Point-of-care oral-based diagnostics.

Many of the target molecules that reside in blood are also present in oral fluids, albeit at lower concentrations. Oral fluids are, however, relatively easy and safe to collect without the need for specialized equipment and training. Thus, oral fluids provide convenient samples for medical diagnostics. Recent advances in lab-on-a-chip technologies have made minute, fully integrated diagnostic systems practical for an assortment of point-of-care tests. Such systems can perform either immunoassays or molecular diagnostics outside centralized laboratories within time periods ranging from minutes to an hour. The article briefly reviews recent advances in devices for point-of-care testing with a focus on work that has been carried out by the authors as part of a NIH program.

Effect of varying dietary fat levels on rat growth and oxidative DNA damage.

Dietary fat has previously been shown to have somewhat complicated relationships to levels of oxidative stress in rats. In this study, we examined the effects of five different dietary fat intakes on levels of oxidative DNA damage in rats. Animals fed diets containing 3%, 5%, 10%, or 15% corn oil had body weights that were similar after 20 weeks. Animals fed a 20% fat diet, however, had significantly higher mean body weight than any other group. Levels of 5-hydroxymethyl-2'-deoxyuridine, one marker of oxidative DNA damage, had different relationships to dietary fat in blood and mammary gland. In blood, levels increased with dietary fat levels, and the highest levels were observed with the 20% fat diet (65% higher levels than with the 3% fat diet). In mammary gland, a plateau-type effect was observed, with maximal levels of oxidative DNA damage being obtained using 10% fat (representing a 68% increase relative to the 3% fat diet). This could be a result of induction of compensatory mechanisms in response to a high-fat diet in mammary gland but not in the short-lived nucleated blood cells. Oxidative DNA damage levels in blood thus appear to be a marker of dietary fat intake. In mammary gland, however, levels of DNA damage are consistent with previously observed promotional effects of dietary fat on mammary gland tumorigenesis at lower levels of fat intake with little or no incremental promoting effects at higher levels of fat intake.

Does caloric restriction induce hormesis?

The question of whether caloric restriction (CR) is hormetic is addressed in terms of two common definitions of the term. In terms of the older definition, i.e., a growth-stimulatory effect when lower doses of a compound which resulted in growth inhibition at higher doses, CR is better characterized as a co-hormetic (i.e., a paradigm which at relatively "low doses," in combination with some stimulus, will evince increased growth (proliferation) and at higher "doses" will inhibit this increased proliferation) rather than a hormetic agent. Mechanisms such as cellular selection of cellular subpopulations, increases in receptor efficiency, and preservation of cellular proliferative potential can interact with agents and produce increased growth as long as the CR is not too severe. In terms of a broader definition, i.e., nonmonotonic dose-response behavior of a compound for any adverse response, CR appears to be hormetic, both as a result of body weight (BW) loss and other potential mechanisms. The impact of changes in BW, or frank CR, can be considered a component of every test for hormesis, and is thus capable for interaction with any other agent. The changes that BW loss (or CR) induce are so profound that any aspect of an agent's action - metabolism, pharmacokinetics, pharmacodynamics - can modulate the response of an organism to an agent. Similarly, other effects of a chemical that induce BW loss, e.g., physical activity or temperature dysregulation, can also induce dose-response curves that appear hormetic. The interaction of the hormetic agents of BW loss and CR can influence agent tests. Controlling these factors may make it possible to dissect the key components of a hormetic response. In addition, the effects of CR or BW loss appear to extrapolate well across species [Colman R, Kemnitz JW. Aging experiments using nonhuman primates. In: Yu BP (Ed), Methods in Aging Research. CRC Press, Boca Raton, FL, 1999, pp. 249-267]. Thus there is some reason to believe that these hormetic factors may be important for humans, and may already be a factor for tests of potentially adverse agents already conducted in humans.

The effect of dietary fat on malondialdehyde concentrations in Fischer 344 rats.

