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The United States Department of Health and Human Services (HHS) is fully committed to the development of medical countermeasures to address national security threats from chemical, biological, radiological, and nuclear agents. Through the Public Health Emergency Medical Countermeasures Enterprise, HHS has launched and managed a multi-agency, comprehensive effort to develop and operationalize medical countermeasures. Within HHS, development of medical countermeasures includes the National Institutes of Health (NIH), (led by the National Institute of Allergy and Infectious Diseases), the Office of the Assistant Secretary of Preparedness and Response/Biomedical Advanced Research and Development Authority (BARDA); with the Division of Medical Countermeasure Strategy and Requirements, the Centers for Disease Control and Prevention, and the Food and Drug Administration as primary partners in this endeavor. This paper describes various programs and coordinating efforts of BARDA and NIH for the development of medical countermeasures for radiological and nuclear threats.

Apoptosis is responsible for the removal of potentially autoreactive or useless T cells during thymic selection and activated T cells in the periphery. Specific families of receptors, kinases, transcription factors, and cysteine proteases, termed caspases, are involved in the apoptotic cascade leading to proteolysis of specific substrates and to morphological changes associated with programmed cell death. Although common members of the apoptotic cascade are shared between different cell types, it appears that cell-specific factors can influence the response to a given apoptotic stimuli. Characterization and understanding of the basic mechanisms involved in the different pathways protecting or leading to cell death may provide novel ways to control inappropriate apoptosis involved in several diseases.

Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

Drug concentrations in cerebrospinal fluid have been assumed to be a natural surrogate for total drug exposures in the central nervous system. The present communication reports a data set from a study of 30 compounds in mice. An attempt was made to correlate cerebrospinal fluid and unbound plasma drug concentrations via incorporation of in vitro P-glycoprotein (Pgp)-mediated transport data. 2. Pgp-deficient (Pgp -/-) and wild-type mice were dosed with compounds of interest by oral gavage (orally) at 5 mg kg −1 . Plasma and cerebrospinal fluid samples were collected at 1 h post-dosing, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for drug concentrations. Mouse and human Pgp-mediated transport were evaluated in vitro by a bi-directional (B to A and A to B) transport assay using LLC-PK1 cells expressing mouse (mdr1a) and human (MDR1) forms of Pgp, respectively. 3. Compounds with B to A/A to B transport ratios < 2 were defined as non-substrates of Pgp, whereas those exhibiting B to A/A to B transport ratios ≥2 were considered Pgp substrates. Plasma protein bind- ing was also determined in vitro via equilibrium dialysis. Of the 30 compounds, 13 were identified to be mouse Pgp substrates, all of which were also human Pgp substrates, demonstrating a good agreement between mouse and human data. 4. In Pgp wild-type mice, the unbound plasma and cerebrospinal fluid concentrations of the non-Pgp sub - strates correlated well, with a regression slope of approximately 1.0. A similar relationship existed for Pgp substrates in Pgp -/- mice. On the other hand, an improved correlation of cerebrospinal fluid and systemic exposures of the Pgp substrates in Pgp wild-type mice was observed when the unbound plasma concentrations were normalized to the corresponding B to A/A to B transport ratios. 5. These results reinforce the premise that a combined use of unbound plasma drug concentrations and in vitro Pgp transport data may be of value for the estimation of central nervous system exposures.

BACKGROUND Epidemiologic and animal model studies suggest that consumption of soy isoflavones may be associated with reduced risk of prostate cancer (PC). In addition, animal model studies suggest that conjugated linoleic acid (CLA), a natural positional isomer of linoleic acid, inhibits tumor growth in various models, including models of PC. METHODS Based on the above-mentioned data, the objective of the present study was to test the hypothesis that supplementation of the diet with combinations of isoflavone-rich soy protein isolate and CLA would act to inhibit the growth of androgen-independent R-3327-AT-1 rat prostate tumor cells inoculated ectopically into male Copenhagen rats. RESULTS The results of this study indicate that neither an isoflavone-rich soy protein isolate (SPI), nor CLA inhibit the in vivo growth and development of prostate tumor cells when administered in the diet either singly or in combination. Moreover, at the highest concentrations SPI and CLA (i.e., 20% SPI, 1% CLA), there was a statistically significant increase in tumors volume over controls. Administration of SPI at 10% in the diet also enhanced tumor growth, whereas at 5%, SPI exerted no measurable effect. CLA administration alone had no observable effects on AT-1 tumor growth. CONCLUSION These results, in an established rat model, suggest caution in using isoflavone-rich SPI in human studies involving advanced hormone-refractory prostate cancer until further investigation of these effects are completed. Prostate 54: 169–180, 2003. © 2002 Wiley-Liss, Inc.

The N-nitroso-N-methylurea-induced rat mammary tumor model was used to conduct two types of studies: a prevention study designed to test the ability of the novel selective estrogen receptor modulator lasofoxifene (LAS) to inhibit the development of mammary tumors, and a treatment study designed to test the inhibitory effect of LAS on the growth of established tumors. The prevention study indicated that LAS markedly delayed the emergence of N-nitroso-N-methylurea-induced tumors to an extent similar to that obtained by the established antiestrogen tamoxifen (TAM). At the highest dose administered, both TAM and LAS reduced tumor incidence by 75% and total tumor number by 90% relative to the controls. LAS also reduced the multiplicity of tumors, i.e., the mean number of tumors per rat, and resulted in substantially smaller total tumor burden. In the treatment study, LAS significantly inhibited tumor growth compared with the controls. In addition, whereas none of the untreated tumors regressed completely over the experimental period, 40% of LAS-treated tumors regressed by50% at the highest dose (10 mg/kg daily). The results of this study in a rat mammary tumor model indicate that LAS has both chemopreventive and chemotherapeutic effects quantitatively comparable with those of TAM.

