survival (24.1%) occurred between AL and 10% DR, indicating that very low levels of DR have a significant effect on survival. Whole-body, liver, prostate, and epididymis weights and body length were decreased by DR, whereas brain weight, testicular weight, and skull length were not altered by DR. Rats from the NCTR colony were found to be ideal for chronic studies because they are much longer-lived than other SD stocks. Although the 104-week survival rate for these SD, non-obese AL rats exceeds the FDA's "Redbook" survival guideline (> 50%) for chronic bioassays, the use of DR is advocated because it reduces individual variability in body weight. more...

Bisphenol A (BPA) is a high production volume industrial chemical to which there is widespread human oral exposure. Guideline studies used to set regulatory limits detected adverse effects only at doses well above human exposures and established a no-observed-adverse-effect level (NOAEL) of 5 mg/kg body weight (bw)/day. However, many reported animal studies link BPA to potentially adverse effects on multiple organ systems at doses below the NOAEL. The primary goals of the subchronic study reported here were to identify adverse effects induced by orally (gavage) administered BPA below the NOAEL, to characterize the dose response for such effects and to determine doses for a subsequent chronic study. Sprague Dawley rat dams were dosed daily from gestation day 6 until the start of labor, and their pups were directly dosed from day 1 after birth to termination. The primary focus was on seven equally spaced BPA doses (2.5–2700 μg/kg bw/day). Also included were a naïve control, two doses of ethinyl estradiol (EE2) to demonstrate the estrogen responsiveness of the animal model, and two high BPA doses (100,000 and 300,000 μg/kg bw/day) expected from guideline studies to produce adverse effects. Clear adverse effects of BPA, including depressed gestational and postnatal body weight gain, effects on the ovary (increased cystic follicles, depleted corpora lutea, and antral follicles), and serum hormones (increased serum estradiol and prolactin and decreased progesterone), were observed only at the two high doses of BPA. BPA-induced effects partially overlapped those induced by EE2, consistent with the known weak estrogenic activity of BPA. more...

Almost 40 y ago the scientific community was taking actions to control environmental factors that contribute to variation in the responses of laboratory animals to scientific manipulation. Laboratory animal diet was recognized as an important variable. During the 1970s, the American Institute of Nutrition, National Academy of Science, Institute of Laboratory Animal Resources, and Laboratory Animals Centre Diets Advisory Committee supported the use of ‘standard reference diets’ in biomedical research as a means to improve the ability to replicate research. As a result the AIN76 purified diet was formulated. During this same time, the laboratory animal nutritionist at the NIH was formulating open-formula, natural-ingredient diets to meet the need for standardized laboratory animal diets. Since the development of open-formula diets, fixed-formula and constant-nutrient–concentration closed-formula laboratory animal natural ingredient diets have been introduced to help reduce the potential variation diet can cause in research. more...

Daidzein (4',7-dihydroxyisoflavone), a soy phytoestrogen, is a weakly estrogenic compound that may have potential health benefits. Biotransformation of daidzein by the human gut microflora after ingestion converts it to either the highly estrogenic metabolite equol or to nonestrogenic metabolites. We investigated the metabolism of daidzein by colonic microflora of rats. Fecal samples, obtained before and after rats were exposed to daidzein at 250 or 1000 parts per million, were incubated in brain-heart infusion (BHI) broth with daidzein under anaerobic conditions. Samples were removed from the cultures daily and analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. The fecal bacteria of all rats, regardless of prior daidzein exposure, metabolized the added daidzein to dihydrodaidzein. Both compounds disappeared rapidly from BHI cultures incubated for more than 24 h, but no other daidzein metabolites were detected. Only daidzein and dihydrodaidzein were found in a direct analysis of the feces of rats that had consumed daidzein in their diets. Unlike the fecal bacteria of humans and monkeys, the rat flora rapidly metabolized daidzein to aliphatic compounds that could not be detected by HPLC or mass spectral analysis. more...