The effects of dietary fat and age on the level of malondialdehyde (MDA), a product of lipid peroxidation, were investigated in cerebellum, kidney, and liver tissues of female Fischer 344 rats. Groups of rats were fed diets containing various levels of corn oil (3, 5, 10, 15, or 20%), starting at 57 days of age, for a duration of 2, 10, or 20 weeks. High fat diets are thought to promote tumor formation, diabetes, and cardiovascular diseases via induction of oxidation stress, and this can begin early in the lifespan. However, it was observed that rats chronically consuming 3 and 5% corn oil diets yielded significantly higher levels of MDA, as analyzed by high-performance liquid chromatography, compared with those fed higher fat diets. After 20 weeks of feeding, the concentration of MDA in each of the three organs studied showed no significant differences among rats consuming diets containing 10, 15, or 20% corn oil. The levels of MDA were highest in the cerebellum, followed by kidney, and lowest in liver. Over the 20-week feeding period, a decrease in MDA level in both cerebellum and liver was observed.

Effects of humidification on nasal symptoms and compliance in sleep apnea patients using continuous positive airway pressure.

STUDY OBJECTIVES: To evaluate the effects of humidification on nasal symptoms and compliance in sleep apnea patients using continuous positive airway pressure (CPAP). DESIGN: A randomized, crossover design was employed. SETTING: The study was conducted at two suburban community-based hospital sleep laboratories. PATIENTS: Data were collected on 38 obstructive sleep apnea patients (mean age, 44.1 years) in whom CPAP was a novel treatment. INTERVENTIONS: The interventions were heated humidity, cold passover humidity, and a washout period without humidity. MEASUREMENTS AND RESULTS: Patients were titrated with heated humidity or cold passover humidity in the laboratory and subsequently initiated on humidity. Objective compliance, self-report of factors affecting CPAP use, satisfaction with CPAP, feeling upon awakening, and daytime sleepiness were assessed at the completion of each 3-week treatment period and a 2-week washout period. Outcome measures were assessed with one-way analysis of variance followed by Scheffe post hoc comparisons. Significant main effects were observed for compliance (F2,37 = 5.2; p = 0.008), satisfaction with CPAP (F2,37 = 4.5; p = 0.01), and feeling refreshed on awakening (F2,37 = 4.4; p = 0.02). A significant decrease in daytime sleepiness was observed between baseline and each of the conditions (F3,37 = 55.5; p<0.0001), but Epworth sleepiness scale scores did not differ between conditions (all p values >0.56). CPAP use with heated humidity (5.52+/-2.1 h/night) was greater than CPAP use without humidity (4.93+/-2.2 h/night; p = 0.008). Compliance differences were not observed between CPAP use with cold passover humidity and CPAP use without humidity. Patients were more satisfied with CPAP when it was used with heated or cold passover humidity (p< or =0.05). However, only heated humidity resulted in feeling more refreshed on awakening (p<0.05). No significant differences were observed among the three groups on the global adverse side effect score (F2,37 = 2.5; p = 0.09). Specific side effects such as dry mouth or throat and dry nose were reported less frequently when CPAP was used with heated humidity compared to CPAP use without humidity (p<0.001). CONCLUSIONS: Compliance with CPAP is enhanced when heated humidification is employed. This is likely due to a reduction in side effects associated with upper airway symptoms and a more refreshed feeling upon awakening. Compliance gains may be realized sooner if patients are started with heated humidity at CPAP initiation.

Adaptive role of caloric intake on the degenerative disease processes.