Various thiol-containing compounds have been shown to inhibit chemically-induced tumors in animal models. Two thiol-containing compounds derived from vegetables, namely 1,2 dithiol-3-thione (DTT) and S-methylmethane thiolsulfonate (MMTS), were tested for their chemopreventive activity in the N-methylnitrosourea (NMU)-induced rat mammary tumor model. Each compound was incorporated into the grain-based Teklad 7001 diet and fed to the rats one week prior to initiation with NMU until termination 18 weeks post NMU. DTT was fed at 166 and 500 ppm and MMTS at 200 and 800 ppm. Neither compound exerted a significant inhibitory effect on any index of tumor development including incidence, total tumor, tumor multiplicity, volume or latency. Serum levels of DTT assessed at termination in the 500 ppm DTT group ranged from 10-30 microg/ml. MMTS was undetectable in serum from either MMTS-fed group. The results of this study, using the direct acting carcinogen, NMU, suggest that the chemopreventive effect of thiol-containing compounds may be confined to animal models using carcinogens that require host activation.

There has been increased interest in the ability of the adult human nervous system to reorganize and adapt to environmental changes throughout life. This ability has been termed "plasticity." Plastic changes in the cerebral cortex have been studied: (a) as modifications of sensory or motor cortical representation of specific body parts (cortical maps, body representation level); and (b) as changes in the efficacy of existing synapses or generation of new synapses (neuronal or synaptic level). In this review, we describe paradigms used to study mechanisms of plasticity in the intact human motor system, the functional relevance of such plasticity, and possible ways to modulate it.

Epidemiological studies suggest an inverse relationship between the intake of dietary fiber, particularly fiber from cereal grains, and colon cancer risk. Animal model assays have demonstrated that the protective effects of dietary fiber on colon cancer development depend on the nature and source of the fiber. Wheat bran (WB) appears to inhibit colon tumorigenesis more consistently than do oat bran or corn bran. This study was designed to determine whether specific WB fractions such as WB fiber, WB lipids, or phytic acid differentially affect colon carcinogenesis in a well-established colon cancer model. In addition, the modulating effect of specific fractions of WB on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 and COX-2 enzymes were assessed in colon tumors as those have been shown to play a role in tumor progression. At 5 weeks of age, groups of male F344 rats were assigned to one of six diets: a high-fat diet containing 10% WB (control diet) and experimental high-fat diets containing 10% dephytinized WB (WB-P), 10% defatted WB (WB-F), 10% dephytinized and defatted WB (WB-PF), 10% WB-PF fortified with 2% bran oil and/or with 0.4% phytate. At 7 weeks of age, all eats except those in the vehicle-treated groups were given two weekly s.c. injections of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight/week. They continued to receive their respective diets until 50 weeks after carcinogen treatment and were then killed. Colon tumors were analyzed for iNOS, COX-1, and COX-2 expression and enzymatic activities. Colon tumors were evaluated histopathologically and classified as adenomas and adenocarcinomas. We found that removal of phytic acid (WB-P) or lipids (WB-F) from WB had no significant effect on colon tumor incidence (% animals with tumors) or multiplicity (tumors/ animal), whereas removal of both phytate and lipids from WB (WB-PF) significantly increased colon tumor multiplicity and volume. Interestingly, WB-PF fortified with excess bran oil or with bran oil plus phytate significantly inhibited colon tumor incidence, multiplicity, and volume; but supplementation of WB-PF with phytate alone had no significant effect on colon tumorigenesis in rats suggesting that lipid fraction of WB possesses tumor-inhibitory properties. Moreover, feeding WB-PF diet significantly increased iNOS, total COX and COX-2 enzyme activities, and iNOS protein expression in colon tumors as compared with wheat bran control diet. Feeding the WB-PF that was fortified with excess bran oil alone or with bran oil plus phytate significantly suppressed the activities of iNOS and COX-2 as well as the expression of iNOS and COX-2 in colon tumors compared with that in rats fed the WB diet or WB-PF diet. The study demonstrates for the first time that the lipid fraction of wheat bran has strong colon tumor inhibitor properties. The exact mechanism(s) by which the lipid fraction of WB inhibits colon carcinogenesis in addition to alteration of iNOS and COX activities remains to be elucidated. Additional studies are warranted to identify biologically active constituents of lipid fraction of WB and their relative role in colon tumor inhibition.

Several epidemiological and animal model studies suggest that consumption of olive oil, which is rich in the monounsaturated fatty acid, oleic acid (OA, C18:, n-9) may reduce the risk of breast cancer. There are however, a wide variety of olive oils in the marketplace with levels of OA ranging from a low of 50% to a high of 80% OA. The purpose of this rodent model study was to determine whether the level of OA in olive oil is a key determinant of its protective effects. We compared the inhibitory effects among three different types of olive oil containing 54, 70 and 80% OA and 20, 15 and 5% linoleic acid (LA), respectively, corn oil and a store bought olive oil, using the NMU-induced rat mammary tumor model. While little difference was found in total mammary tumor yields, a differential effect was found in the histological type of tumors formed. Olive oil containing 80% OA and 5% LA exhibited the lowest level of adenocarcinomas and the highest level of the more benign adenocarcinoma arising from within a fibroadenoma. While the reasons for this effect remain to be clarified, these results suggest that future studies on the health benefits of olive oil should take into account the type as well as the amount of olive oil.