Background and aims: The primary purpose of this study was to evaluate the effects of varied levels of dietary restriction (DR) on neoplastic pathologies in rodents at 58 and 110 weeks of age. Methods: Male Sprague-Dawley (SD) rats were divided into four nutritional groups; an ad libitum (AL) control group, and three dietary restricted (DR) groups that were fed the NIH-31 diet reduced in amount by 10, 25, and 40%. Results: At 110 weeks of age, compared to AL rats, the incidence of benign tumors was significantly lower in all DR groups while primary tumors were significantly lower in the 10 and 40% DR groups; no malignant tumors were detected in the 10% DR group. Most defined mortalities were caused by neoplastic lesions. All levels of DR reduced the percentage of tumor-bearing animals, the incidence of skin tumors (combined), and the total number of tumors. Pituitary, skin, and pancreatic tumors were the most prolific lesions; pituitary and skin tumors were the most fatal. Compared to AL rats, the time to onset of skin and pancreatic tumors was longer in all of the DR groups. Conclusion: In many cases, the incidences of neoplastic lesions were similar among the DR groups, clearly indicating that the DR effect is not linear and that even a very low level of DR (10%) can have a significant effect on many important neoplastic lesions and tumor burden. The main effect of DR was to decrease the incidence of some neoplastic lesions and to increase the time to onset and/or decrease the progression of tumors, thereby increasing the 110-week survival rate of DR rats. more...

Caloric restriction has previously been shown to decrease levels of oxidative stress in rats. In this study, we examined the effects of 5 different caloric intake levels on one type of oxidative DNA damage in rat mammary gland, blood, and liver. Animals were fed modified AIN-93G diets to accommodate 10, 20, 30, or 40% calorie restriction (CR), relative to ad libitum (AL) consumption. The intakes of fat, protein, vitamins, and minerals thus remained constant, but total carbohydrate intake decreased. Body weights of the animals at 20 weeks reflected the degree of restriction, but in the first 10 weeks, weight gain in the 10% CR group was not reduced relative to animals fed ad libitum. Levels of 5-hydroxymethyl-2'-deoxyuridine increased with time in mammary gland and nucleated blood cells regardless of CR level, indicating an effect of animal age, despite the fact that the animals were only 7 months old after the 20-week dietary study. In liver, however, there was a trend towards decreased DNA damage levels with time. The effect of diet on levels of 5-hydroxymethyl-2'-deoxyuridine was not statistically significant, indicating no protective effect of restricted dietary carbohydrate. This dietary study differed from previous work in that the modified AIN-93G dietary formulation contains relatively higher levels of fat and vitamins K, E, and B(12), and it has certain added trace minerals. This data raises the question of whether the previously reported effects of calorie restriction on preventing oxidative stress in mammary gland are dependent on the type of dietary formulation used. more...

The collaborative Interagency Agreement between the National Center for Toxicological Research (NCTR) and the National Institute on Aging (NIA) was aimed at identifying and validating a panel of biomarkers of aging in rodents in order to rapidly test the efficacy and safety of interventions designed to slow aging. Another aim was to provide a basis for developing biomarkers of aging in humans, using the assumption that biomarkers that were useful across different genotypes and species were sensitive to fundamental processes that would extrapolate to humans. Caloric restriction (CR), the only intervention that consistently extends both mean and maximal life span in a variety of species, was used to provide a model with extended life span. C57BI/6NNia, DBA/2JNia, B6D2F1, and B6C3F1 mice and Brown Norway (BN/RijNia), Fischer (F344/NNia) and Fischer x Brown Norway hybrid (F344 x BN F1) rats were bred and maintained on study. NCTR generated data from over 60,000 individually housed animals of the seven different genotypes and both sexes, approximately half ad libitum (AL) fed, the remainder CR. Approximately half the animals were shipped to offsite NIA investigators internationally, with the majority of the remainder maintained at NCTR until they died. The collaboration supplied a choice of healthy, long-lived rodent models to investigators, while allowing for the development of some of the most definitive information on life span, food consumption, and growth characteristics in these genotypes under diverse feeding paradigms. more...