Carcinogenicity and aging are characterized by a set of complex endpoints, which appear as a series of molecular events. Many of these events can be modified by caloric intake. Since most of these processes determine an organism's ability to cope with various environmental stressors, it is not surprising that a relationship (in the presence of a constant nutrient density) exists between caloric intake and time-to-tumor and/or life span. Our studies have clearly shown that generally, the greater the caloric intake, the greater the body weight, the higher the incidence of spontaneous tumor occurrence, the greater the susceptibility to chemical carcinogens, and the shorter the life span. It is also recognized that variables other than body weight influence the life span and carcinogenesis. We have focused our attention on the questions of how and to what extent caloric intake modifies those homeostatic processes believed to be critical in determining the ability of an organism to cope with endogenous and exogenous stresses such as chemical, physical, and biological carcinogens. The response of an organism to its environment can be divided into four categories--physiological, metabolic, molecular, and cellular. We have found that, from a physiological perspective, decreasing caloric intake causes body temperature in rodents to be decreased by 0.5 to 1.8 degrees C and water consumption to be increased by 80%, as is running activity. However, metabolic output per gram of lean body mass is not altered. Reproductive capacity declines, whereas the ECG waveform is preserved as caloric intake decreases. Alterations in these and other physiological functions suggests that energy intake serves as a signal to up-regulate or down-regulate functions related to the flight-or-fight response observed in placental mammals. A number of key metabolic pathways are altered as a function of lowered caloric intake, even though the rate of food consumption per gram of lean body mass remains steady during body weight decreases caused by decreasing caloric intake. Pharmacological compartmentalization, however, is altered. As caloric intake declines, changes occur in the expression of a number of drug-metabolizing enzymes, with the most striking effect seen in sex-specific growth hormones and liver-dependent phase I and phase II enzymes. Additionally, oxidative stress (free-radical and mediated damage to macromolecules) appears to decrease as a function of reduced caloric intake. A number of molecular processes also change with changes in energy consumption. Our studies have shown that, regardless of the source and nature of DNA damage, DNA repair is better preserved and/or enhanced when caloric consumption decreases. In addition, the fidelity of DNA replication increases and oncogene expression is stabilized, P53 gene expression is increased, and apoptosis is elevated by up to 500% with decreased caloric intake. At the cellular level, cell proliferation is decreased in direct proportion to lower energy intake in some but not all tissues. Studies have also shown an enhancement in immune capacity, changes in IGF1, and accelerated rates of wound healing proportionate to declines in energy consumption. Our most recent findings, however, have shown that the benefits associated with decreases in caloric intake only occur in the presence of sufficient nutrient quality and density. In the absence of proper nutrition, however, sensitivity to carcinogens and toxic substances appears to be enhanced. These findings are supported by independent studies. These observations have led us to conclude that, in certain organisms, when caloric intake is decreased, there is an up-regulation of those processes that modulate the responses to a wide range of environmental stressors. This response allows for a better survival rate and a down-regulation of reproductive activity. It is our belief that, during periods of environmental stress, these systems may be essential to perpetu

Growth curves and survival characteristics of the animals used in the Biomarkers of Aging Program.

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.

Hormesis--implications for risk assessment caloric intake (body weight) as an exemplar.

Hormesis can be considered as a parameter which has a non-monotonic relationship with some endpoint. Since caloric intake is such a parameter, and the impact of this parameter on risk assessment has been fairly well characterized, it can provide clues as to how to integrate the information from a hormetic parameter into risk assessments for toxicants. Based on the work with caloric intake, one could: (a) define a biomarker for hormetic effect; (b) integrate specific information on when in the animals lifespan the parameter is active to influence parameters such as survival; (c) evaluate component effects of the overall hormetic response; and (d) address the consequences of a non-monotonic relationship between the hormetic parameter and endpoints critical for risk assessment. These impacts on risk assessments have been characterized for chronic tests, but are also true for short-term tests. A priority is the characterization of the dose-response curves for hormetic parameters. This quantification will be critical in utilizing them in risk assessment. With this information, one could better quantitatively address the changes one expects to result from the hormetic parameter, and limit the uncertainty and variability which occurs in toxicity testing.

Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils.

BACKGROUND: As assessed by flow cytometry, the increase in hydrogen peroxide in individual neutrophils from old volunteers was significantly greater than in neutrophils from young volunteers. To explain the discrepancy in previous reports that showed reduced superoxide generation with age and our finding, we measured the kinetics of antioxidative enzymes. METHODS: Neutrophils were obtained from young (ages 21-34) and old (ages over 65) volunteers. The increase in hydrogen peroxide following stimulation with formyl peptide in individual neutrophils was assessed by flow cytometry by using dihydrorhodamine 123. The enzyme kinetics was determined from the best fit curve using Michaelis-Menten equations. RESULTS: Aging was associated with a significant reduction in the Vmax for glutathione peroxidase. The decreased activity was not due to selenium deficiency as the serum and neutrophil concentrations were identical with age. Following activation, a significant increase in the Km was noted in neutrophils from young but not from old volunteers. CONCLUSIONS: These results account for the increased intracellular accumulation of hydrogen peroxide as a function of age in stimulated neutrophils. These results provide evidence in humans of an age-related impairment in antioxidative defense mechanisms that support the free radical theory of aging.