Hybrid Polar Cytodifferentiation (HPC) agents represent a novel class of anticancer compounds which act by inducing terminal differentiation and/or apoptosis rather than by cytotoxic action. Among these are HPC agents such as hexamethylenebisacetamide (HMBA) and more potent 2nd generation hybrid/polar compounds such as suberanilohydroxamic acid (SAHA). As of the present, most studies on HPC agents have focused on cancers of the hematopoietic system rather than solid epithelial tumors. The objective of the present study therefore was to assess the chemopreventive action of these two related compounds in the N-methylnitrosourea (NMU)-induced rat mammary tumor model. Female Sprague-Dawley rats were fed diets containing 450 and 900 ppm, SAHA and 1000 and 2000 ppm HMBA, starting one week prior to NMU administration and continued for a period of 18 weeks. Mammary tumor development was monitored by palpation throughout the study, and at termination tumor incidence, number, multiplicity, latency and volume were determined. Weight gain was measured biweekly throughout the study. The salient results were as follows: SAHA at 900 ppm reduced NMU-induced mammary tumor incidence by 40%, total tumors by 66%, mean tumor multiplicity by 43% and mean tumor volume by 78%, with no detectable toxic side effects. HMBA exerted no tumor inhibiting effects at either concentration. This study represents the first demonstration that an HPC agent, namely SAHA, can inhibit the development of a chemically-induced, solid, epithelial tumor, at a relatively low dose (approximately 13 mgs/rat/day) without untoward side effects.

The Fiber Hypothesis which had its origins in the work of Burkitt and others in the early 1970's, focussed largely on fiber's beneficial effects on colon cancer and disorders of the gastric intestinal tract. In the 1980's it was proposed that fiber may also have beneficial effects on breast cancer and a rational for this was proposed involving modulation, by fiber, of the enterohepatic recirculation of estrogens. In the following the evidence from epidemiology, clinical interventions and animal model studies, supporting a role for fiber in breast cancer is critically reviewed. Evidence from animal model studies support the notion that supplementary fiber inhibits chemically-induced mammary tumorigenesis but do not support an estrogen-based mechanism. Some studies in human populations suggest modulation by estrogens and some do not. The aggregate data point to minor constituents present in fiber, such as isoflavones and phytate as the biologically active components of fiber which may be responsible for its anti cancer effects.

Epidemiological and experimental studies suggest that consumption of garlic may protect against several types of cancer. Moreover, a plausible hypothesis has been proposed that the biological effects of garlic can be attributed to the enhancing action of a variety of organosulfur compounds, present in garlic, on hepatic phase II carcinogen detoxification enzymes. We have used the N-methylnitrosourea (NMU)-induced rat mammary tumor model to test the chemopreventive effects of a water-soluble organosulfur constituent derived from aged garlic, S-allylcysteine (SAC). Rats were fed diets supplemented with 666 and 2,000 ppm SAC beginning seven days before initiation with NMU (55 days of age) to termination (18 wk post-NMU), at which time mammary tumors were enumerated. At neither dose did SAC exert an inhibitory effect on any index of tumor development, including incidence, latency, multiplicity, or volume, compared with untreated controls. Weight gains in all groups were similar. Assay of serum SAC levels in supplemented groups indicated that SAC concentrations were beneath the limits of detection of the high-performance liquid chromatography system used. These results contradict previous animal model studies indicating that SAC acts as an inhibitory agent in experimental mammary tumorigenesis; reasons for this discrepancy include the possibility that SAC may exhibit nonlinear dose effects.

Optical monitoring of activity provides new kinds of information about brain function. Two examples are discussed in this article. First, the spike activity of many individual neurons in small ganglia can be determined. Second, the spatiotemporal characteristics of coherent activity in the brain can be directly measured. This article discusses both general characteristics of optical measurements (sources of noise) as well as more methodological aspects related to voltage-sensitive dye measurements from the nervous system.

Studies in laboratory animals and epidemiological surveys suggest a relationship between the type and amount of dietary fat and mammary cancer. One mechanism proposed to explain this relationship is modulation by dietary fat, of mammary tumor eicosanoid levels through action at the rate limiting enzyme in eicosanoid synthesis, cyclooxygenase (COX). Until recently there have been no studies which have examined COX gene expression in human breast or rodent mammary tissues. In this study we have demonstrated the presence of two immunoreactive isoforms of cyclooxygenase (COX-1 and -2), and the modulating effects of n-3 fatty acids on their expression, in N-nitrosomethylurea (NMU)-induced rat mammary tumors. Three different high fat diets were compared namely, corn oil (CO) 23%; CO 18% menhaden oil (MO) 5%; CO, 5%/MO 18%; low fat corn oil (5%) served as a control. It was found that immunoreactive COX-2 protein levels were approximately 3x higher than COX-1 levels in NMU-induced mammary tumors. Moreover, the high menhaden oil diet (rich in n-3 fatty acids) significantly suppressed both COX-1 (-28%) and COX-2 (-36%) protein levels when compared to the high corn oil diet. No differences were found among the other treatment groups when compared pair-wise or with low-fat control. The mechanism(s) by which n-3 fatty acids suppress COX-1 and COX-2 remain to be determined.

Sodium hyaluronate has potential uses as an electrode lubricant in cochlear implant surgery. Deeper electrode insertions were achieved in six temporal bones implanted with sodium hyaluronate, compared to 22 patients implanted without sodium hyaluronate. Preliminary biosafety studies measuring hearing thresholds of six cats 4 months after implantation with dummy electrodes did not show evidence of ototoxicity caused by sodium hyaluronate. Until the biosafety studies are complete, we cannot recommend sodium hyaluronate for routine use, but the results so far are promising.

