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

Identification of enzymes responsible for the metabolism of heme in human platelets.

The major enzymes involved in the degradation of heme were identified in human platelets. It was determined that heme oxygenase activity levels in umbilical cord blood platelets were higher, whereas biliverdin reductase activity levels were comparable with that found in platelets from adults. In membranes prepared from adenosine diphosphate-activated platelets, UDP-glucuronic acid-dependent bilirubin conjugation was detected, whereas activity was negligible in unactivated platelets and undetected in serum and heat-inactivated platelets, and in platelets prepared from umbilical cord blood. Platelet fractions were analyzed by Western blot and shown to express heme oxygenase, biliverdin reductase, and UDP-glucuronosyltransferases, and there was concordance with known developmental profiles found in other tissues. Heme oxygenase expression was higher, whereas UGT expression was lower, in neonatal compared with adult platelets. These data suggest that platelets are involved in multiple steps of heme and bilirubin metabolism and that developmental regulation of these enzymes may be similar to that in other human tissues.

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.

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.

Increased effective activity of rat liver catalase by dietary restriction.

While dietary restriction (DR) increases maximum life span in many animal species, the mechanisms by which this is achieved remain unclear. One possibility is that DR may act in part to reduce free radical levels by retarding age-related declines in rat liver catalase activity. We measured liver cytosolic catalase activity at various times of day in 9-12 month old male (BN X F344)F1 rats fed ad libitum (AL) or subjected to a 30% DR from 14 weeks of age. Catalase activity (expressed as µmol·min(-1)·g liver(-1)) in both diet groups reached minimums at 0600 h but activity was 26% higher in DR as compared to AL rats. This traditional expression of catalase activity did not significantly differ between diet groups at other times of day. One must be careful in the interpretation of such data, however, since catalase is rapidly inactivated by its substrate (H2O2), thus displaying abnormal enzyme kinetics. In order to avoid this difficulty we evaluated the time period during which the reaction remained linear and multiplied it by its activity to yield the effective catalase activity. Using this method we found a significant increase in catalase activity in DR animals at several H2O2 concentrations during the light span. At 1800 h (the beginning of the dark span when the controls initiated peak food intake), fewer and smaller dietary differences were observed and no dietary effects were observed at 2400 h. These data suggest that DR reduces the rate of accumulation of inactive catalase and may contribute to an increased capacity in DR animals to remove free radicals.

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 sponsor's submission or in risk assessments. The outcome of that conference was the FDA's commitment to develop Points-to-Consider documents that address the issue of dietary control in chronic toxicity and carcinogenicity studies.

Effects of caloric restriction on expression of testicular cytochrome P450 enzymes associated with the metabolic activation of carcinogens.

Previous work demonstrated that microsomal cytochrome P4502A1 (CYP2A1) is expressed in rat testicular Leydig cells. The present study investigates the effects of diet, age, and strain on rat testicular CYP2A1 expression and assesses the potential role of testicular CYP2A1 in the metabolic activation of carcinogens. In ad libitum-fed 18-week-old Fischer 344 rats, testicular CYP2A1 immunoreactive protein and testosterone 7alpha-hydroxylase activity (7alpha-TOHase) exhibited a circadian variation with a daytime maximum and a night-time minimum (82.2 +/- 42.0 and 21.9 +/- 4.5 pmol 7alpha-hydroxytestosterone/min/mg protein, respectively). Caloric restriction (to 60% of ad libitum consumption), which reduces the severity of Leydig cell tumors in rats, decreased expression of both CYP2A1 and testicular 7alpha-TOHase >80% and eliminated their circadian variation. Conversely, caloric restriction induced a circadian rhythm in testicular 7-benzyloxyresorufin-O-dealkylase activity. Testicular microsomes from ad libitum-fed rats having peak diurnal 7alpha-TOHase activity had significantly greater (30%) microsome-mediated aflatoxin B1-DNA binding activity compared to microsomes prepared from nocturnal phase ad libitum-fed or calorically restricted rats which expressed low 7alpha-TOHase activity. In 12-month-old Fischer 344 rats, high CYP2A1 expression was correlated with severe Leydig cell hyperplasia (r = 0.80), whereas CYP2A immunoreactive protein and 7alpha-TOHase were expressed at lower levels in Sprague-Dawley than in Fischer 344 rats and were undetectable in pig, monkey, and human testes. These are strains/species that do not exhibit significant Leydig cell hyperplasia. This suggests that caloric intake, strain, and circadian factors may all mediate testicular CYP2A1 expression in the rat and that CYP2A1 may in turn influence carcinogen activation and pathological status in the testis.