Caloric restriction as a mechanism mediating resistance to environmental disease.

It has been observed that susceptibility to many degenerative diseases increases concurrently with industrialization and rising living standards. Although epidemiologic studies suggest that specific environmental and dietary factors may be important, caloric intake alone (as reflected in body size) may account for much of the differential risk observed among diverse human populations. It has been suggested from animal studies that caloric intake may be the primary effector for many hormonal, metabolic, physiologic, and behavioral responses that coordinate reproductive strategy to apparent availability of food. When caloric intake is excessive, particularly at critical developmental stages, physiologic priorities are set for body growth and fecundity rather than for endurance and longevity. The converse occurs during periods of famine, thus increasing the probability that sufficient individuals survive to restore the population when conditions improve. Calorically restricted rodents have significantly longer reproductive and total life spans than their ad libitum-fed controls and exhibit a spectrum of biochemical and physiologic alterations that characterize their adaptation to reduced intake. These include reduced stature, hypercorticism in the absence of elevated adrenocorticotropic hormone levels, increased metabolic efficiency, decreased mitogenic response coupled with increased rates of apoptosis, reduced inflammatory response, induction of stress proteins and DNA repair enzymes, altered drug-metabolizing enzyme expression, and modified cell-mediated immune function. The overall profile of these changes is one of improved defense against environmental stress. This has been suggested as the mechanistic basis for the protective effects of low body weight on radiation and chemically induced cancers in experimental animals. It may also explain the significantly higher thresholds of acute toxicity observed when calorically restricted rodents are exposed to certain test compounds.

Liver tumors induced in B6C3F1 mice by 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene contain K-ras protooncogene mutations.

We previously examined the tumorigenicity of 7-chlorobenz[a]anthracene (7-Cl-BA) and 7-bromobenz[a]anthracene (7-Br-BA) in the neonatal mouse bioassay and found that 7-Cl-BA and 7-Br-BA induced hepatocellular adenoma in 92 and 96% of the mice and hepatocellular carcinoma in 100 and 83% of the mice, respectively. In the present study, mRNA was isolated from each of the liver tumors induced by the two compounds and reverse-transcribed to cDNA. Portions of the K- and H-ras oncogene coding sequences were then amplified and analyzed for DNA sequence alterations. Eighty-three percent (20/24) of 7-Cl-BA-induced and 91% (20/22) of 7-Br-BA-induced liver tumors had activated ras protooncogenes. In contrast to the general finding of H-ras mutations in B6C3F1 mouse liver tumors, both compounds had 95% (19/20) of the mutations located at the first base of K-ras codon 13, resulting in a pattern of GGC --> CGC. Thus, our results demonstrate that 7-Cl-BA and 7-Br-BA induce a unique type of ras (K-ras) oncogene activation in liver tumors of B6C3F1 mice.

Evolution and dietary restriction.

Placing lifespan in the context of the life history of an organism, Alex Comfort's work has stimulated the analysis of dietary restriction (DR) and its effects on lifespan in an evolutionary context. DR results in the curtailment of energy-intensive nonfood-gathering activities, increased efficiency of food utilization, an increase in food acquisition activity, an increase in the reproductive lifespan, and an increase in the protection of genomic integrity. These result in further refinement of the Adaptive-Longevity Related Process Theory of the effects of dietary modulation to include increased protection of the genomic integrity of cells that result from delayed reproduction, and increased ability to compete for available food. These effects are discussed in the context of the "spacecraft" metaphor for the evolution of senescence. Also, the apparent paradox that increased body weight seems to be directly correlated to survival across species and inversely correlated to survival within a species is discussed in light of the importance of the cephalization index (a modified ratio of brain weight to body weight) for longevity.

P53 synthesis and phosphorylation in the aging diet-restricted rat following retinoic acid administration.