A new model for studying ileal reservoirs was used to compare J and W pouches. Sixteen female piglets were operated on between 62 and 67 days of age (median = 65). An approximately 50 cm vascularized segment of terminal ileum was isolated from the faecal stream and intestinal continuity restored. The distal 40 cm of the isolated loop was opened along its antimesenteric border and the pouch constructed (J = 8, W = 8). The remaining proximal length of ileum was brought out through the abdominal wall as a mucous fistula. One piglet in the W pouch group died post-operatively and in another, spontaneous closure of the mucous fistula prevented access to the W pouch for testing leaving six W and eight J pouches for comparison. Compliance was tested at 4 weeks by measuring intrapouch pressure during continuous distension by water. The volumes instilled at pressures of 20, 40, 60 and 80 cm of water were compared using the Mann-Whitney U-Test. Volumes varied widely at all pressures within both study groups although W pouches tended to be of larger volume for any given pouch pressure (P = NS). The failure of this study to demonstrate more clearly a difference between J and W pouches may be due to the small numbers involved but the wide range of volumes seen raises doubts about the usefulness of this pouch model.

Cyclin B, a positive regulatory subunit of the cdc2 protein kinase complex, is synthesized across the cell cycle and then rapidly degraded at the end of mitosis. Degradation of cyclin B is triggered by increased levels of active cdc2 and is required for exit from mitosis. It was shown previously that cyclin degradation is carried out by the ubiquitin system, but the components responsible for the specificity and regulation of cyclin-ubiquitin ligation have not been identified. The formation of ubiquitin-protein conjugates usually requires the sequential action of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3). In this work we employed a fractionation approach to identify the components of a clam oocyte system responsible for specific ubiquitination of cyclin and to determine which components are regulated by cdc2. Experimental conditions were established under which a fusion protein containing an amino-terminal fragment of cyclin B is ligated to ubiquitin only in extracts from M-phase but not from interphase cells. Fractionation of M-phase extracts by DEAE-cellulose and high speed centrifugation yielded three fractions that were all required for cell cycle stage-specific cyclin-ubiquitin ligation. Only one of these fractions could be replaced by a previously known enzyme of the ubiquitin system, E1. A second fraction contained a novel species of E2, termed E2-C, which acts in the ligation of ubiquitin to cyclin but not to other endogenous proteins. A third component is associated with particulate material. Whereas E2-C from either M-phase or interphase extracts is active, the particulate component is active only in M-phase. Incubation of the particulate fraction from interphase cells with the protein kinase cdc2 activates it for cyclin-ubiquitin ligation, after a lag of about 30 min. These findings suggest that the particulate fraction may contain an E3 enzyme that acts on cyclin, as well as additional factors activated by cdc2.

To reevaluate the risk associated with in utero exposure to lithium.Data were obtained from all published studies, in multiple languages, referenced in MEDLINE, Toxline, and the Lithium Information Center databases. Unpublished studies were not included. The search terms were lithium, pregnancy, teratogen, abnormalities (drug induced), Ebstein's anomaly, and adverse effects.In the 1970s a very strong association was suggested between maternal lithium treatment during pregnancy and Ebstein's anomaly of the heart in the offspring. The relative risk for Ebstein's anomaly among such children was estimated to be 400 on the basis of data collected from a registry of voluntarily submitted cases. More recent controlled epidemiologic studies have consistently shown a lower risk. No women who took lithium during pregnancy were found among four case-control studies of Ebstein's anomaly involving 25, 34, 59, and 89 affected children, respectively. In two cohort studies, risk ratios of 3.0 (95% confidence interval [CI], 1.2 to 7.7) and 1.5 (95% CI, 0.4 to 6.8) for all congenital anomalies have been observed. The risk ratios for cardiac malformations in these studies were 7.7 (95% CI, 1.5 to 41.2) and 1.2 (95% CI, 0.1 to 18.3), respectively.While initial information regarding the teratogenic risk of lithium treatment was derived from biased retrospective reports, more recent epidemiologic data indicate that the teratogenic risk of first-trimester lithium exposure is lower than previously suggested. The clinical management of women with bipolar disorder who have childbearing potential should be modified with this revised risk estimate.

Hepatic lipid and apolipoprotein synthesis is increased in the nephrotic syndrome. Catabolism of triglyceride-rich lipoproteins is impaired in nephrotic syndrome but not in rats with hereditary analbuminemia (NA), suggesting that lipid synthesis should be increased by analbuminemia in the absence of proteinuria. In this study the rate of cholesterol and fatty acid synthesis in liver and extrahepatic tissue was measured in female NA and control Sprague-Dawley (SD) rats to determine whether lipid synthesis was indeed increased in isolated analbuminemia and to identify the site(s) of increased lipogenesis. We also measured the concentrations of apolipoproteins (apo) AI, B, and E in plasma, as well as the levels of the respective mRNAs in liver. Plasma cholesterol, triglycerides, and apo AI, B, and E were all increased severalfold in the NA rat (P < 0.001). Although liver apolipoprotein mRNA content was significantly increased (P < 0.001) for apo AI (643%), B (273%), and E (299%), 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in liver microsomes and hepatic cholesterol synthesis were not significantly increased in the NA rats. Hepatic fatty acid synthesis and intestinal cholesterol synthesis were not increased in the NA rats. Surprisingly, intestinal fatty acid synthesis was elevated by 60% (P < 0.01). The NA rats demonstrated approximately fourfold increases in the incorporation of 3H2O into circulating cholesterol and fatty acids (P < 0.001). A 56% increase in the synthesis of total nonsaponifiable lipid was found in the extravisceral carcass (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