P48: a novel nuclear protein possibly associated with aging and mortality.

The synthesis ([35S]-incorporation) of stress proteins (sps, i.e., 24, 25, 70, 90 Mr) and of nuclear protein 48 (p48) was investigated in the heart and bone marrow cells of three groups of male Fischer 344 rats following administration of isoproterenol (IPR). Two groups of rats, young ad libitum (Y/AL-3 1/2 months) and old/AL (O/AL-28 months), had full access to rat chow; a third group of old diet restricted (O/DR-28 months) rats was maintained on a diet restricted intake of 40% of the Y/AL animals. Sp synthesis in the bone marrow (25, 70, 90 Mr) and heart (24, 70, 90 Mr) nuclei of O/AL was significantly reduced, as compared with Y/AL and O/DR rats, following their induction with IPR. A unique sp24 was expressed in heart following IPR dosing. A 1 mg/kg dose of IPR was lethal for O/AL, but not for Y/AL or O/DR animals. This lethal dose induced synthesis of p48 in heart and bone marrow nuclei of O/AL rats only. P48 existed in isoform states in bone marrow, and when a lethal dose of IPR was administered in this tissue, it was expressed in O/AL rats in a cell-cycle regulated pattern. Stress proteins and other non-sps were seen as cell cycle regulated following IPR administration. P48 in bone marrow and heart nuclei from O/AL rats showed an antigenic response identical to that of p48 in HL60 nuclei. The presence of p48 is correlated with mortality and with an ad libitum diet in old rats, since it is absent in old diet restricted animals; therefore, DR may impede the expression of p48 through a mechanism(s) that is undisclosed at this time.

The effect of aging and dietary restriction on the retinoylation of nuclear matrix proteins in rats.

The labeling in vivo of young ad libitum (Y/AL) and old diet restricted (O/DR) rats with [3H]retinoic acid (RA) for 6 hours, and the exposure of electrophoretically separated nuclear matrix proteins from bone marrow tissue on film for 48 days revealed the presence of eleven retinoylated proteins. Dosing with RA (100 mg/kg body weight) for 96 hours and exposure to [3H]RA enhanced the levels of radioactive incorporation of several nuclear matrix proteins, including p51, and p55, similarly in Y/AL and O/DR rats. Dosing of old ad libitum (O/AL) rats with [3H]RA for 6 hours showed the incorporation of six proteins following 48 days of exposure on film. Long-term dosing of RA (96 hours) did not increase [3H]RA incorporation in these proteins, including p51 and p55, in O/AL rats. Increasing the level of RA by two-fold (200 mg/kg body weight) in Y/AL and O/DR rats elicited an increase in the incorporation levels of [3H]RA in five proteins. This dose response following increased levels of RA was not seen in the retinoylated proteins of O/AL animals. Analysis by the Western blotting technique showed p51 and p55 from rat bone marrow cells to have the same immunochemical determinates with proteins of identical molecular masses in HL60 cells. The levels of retinoylation of nuclear matrix proteins in O/DR animals, altered by age- and diet-dependent factors, suggests a condition that is more reminiscent of Y/AL than of O/AL animals.

The use of transgenic cell lines for evaluating toxic metabolites of carbamazepine.

Human lymphoblastoid cell lines transgenic for human CYP450s were evaluated for the identification of toxic metabolites of the anticonvulsant drug carbamazepine (CBZ). Human CYP450 isoforms expressed by these cell lines included 1A1, 1A2, 2E1, 2A6, and 3A4. A dose-dependent inhibition of population growth from 50-200 micrograms/ml CBZ was detected by measuring cell number and respiration. The inhibition increased with the growth rate of the various lines, which correlated inversely with the presence of CYP450s, and may have been caused by CBZ itself. Cytotoxicity was observed only at the highest dose and in the line lacking transfected CYP450s. Microsomal preparations from hCYP3A4/OR cells converted CBZ into its principal oxidative metabolite, carbamazepine-10,11-epoxide (CBZ-E), at a rate of 630 pmol/min per mg protein, confirming a major role of CYP3A4 in this reaction. However, no CBZ-E (or any metabolite) was recovered from any whole-cell incubation even though hCYP3A4 cells readily converted testosterone to 6 beta-hydroxytestosterone. This suggests that differences exist between whole-cell and microsomal preparations of lymphoblastoid cells in their ability to metabolize CBZ.

Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics.

The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S-transferase, UDP-glucurono-syltransferase, UDP-glucosyltransferase, and N-acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min-1 mg-1), UDP-glucosyltransferase (0.27 nmol min-1 mg-1) and glutathione S-transferase (20.8 nmol min-1 mg-1). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min-1 mg-1) and N-demethylation (0.17 nmol min-1 mg-1) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min-1 mg-1) and the microsomes (0.13 nmol min-1 mg-1). N-Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.

Caloric restriction and toxicity.

The modulatory effects of caloric intake on the rate and extent of both spontaneous and induced disease incidence is well known, but the significance of these effects in the interpretation of testing data has only recently become appreciated. This is especially true relative to the impact of caloric intake on both survival and background incidence for common tumors. In order to enhance the health and survival of animals ongoing chronic toxicity testing it has been suggested that such tests should restrict food consumption. Although this restriction will result in increasing survival of the test animals, it may also effect the expression of toxicity by altering agent metabolism and disease progression. Focus in this symposium is on the necessity to control dietary consumption in toxicity tests (dietary control), and if such a need does exist to what level of consumption should be diet be focused (caloric restriction).

Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp.

A constitutively expressed aliphatic amidase from a Rhodococcus sp. catalyzing acrylamide deamination was purified to electrophoretic homogeneity. The molecular weight of the native enzyme was estimated to be 360,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500. The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0. The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively. Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme. No labile sulfide was detected. The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+). However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents. The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp. strain R312.

Effects of caloric restriction on rodent drug and carcinogen metabolizing enzymes: implications for mutagenesis and cancer.

Caloric restriction in rodents results in increased longevity and a decreased rate of spontaneous and chemically induced neoplasia. The low rates of spontaneous neoplasia and other pathologies have made calorically restricted rodents attractive for use in chronic bioassays. However, caloric restriction also alters hepatic drug metabolizing enzyme (DME) expression and so may also alter the biotransformation rates of test chemicals. These alterations in DME expression may be divided into two types: (1) those that are the direct result of caloric restriction itself and are detectable from shortly after the restriction is initiated; (2) those which are the result of pathological conditions that are delayed by caloric restriction. These latter alterations do not usually become apparent until late in the life of the organism. In rats, the largest direct effect of caloric restriction on liver DMEs is an apparent de-differentiation of sex-specific enzyme expression. This includes a 40-70% decrease in cytochrome P450 2C11 (CYP2C11) expression in males and a 20-30% reduction of corticosterone sulfotransferase activity in females. Changes in DME activities that occur late in life in calorically restricted rats include a stimulation of CYP2E1-dependent 4-nitrophenol hydroxylase activity and a delay in the disappearance of male-specific enzyme activities in senescent males. It is probable that altered DME expression is associated with altered metabolic activation of chemical carcinogens. For example the relative expression of hepatic CYP2C11 in ad libitum-fed or calorically restricted rats of different ages is closely correlated with the amount of genetic damage in 2-acetylaminofluorene- or aflatoxin B1-pretreated hepatocytes isolated from rats of the same age and caloric intake. This suggests that altered hepatic drug and carcinogen metabolism in calorically restricted rats can influence the carcinogenicity of test chemicals.

Synthesis and stability of isotopically labeled p-chloro-m-xylenol (PCMX).

The synthesis, reaction kinetics, and pH stability of isotopically labeled p-chloro-m-xylenol (PCMX) were evaluated. While base catalysis was more rapid than acid catalysis, the latter allowed the use of a cosolvent for deuterium and tritium labeling using as little as 250 microL labeled water. Both acid and base catalysis were markedly more rapid than that reported previously for the deuteration of PCMX and related phenols. Isotopic labeling occurred only at the 2 and 6 ring positions, ortho to the phenolic group of PCMX. No deuterium loss was observed after storage for 21 days at 37 degrees C over a pH range of 2-14. Isotopic loss was observed only below pH 2. The prepared 3H-labeled PCMX had a specific activity of 1.18 mCi/mmol, a radiochemical purity of 99.0%, and a chemical purity exceeding 99.0%, with a high stability during prolonged cold storage.