Multiple doses of retinoic acid (RA) were administered intraperitoneally to three groups of male Fischer 344 rats over a 36 h period. The p53 isoforms from bone marrow nuclei in these three groups of rats were analyzed over time by two-dimensional polyacrylamide gel electrophoresis (PAGE) and fluorography for the incorporation of [35S]methionine (p53-synthesis) and [32P]phosphate (p53-phosphorylation). Two groups of rats, young (3.5 months) ad libitum (Y/AL) and old (28 months) ad libitum (O/AL), had free access to Purina rat chow; a third group of old (28 months) diet-restricted rats (O/DR) were maintained on a restricted caloric intake (60% of the AL diet) from 3 months of age. After 36 h of RA dosing, the PAGE patterns of p53 synthesis and phosphorylation in Y/AL and O/DR rats were very similar. In both groups, an increase in complexity was observed with labeling of additional isotypes possessing more acidic isoelectric values. In contrast, the O/AL animals showed a pattern of p53 isoform synthesis and phosphorylation that was considerably less complex and lacked the pronounced shift to more acidic forms following RA dosing. The p53 isoforms of O/AL rats as recognized by wild type (wt) Pab 246 antibody, were also much less dramatic in their increase to more acidic forms. Two-dimensional phospho-tryptic maps of Y/AL and O/DR rats were also very similar, both exhibiting two additional minor 32P-labeled fragments after RA dosing. The maps of O/AL rats did not show the two additional fragments following RA administration. After RA dosing, cyclin protein inhibitors (p16, p21, p27) revealed robust labeling with their respective antibodies in Y/AL and O/DR rats as analyzed by Western blotting. The O/AL animals showed marginally detectable antibody recognition of the cyclin inhibitors after RA dosing. Taken together, these data suggest that the biosynthesis and phosphorylation of p53 isoforms and the expression of cyclin dependent kinase inhibitor proteins is not significantly different between Y/AL and O/DR rats. Further, these results confirm and extend our previous observations that chronic diet-restriction attenuates the age related decline in the metabolic activity of nuclear protein products.

Dietary restriction modulated carcinogen-DNA adduct formation and the carcinogen-induced DNA strand breaks.

Dietary restriction (DR) alters the activities of hepatic drug metabolizing enzymes and modulates the formation of carcinogen-DNA adducts in carcinogen treated animals. Our previous results showed that a 40% restriction of diet (60% of ad libitum (AL) food consumption) reduced the hepatic metabolic activation of aflatoxin B1 (AFB1) but increased the activation of benzo[a]-pyrene (BaP) in both rats and mice. In this study, the focus was directed toward the levels of carcinogen-DNA adducts formation and the carcinogen-induced DNA strand breaks in mouse kidney and liver DNA. DR significantly inhibited both AFB1-DNA adduct formation and AFB1-induced DNA strand breaks in kidney DNA of mice that received a single dose of [3H]AFB1 (5 mg/kg). The levels of AFB1-DNA adduct formation in mouse kidney DNA correlated well with increased AFB1-induced DNA strand breaks. The correlation between the levels of AFB1-DNA-adducts formed and DNA strand breaks in kidney DNA of DR-mice was less linear than between its AL-counterpart suggesting that other factors, such as different rates of DNA repair, may be involved. In addition, DR enhanced hepatic BaP- and 6-nitrochrysene (6-NC)-DNA adduct formation in the mice treated with BaP and 6-NC, respectively. The formation of the specific BaP-adduct, 10-(N2-deoxyguanosinyl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (N2-dG-BaP), in mouse liver was proportional to the dose, and was compatible to the BaP-induced DNA strand breaks affected by DR. The enhancement of the total 6-NC-DNA adduct formation in DR-mouse was also in correlation with the increased 6-NC-induced DNA strand breaks. The activity of mouse liver microsomal nitro-reductase increased by 2-fold in response to DR indicating that the nitroreduction may contribute to the increase of the metabolic activation of 6-NC. Our present results indicate that the effect of DR on the carcinogen activation is dependent upon the DR-modulated carcinogen metabolizing enzyme activities.

Dietary restrictions and cancer.