In this study, the anti-promoting effect of voluntary (wheel) exercise on 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumorigenesis was investigated. All rats were fed high fat diets (23% of calories as fat) to mimice the typical western diet. Two doses of DMBA were used to determine if the antipromoting effects of exercise were dependent on the strength of the initiating agent. In addition, tumor estrogen receptors were assayed to determined whether exercise, through an estrogen-suppressing mechanism, selects for estrogen receptor-negative tumors. Female Sprague-Dawley rats were fed a semi-purified 23% fat (corn oil) diet (AIN-76A) and, on day 50 of age administered DMBA by gavage at 5 or 10 mg/rat. Rats were then randomized into 4 groups (n = 30) as follows: 1) low DMBA/sedentary; 2) low DMBA/exercise; 3) high DMBA/-sedentary; and 4) high DMBA/exercise. Active rats were placed in wheel-cage units, which allowed voluntary access to an activity wheel for 133 (low DMBA) and 77 (high DMBA) days, respectively, Sedentary rats were placed in conventional cages. Both active groups exhibited significantly lower total tumor numbers than their sedentary controls: 75 vs 102 (low DMBA) (p0.05) and 90 vs 160 (high DMBA) (p0.001). Compared to sedentary controls, latency was significantly lengthened in the low but not the high DMBA active groups; multiplicity, in contrast, was significantly decreased in the high, but not the low DMBA exercised group. Exercise had no effect on overall tumor incidence. When segregated into exercise tertiles, total tumor active compared to the least active tertile, particularly in the high DMBA group.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibers in foods are complex carbohydrates. There are several types of fiber, but, for the purpose of mechanistic insight into their mode of protective action in carcinogenesis, classification into two broad types, soluble and insoluble fibers, is warranted. Soluble fibers are present in fruits, vegetables, and certain grains like oats. This type of fiber undergoes metabolism in the small intestine and especially in the large intestine through bacterial enzymes, converting it to products that increase stool size only moderately. But, they have appreciable effects in modifying the metabolism of colon carcinogens like azoxymethane to yield detoxified products and, thus, reducing colon carcinogenesis. In contrast, insoluble fibers present in sizeable amounts in bran cereals, like wheat or rice, are not significantly metabolized by enzymes in the intestinal flora. Such fibers increase stool size substantially through several mechanisms, including higher water retention. The larger bulk dilutes carcinogens, especially tumor promoters such as secondary bile acids, resulting in lower risk of colon cancer in animals and in humans. Evidence in animal models and in humans also indicates that fiber may lower the risk of breast cancer, possibly via an endocrine mechanism. Based on these concepts, increased intake of total fiber, but especially of wheat bran cereal fiber, to yield a daily stool in adults of about 200 grams can significantly reduce the risk of colon cancer and, to a lesser but definite extent, of breast cancer. Thus, adequate fiber intake from cereals, fruits, and vegetables can help prevent important types of human cancer.

We previously reported that supplementary dietary wheat bran significantly reduced the incidence of N-nitrosomethylurea (NMU) induced mammary tumors in rats fed a high fat (HF) diet and reduced to a lesser extent the incidence in rats fed a low fat (LF) diet compared to their unsupplemented controls. Using the same cohort of experimental animals, we here report the results of biochemical analyses designed to investigate the effect of supplemental dietary fiber on estrogen metabolism and lipid excretion. Serum, hepatic tissue, urine, and feces were obtained from rats which had been fed a HF (23.5% corn oil) diet, a HF plus fiber (HF + F, 10% soft white wheat bran, SWWB) diet, a LF (5.0% corn oil) diet, or a LF plus fiber (LF + F) diet for 15 weeks beginning 3 days after a single dose of NMU. Our working hypotheses to explain how dietary fiber protects against mammary tumorigenesis were that fiber may: 1) act as an antiestrogen; 2) decrease circulating estrogens; 3) alter the enterohepatic recirculation of estrogens; and 4) physically bind to lipid and remove it in the feces. Therefore, various indices of estrogen metabolism were assessed including: 1) circulating estradiol (E2) and progesterone; 2) hepatic estrogen receptor (ER) protein; 3) excretion of estrogen in the urine and feces; 4) the in vitro estrogen binding properties of SWWB; 5) fecal lipid content; and 6) the in vitro lipid binding capacity of SWWB. Serum 17 beta-estradiol and progesterone remained unchanged as did hepatic cytosolic (cER) and nuclear (nER) estrogen receptor protein content, in the fiber supplemented groups compared to their respective controls.(ABSTRACT TRUNCATED AT 250 WORDS)

We synthesized a novel organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (XSC), possessing low toxicity by comparison with inorganic Na2SeO3, and several other synthetic organoselenium compounds (K. El-Bayoumy, Cancer Res., 45: 3631-3636, 1985). We tested the effect of XSC treatment during the initiation phase on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma formation. A semipurified high-fat diet containing 80 ppm of XSC (40 ppm as selenium) was fed to 6-wk-old virgin female Sprague-Dawley rats for 2 wk, starting 1 wk before and ending 1 wk after carcinogen treatment. At 7 wk of age, rats were given a single dose of DMBA (5 mg) in 0.2 ml of olive oil by gastric intubation; the experiment was terminated 16 wk later. The development of mammary tumors in those rats that received XSC-supplemented diets was significantly inhibited when compared with the control group (fed the same diet without XSC supplements). This was evident from tumor incidence (percentage of tumor-bearing rats, 88 versus 20) and multiplicity of tumors (mean number of tumors/rats, 3.96 versus 0.28). The finding that XSC acts as a chemopreventive agent in the DMBA mammary tumor model prompted us to examine the effect of dietary XSC on DMBA-DNA binding in both the liver and mammary tissue under conditions identical to those described above for the bioassay. Rats (four/group) were killed 6, 24, 48, and 168 h after [3H]DMBA (5 mg/rat; specific activity, 51.2 mCi/mM) administration. Liver and mammary tissue were obtained and DNA was isolated. Dietary XSC was found to inhibit total DMBA-DNA binding in the mammary tissue, but not in the liver. The most profound effect was observed at early time points, i.e., 24 to 48 h after [3H]DMBA administration. The inhibition in total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol-epoxides of DMBA; these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine adducts on the basis of their chromatographic characteristics on high-pressure liquid chromatography and on a boronate affinity column. The inhibition of the DMBA-DNA binding in the target tissue provides a plausible explanation for the chemopreventive effect of XSC during the initiation stage of carcinogenesis.

The results of these studies indicate that voluntary activity suppresses the development of chemically and virally induced primary mammary tumors in rats and mice fed high-fat diets. These diets were chosen to mimic the current U.S. fat consumption of approximately 40% of calories as fat. It remains to be seen if activity exerts a similar suppressive effect on animals fed their customary low-fat diet (10% calories as fat). In general, the activity profiles of the female Fischer F-344 and Sprague-Dawley rat and the C3H/o mu j mouse exhibited a similar pattern with an early peak followed by a gradual plateau over time. The effects of activity on body fat composition showed a trend toward a decreased percent of body fat when compared to sedentary animals but a statistically significant decrease was found only in the F-344 female rat. In the DMBA model, carcinogen dose did alter outcome parameters. For example, time to first tumor was extended under low- but not high-DMBA conditions, and, conversely, tumor multiplicity was significantly decreased in the high- but not low-DMBA group. In the NMU model, an inverse association was found between the amount of activity and tumor incidence. A similar association was not found with the DMBA model. The reason for this is uncertain, but further analysis in terms of other parameters such as total tumor number may shed more light on this discrepancy. The suppressive effect of activity on the MMTV-induced mouse mammary tumor is of particular interest since it raises the possibility that activity may exert effects on the process of provirus insertion, and/or oncogene activation--an area of great potential promise in cancer prevention. Activity appeared to enhance the volume and to a lesser degree the number of metastatic foci in the lungs of F-344 retired breeders under high-fat but not medium-fat conditions. In addition, the most active animals in the high-fat group exhibited the greatest volume of metastases. These results, together with those in the NMU model, point to the critical importance of the quantity of voluntary activity an animal engages in and its relation to both primary and secondary cancer prevention. They imply that beyond a certain point of either frequency or intensity, the beneficial effect of exercise may be nullified by competing deleterious effects. The metastases study has also brought to light the importance of dietary fat as a potential intervening variable.(ABSTRACT TRUNCATED AT 400 WORDS)

The tissue distribution and time course of incorporation into acid insoluble (bound) and acid soluble (free) fractions of [3H]2-fluorohistidine is compared to that of U[14C]Histidine in mouse tissues in vivo. The cycloheximide-sensitive incorporation of 2-FHis is between 9 and 17 percent of that of His. Unlike [14C]His a major fraction, approximately 90% at 72 hrs, of isotope derived from [3H]2-FHis remains in tissues for a prolonged period in an acid soluble form. The excretion of isotope derived from [14C]His (T1/2 = 5 hr) is more rapid than from [3H]2-FHis (T1/2 = 11.4 hrs). 2-FHis, at doses from 100 to 250 mg/kg produce a reversible inhibition of growth in mice.

A derivative of ubiquitin in which amino groups were blocked by reductive methylation was used to study the possible role of the ubiquitin pathway in the cell cycle-programmed degradation of cyclin. It was shown previously that methylated ubiquitin can be efficiently ligated to protein substrates, but cannot form polyubiquitin chains. In the well-characterized ubiquitin-dependent proteolytic system from reticulocytes, it was found that rates of protein breakdown obtained with methylated ubiquitin are generally slower than those with ubiquitin; and thus, this derivative can be used, in some cases, as an inhibitor of ubiquitin-dependent protein degradation. The addition of methylated ubiquitin to a cell-free system from fertilized clam oocytes inhibited the degradation of both cyclins A and B. That this was due to specific interference with ubiquitin function was indicated by the observation that the supplementation of excess ubiquitin completely overcame the inhibitory action of methylated ubiquitin on cyclin degradation. These findings suggest that polyubiquitin chain formation is required for cyclin degradation.

The effect of dietary fat and exercise on N-nitrosomethylurea [NMU:CAS:684-93-5]-induced mammary tumorigenesis in female F344 rats was investigated. Rats were fed the NIH-07 diet until NMU administration on day 50 of age, when they were transferred to four treatment groups. Three sedentary groups were fed either high-fat (20% wt/wt), medium fat (10%) or low fat (5%) diets (HF, MF, LF, respectively), and a fourth group was fed a HF diet but allowed free access to an activity wheel (HFEX). Tumor yields among the three sedentary groups were significantly greater in the HF and MF groups when compared to the LF group. Voluntary exercise reduced tumor yields and delayed time of tumor appearance in HFEX animals to levels similar to those found in LF sedentary animals. Animals with voluntary access to exercise wheels averaged between 1.03 and 2.85 miles/day, consumed more food (+ 18%) and exhibited greater weight gain (+ 13%) than their sedentary counterparts. No differences in weight gains were detected among the HF, MF, and LF groups, despite widely varying amounts of fat intake. Body composition studies indicated that body fat content was not influenced by the quantity of fat consumed in the diet, but was significantly reduced by voluntary exercise (-20%). Since exercise and fat intake have been associated with alterations in endocrine status, circulating bioactive and immunoactive prolactin were assessed at termination. No significant changes were found in either form of prolactin among the four experimental groups, casting doubt on mediation by this pituitary hormone.