Dietary restriction (DR) alters a significant environmental factor in carcinogenesis, dietary intake, thus inhibiting both spontaneous and induced tumorigenesis. Potential mechanisms for the inhibition of spontaneous cancer may include the effects of DR to do the following: decrease body weight, which decreases cellular proliferation and increases apoptosis in a number of organs that increase and decrease with body size; decrease body temperature, thereby lowering the amount of endogenous DNA damage temperature generates; decrease oxidative damage, by increasing antioxidant damage defense systems; decrease, generally, cellular proliferation; and protect the fidelity of the genome by decreasing DNA damage, increasing DNA repair, and preventing aberrant gene expression. Potential mechanisms for reducing induced tumor incidence include lowering agent activation, changing agent disposition, decreasing the adducts most associated with agent toxicity, and inhibiting tumor progression through mechanisms similar to those that can effect spontaneous tumorigenesis. As a method to control a major source of environmental cancer, and as the major modulator of the agent induction of this disease, understanding how DR works may significantly contribute to the efforts to explain how diet impacts on development of cancer in the United States, and may suggest methods to reduce the adverse impacts of other environmental agents on the disease.

Age and temperature related changes in behavioral and physiological performance in the Peromyscus leucopus mouse.

Age-related and ambient temperature-related changes in motor activity, body temperature, body weight (b.w.), and food consumption were studied in the long-lived Peromyscus leucopus mouse at environmental temperatures of 29 and 21 degrees C. Major changes in physiological performance were observed between the young (6 months) and old (60-72 month) age groups. The number of daily activity episodes, and total activity output was significantly lower in old mice. Maximum, average and minimum daily body temperature was lower in the old mice and a significant ambient temperature-by-age interaction was found. Maximum, minimum, and average daily b.w. was higher in old mice. Motor activity was evenly distributed over the active (night) phase in young mice but in old mice activity was significantly greater in the late night partition of the active cycle than in the early night partition. Both groups were significantly more active at night than during the day. Most of the food consumption in both groups occurred at night, but young mice consumed significantly more during the late night partition than the early night partition, and the consumption rates for old mice were not significantly different between early and late night partitions. The percentage of activity episodes involved with food consumption in both groups was significantly higher during the night partition, but the percentage during the early night partition was significantly higher in old mice than in young mice. Significant episodes of circadian torpor occurred in a high percentage of old mice at 06:00, on consecutive days, at both environmental temperatures, but young mice expressed no evidence of torpor.

Mechanistic studies on genotoxicity and carcinogenicity of salicylazosulfapyridine an anti-inflammatory medicine.

Salicylazosulfapyridine (SASP), which has been in clinical use for over 50 years, was reported by the National Toxicology Program to increase rat (F344 strain) urinary bladder and mouse (B6C3F1 hybrid) liver tumours under ad libitum (AL) feeding conditions, while under a feed restriction (FR) regimen, these tumours were not increased. The present investigations were undertaken to assess the implications of these results for the safety of SASP in humans. SASP and its 2 major metabolites, 5-aminosalicylic acid (ASA) and sulfapyridine (SP) were tested for in vivo induction of micronuclei in mouse bone marrow cells with or without prefolic treatment and for in vivo formation of DNA adducts in rat and mouse liver and urinary bladder. None exhibited mutagenicity or DNA reactivity. SASP and SP have induced sister chromatid exchanges and micronuclei (MN) in cultured human lymphocytes in the absence of liver activation enzymes and in B6C3F1 mice (but not in rats) MN in bone marrow and peripheral RBC. Treatment with folate reduces the frequency of MN. Perhaps the short (28 days) RBC lifespan in mouse underlies the sensitivity of this species. Thus, SASP without folate supplementation is an aneuploidogen. In a 2-year study in AL fed SASP-treated (high dose 337.5 mg/kg) rats, urinary pH was increased and urinary specific gravity was reduced at 60 weeks. At the end, this SASP group showed urothelial hyperplasia and papillomas in the urinary bladders of male rats primarily. In the FR high dose SASP group, the hyperplasia was reduced from 82% to 14%. At the end of 2 years, the incidence of multi-organ leukemia was reduced in both AL and FR high dose SASP groups. Thus, SASP caused intraluminal bladder changes in the rat (especially males) consisting of chronic urothelial stimulation, concretions, hyperplasia which resulted in neoplasia. In the mouse, because of species differences in liver ratios (mouse > rat) and, increasing (3 times higher) liver perfusion in the mouse, compared to the rat, there was hepatocellular toxicity and resulting preneoplasia and neoplasia within 2 years. These findings occurred in all AL SASP groups (flat curve without dose response); but were reduced under FR conditions. In this species, the multiorgan lymphoma incidence was reduced in both AL and FR high dose SASP groups. Thus, SASP and its major metabolites are not genotoxic. Folate deficiency associated with SASP administration is probably responsible for aneuploidy in lymphocytes and erythrocytes. SASP does not induce neoplasia directly in either livers, urinary bladders or other organs. Accordingly, SASP is judged to pose no carcinogenic risk to humans.