The ability of antipili antibody to prevent ascending urinary tract infection was investigated in rats. One group of rats was immunized passively with rabbit antisera to purified pili and challenged by intravesicular inoculation of 5 x 10(7) heavily piliated Escherichia coli. Only 2 of 14 immunized animals developed cortical abscesses as compared to 13 of 15 control rats given normal rabbit serum (P equals 0.0001). The mean log titer of bacteria in the kidneys of the immunized rats was 0.85 vs. 6.08 in the controls (P less than 0.005). A second group was actively immunized with pili. 3 of 16 immunized animals became infected as compared to 10 of 15 controls (P equals 0.01). The mean log titers were 2.13 and 4.54, respectively (P less than 0.01). A third group was passively immunized and challenged with a strain that had different O, K, and H antigens but shared pili antigens. Abscesses occurred in 4 of 15 immunized animals as compared to 13 of 15 controls (P equals 0.001). The mean log titers were 2.37 and 5.63, respectively (P less than 0.005). These results indicate that antipili antibody protects rats against ascending urinary tract infections.

Dynorphin, an opioid peptide, is thought to play an important role in the modulation of nociceptive neural circuits at the level of the spinal cord. In a model of peripheral inflammation and hyperalgesia, an oligodeoxyribonucleotide probe complementary to a portion of preprodynorphin mRNA and antisera to dynorphin A-(1-8) were used to localize changes in dynorphin mRNA and peptide to individual spinal cord neurons. Intraplantar injection in rats of complete Freund's adjuvant resulted in edema and hyperalgesia to radiant heat stimulation of the injected hind paw that reached a peak at 4 days. At the same time, in situ hybridization histochemistry and immunocytochemistry identified an increase in transcription of preprodynorphin mRNA that was paralleled by an increase in dynorphin peptide. These changes were seen in spinal neurons in the medial two-thirds of laminae I and II and in laminae V and VI of lumbar segments receiving innervation from the inflamed paw. Since neurons demonstrating the increase in dynorphin biosynthesis are located in both the superficial and deep dorsal horn laminae, our data provide evidence for opioid modulation of nociceptive neural circuits in these two distinct spinal locations.

Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (less than 0.5 microM) of U18666A, inhibitors of desmosterol delta 24-reductase, of lanosterol 14 alpha-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3 microM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity. In the presence of 30 microM-U18666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.

SummaryThe effects of estradiol and ovine prolactin on the growth of a rat mammary adenocarcinoma cell line were investigated. The action of estradiol was dose-dependent. At ±1 μg/ml it did not influence growth, at 5-10 μg/ml it was inhibitory, and at 20 μg/ml it was toxic. Prolactin alone had little effect on growth. However, the inhibitory effect of estradiol could be counteracted by a high concentration of prolactin. It appears, therefore, that the growth of cultured rat mammary adenocarcinoma cells can be regulated by the ratio of prolactin to estradiol: When the ratio is high growth is favored; when it is low, growth is inhibited.We thank Dr. J. H. Weisburger for his critical review of the manuscript; the Hormone Distribution Program, National Institute of Arthritis, Metabolic and Digestive Diseases for the generous supply of ovine prolactin; and Ms. Michele Soyer and Dr. P. Hill for estrogen assays. Supported in part by Public Health Service Research Grant No. CA 012376 from the National Cancer Inst...

International audience; Fatty acid metabolites (long-chain esters of CoA and carnitine) which collect in ischemic myocardium can form amphiphiles capable of disrupting subcellular performance. It is important to document the role of these amphiphiles in intact tissue. D-Octanoylcarnitine was chosen because of its previously described effects on inhibiting palmitoylcarnitine transferasc (PCT-II) inin vitro andin vivo liver preparations. This inhibition will shift tissue levels of CoA and carnitine intermediates and thus alter amphiphile levels. The compound's actions in cardiac muscle are unknown. Dose response curves were developed in intact hearts to test the influence of D-octanoylcarnitine at pharmacological concentrations. Measurements were obtained in working, extracorporeally perfused, swine hearts. Drug was administered either systemically (IV) or via dircct intracoronary (IC) infusions into the left anterior descending coronary circulation. Excess fatty acids were provided to ensure adequate fatty acid substrate for oxidation. Coronary flow was controlled at aerobic levels. Systemic administration of D-octanoylcarnitine (0.8–6.8 mM) resulted in transient peripheral hypotension which caused correlative decreascs in14CO2 production from labeled palmitate. Infusion of D-octanoylcarnitine (0.5–3.9 mM) IC did not cause appreciable hypotension and was not associated with suppression of fatty acid oxidation. No build-up of carnitine esters was noted in treated hearts but acyl CoA levels were reduced (p

A hypothesis is presented that explains the mammary tumor-promoting effects of high fat diets on the basis of alteration in the hormonal milieu, namely the relative concentrations of circulating prolactin to estrogen. Evidence from in vivo and in vitro studies drawn from work in our laboratory and others is reviewed in light of this hypothesis. It is postulated that mammary tumor cell proliferation is stimulated when the prolactin:estrogen ratio is high and is inhibited when the ratio is low. Chronic high fat intake elevates serum prolactin levels, thus raises the prolactin:estrogen ratio, and thereby promotes mammary tumor cell growth.

Two omega-3 fatty acids present in fish oil are effective inhibitors of some models of mammary and colon tumorigenesis in rodents. The present studies were conducted to determine if eicosapentaenoic and docosahexaenoic acids can modify the growth of DU-145 human prostatic tumor cells in nude mice. Two experimental diets tested contained either 23.52% corn oil or 20.52% fish oil, plus 3% corn oil (w/w). In the fish oil-fed group of mice: (a) tumor growth was significantly inhibited; (b) tumor cells in histological sections were smaller but more connective tissue was present; (c) immunochemical staining for human prostatic acid phosphatase was less intense, and (d) tumor content of PGE2 was smaller than in the 23.52% corn oil-fed group. Fatty acid composition of phosphoglyceride and neutral lipid fractions of liver, prostate, and tumor tissue reflect the dietary intake of omega-3 and omega-6 fatty acids. These results are consistent with a role for omega-3 fatty acids in the inhibition of growth of human prostatic tumor cells in nude mice by dietary modification.

The effects of four halogenated analogs of histidine on in vitro growth of Plasmodium falciparum malaria parasites were monitored by measurement of the incorporation of 3H-labeled amino acids into parasite proteins and by light and electron microscopy. The uptake of [3H]isoleucine was reduced to 50% of the control value by addition of 70 microM 2-fluoro-L-histidine (2-F-HIS) or 420 microM 2-iodo-L-histidine (2-I-HIS). [3H]histidine uptake into acid-insoluble material was affected equally by these two compounds, 50% inhibition resulting at 200 microM concentration. Morphological analysis of parasite development proved a sensitive assay, since development of mature trophozoites was inhibited 50% by 25 microM 2-F-HIS or 100 2-I-HIS. Electron microscopy studies suggested different mechanisms of action of 2-F-HIS and 2-I-HIS on P. falciparum. 2-F-HIS produced a decrease in knob number at the erythrocyte surface and accumulation of electron-dense material under the parasite membrane. 2-I-HIS had no obvious effect on knobs or electron-dense material but affected parasite morphology. Surprisingly, 2-chloro-L-histidine and 2-bromo-L-histidine did not inhibit P. falciparum in vitro, even though their halogen atom substituents are intermediate in size between F and I atoms. 2-F-HIS and 2-I-HIS were tested in vivo against P. falciparum in owl monkeys (Aotus sp.) but were ineffective at doses that were nontoxic.

The effect of dietary fat, energy restriction, and exercise on N-nitrosomethylurea (NMU:CAS:684-93-5)-induced mammary tumorigenesis in female F344 rats was investigated. Rats were fed the NIH-07 diet until N-nitrosomethylurea administration on Day 50 of age, when they were transferred to six treatment groups. Three sedentary groups were fed either high-fat (20%, w/w), medium-fat (10%), or low-fat (5%) diets ad libitum (HFAL, MFAL, LFAL, respectively); two sedentary groups were fed high fat and medium fat diets restricted to 75% of the food consumed by their ad libitum counterparts (HFR, MFR), and one group was fed a HFAL diet but allowed free access to an activity wheel (HFEX). Tumor yields among the three ad libitum sedentary groups were significantly greater in the HFAL and MFAL groups when compared to the LFAL group. Dietary restriction reduced tumor yields by more than 90% of ad libitum controls regardless of fat intake. Voluntary exercise reduced tumor yields and delayed time of tumor appearance in HFEX animals to levels similar to those found in LFAL animals. Animals with voluntary access to exercise wheels averaged between 1.03 and 2.85 miles/day, consumed more food (+18%), and exhibited greater weight gain (+13%) than their sedentary counterparts. Restricted animals exhibited significantly decreased body weight gains (-15%) compared to their ad libitum counterparts, but no differences in weight gains were detected among the HFAL, MFAL, and LFAL groups, despite widely varying amounts of fat intake. Body composition studies indicated that body fat content was not influenced by the quantity of fat consumed in the diet, but was significantly reduced by caloric restriction (-20 to 26%) and exercise (-20%). While the precise mechanisms underlying the tumor-promoting effects of HFAL diets and the antipromoting effects of energy restriction and exercise remain to be elucidated, available evidence suggests that these effects are not due to alterations in energy homeostasis per se, but may instead be exerted indirectly, and perhaps independently via endocrine, paracrine, or neurohormonal mechanisms.

In liver, kidney, and lung of the mouse, two of the major H1 subtypes present at 1 week after birth, H1a and H1b, decline to nearly undetectable levels by 8-16 weeks, and a third subtype, H1d, also diminishes. The amounts of two other H1 subtypes, H1e and H1 degrees, increase markedly and that of another, H1c, also increases. The disappearance of H1a is almost complete by the age of 4 weeks, whereas the decline in H1b occurs largely between 4 and 16 weeks, when most cells are nondividing. A decline in H1a was observed also in cultured mouse embryo fibroblasts after they became quiescent. Furthermore, in the quiescent fibroblasts, the synthesis of H1a and H1b was preferentially reduced. In the thymus, newly formed nondividing cells have as much H1a and H1b as the dividing cells from which they are derived, but circulating lymphocytes have less H1a and H1b and more H1e, indicating that molecules of H1a and H1b are removed from chromatin, and H1e deposited, after cell division ceases. H1 degrees, however, is not present in thymocytes or peripheral blood lymphocytes. Our results indicate that 1) there are no tissue-specific H1 subtypes among the somatic tissues examined; 2) H1a and H1b are synthesized in large amounts only in dividing cells, whereas H1c, H1d, and H1e are synthesized in both dividing and nondividing cells; 3) different H1 subtypes are degraded at different rates in nondividing cells; and 4) H1e always, and H1 degrees sometimes, accumulates in nondividing somatic cells. We conclude that alterations of the H1 composition of chromatin are a general feature of nondividing cell formation; that such alterations are made, in large part, after the nondividing cells are formed and are accomplished both by changes in the pattern of synthesis of the various H1 subtypes and by differential loss of subtypes; and that each H1 subtype differs from the others in some or all of the properties that we have examined, supporting the idea that the individual H1 subtypes differ from each other in some of their functions.