Effect of food restriction on life span and immune functions in long-lived Fischer-344 x Brown Norway F1 rats.

Life-long food restriction is known to slow aging and reduce the rate of occurrence of age-associated disease processes, but the mechanism by which this is accomplished is unknown. In this study we have examined the effect of food restriction on the proliferative response of spleen cells to mitogens and lymphokine production in 6-, 18-, and 30-month-old AL and FR Fischer-344 x Brown Norway (F-344 x BNF1) female rats whose average life span is 137 weeks on an ad libitum (AL) diet and 177 weeks on a food-restricted (FR) diet. In addition, the ability of food restriction to recall antigens was tested in 10-month-old rats by immunizing them with keyhole limpet and hen's egg albumin and measuring proliferative response of draining lymph node cells to these antigens. Our results indicated that the spleen-cell proliferative response to phytohemagglutinin and concanavalin A (Con A) was equal in 6- and 18-month-old rats but declined significantly in 30-month-old AL rats compared to FR rats. Although flow cytometric analyses did not reveal differences for CD4, CD8, and Ig+ cells with age, a significant rise in memory T cells (Ox-22low) in both CD4+ and CD8+ T-cell subset lineage was noted in AL-fed rats at 30 months of age. In FR rats, however, only a minimal shift of naive T cells (Ox-22high) to memory cells was observed. In FR rats, the observed changes in the naive and memory T-cell subsets correlate well with the observed higher levels of the antiinflammatory interleukin-2 (IL-2) and lower levels of the proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha. The ability of food-restricted animals to recall antigens was lower compared to their age-matched controls, though the proliferative response to T-cell mitogen Con A and superantigen staphylococcal enterotoxin B was higher. These findings indicate that food restriction may selectively act to maintain a lower number of antigen-induced memory T cells with age, thereby maintaining the organism's ability to produce higher levels of IL-2 with age. In summary, the increased cell-mediated immune function noted in aged FR rats appears to be due to the presence of a higher number of naive T cells, which are known to produce elevated levels of the antiinflammatory cytokines, which may in part be responsible for reducing the observed age-related rise in disease.

The physiologic, neurologic, and behavioral effects of caloric restriction related to aging, disease, and environmental factors.

Little is known about the mechanisms by which acute and chronic caloric restriction (CR) modulate disease, longevity, and toxicity. To study these endpoints, behavioral parameters such as food and water consumption and physiologic parameters such as motor activity, body temperature, metabolic output (oxygen use), and respiratory quotient (RQ) were continuously monitored in 26-month-old male B6C3F1 mice and Fischer 344 rats fed either ad libitum (AL) or a CR diet (60% of AL). Different dietary regimens were used: rodents were (1) chronically food-restricted using daily feeding starting at 14 months of age, (2) chronically food-restricted using alternate day feeding, or (3) abruptly switched from CR to AL (acute CR). The physiologic and behavioral changes seen with chronic and acute CR were consistent across strains and species. Average body temperature, the number of meals, and the ratio of food/water consumption were significantly lower in CR rodents than in AL rodents. Also, the daily range of body temperature, oxygen metabolism, RQ, average water consumption, and motor activity was significantly higher in CR rodents. CR caused the onset of altered neurobehavioral functions such as abnormal water consumption; increases in motor activity, serum corticosterone, and stress proteins (HSP); and decreases in the basal setpoint for body temperature and brain metabolism. These changes strongly suggest that many beneficial effects of CR are controlled by the hypothalamic-pituitary-adrenal axis via hormonal regulation. This study supports the assertion that nutritional status may be a primary factor of consideration in development of safety standards and assessment of risk.