Subchronic oral toxicity studies of Se-methylselenocysteine, an organoselenium compound for breast cancer prevention.

Se-methylselenocysteine (MSC) is an organoselenium compound being developed for breast cancer chemoprevention. To characterize MSC toxicity, CD rats received daily gavage doses of 0, 0.5, 1.0, or 2.0 mg/kg/day (0, 3, 6, or 12 mg/m(2)/day), and beagle dogs received daily gavage doses of 0, 0.15, 0.3, or 0.6 mg/kg/day (0, 3, 6, or 12 mg/m(2)/day) for 28 days. In rats, MSC induced dose-related hepatomegaly in both sexes; mild anemia, thrombocytopenia, and elevated liver enzymes were observed in high dose females only. Microscopic pathology included hepatocellular degeneration (high dose males, all doses in females); arrested spermatogenesis (high dose males); and atrophy of corpora lutea (middle and high dose females). In dogs, MSC induced mild anemia in middle and high dose males, and in high dose females. Toxicologically significant microscopic lesions in dogs were seen only in the liver (peliosis and vacuolar degeneration in high dose males, midzonal necrosis in males in all dose groups). Based on liver pathology seen in female rats in all dose groups, the no observed adverse effect level (NOAEL) for MSC in rats is <0.5mg/kg/day. Based on alterations in hematology parameters and liver morphology in male dogs in all dose groups, the NOAEL for MSC in dogs is <0.15 mg/kg/day.

Oncogenicity evaluation of resveratrol in p53(+/-) (p53 knockout) mice.

A six-month study was conducted in p53(+/-) mice to evaluate the possible oncogenicity of resveratrol (3,5,4'-trihydroxy-trans-stilbene), a cancer chemopreventive agent present in grapes and other foods. p53(+/-) mice (25/sex/group) received daily gavage exposure to vehicle only (negative control), resveratrol doses of 1000, 2000, or 4000 mg/kg/day, or p-cresidine (400 mg/kg/day; positive control). No mortality was seen in mice receiving the low dose of resveratrol. However, the mid and high doses induced mortality associated with impaction of the test article in the gastrointestinal tract. Resveratrol had no effect on body weight, food consumption, or clinical signs in surviving mice in any dose group, but induced dose-related increases in liver weight and serum cholesterol in both sexes. Mild anemia was seen in male mice at the high dose only; hematologic effects were not seen in females. Histopathology identified the kidney (hydronephrosis) and urinary bladder (epithelial hyperplasia) as target tissues for resveratrol toxicity. The incidences of both benign and malignant tumors in mice exposed to resveratrol were comparable to those in vehicle controls. By contrast, the positive control article, p-cresidine, induced urinary bladder cancer in both sexes. When administered to p53(+/-) mice at its maximum tolerated dose, resveratrol demonstrates no evidence of oncogenicity.

Histological alterations in male A/J mice following nose-only exposure to tobacco smoke.

The incidence and multiplicity of grossly observed and microscopic lesions of the respiratory tract of A/J mice exposed nose-only to mainstream smoke (50, 200, or 400 mg total particulate matter/m3 from 2R4F cigarettes) was compared to those of filtered air controls. Animals were necropsied at the end of exposure (5 mo) or following 4 or 7 mo of recovery. Lungs were visually inspected for tumors at all necropsies and examined histopathologically at 9 and 12 mo. At 5 mo no tumors were recorded. No significant elevations in tumor incidence or multiplicity were recorded although at 9 mo multiplicity was elevated in the mid-exposure group (0.90 versus 0.55 tumors per animal for controls). At 12 mo, multiplicity was increased over the 9-mo necropsy at all exposures except 200 mg/m3; however, there were no dose-related trends in multiplicity or incidence. Histopathological alterations included hyperplasia, metaplasia, and inflammation of the nose and larynx and proliferative lesions of the lungs. At 9 mo, the multiplicity of focal lung lesions was 1.4 per animal in controls but averaged 1.0 among smoke-exposed groups. There was an inverse relation (p < .059) between smoke concentration and the percentage of hyperplastic lesions at 9 mo. At 12 mo the high-exposure group had slightly increased multiplicity of 2.3 lesions compared with 1.6 among controls, while the percentage of hyperplasic lesions was similar between groups. Nose-only inhalation of mainstream tobacco smoke resulted in chronic inflammatory changes of the respiratory tract yet failed to produce statistically significant changes in tumor incidence or multiplicity.

Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation.

The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.

Calcium accumulation by chick intestinal mitochondria. Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3.

We have the evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca2+ accumulation by chick intestinal mitochondria. Ca2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na+-labile pool followed by an ATP-dependent accumulation into an Na+-resistant pool. Ca2+ accumulation is extensive at free Ca2+ concentrations greater than 3 . 10(-6) M in the presence of ATP. Ruthenium red and dinitrophenol block Ca2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca2+, or the rate and extent of ATP-supported Ca2+ accumulation. Intestinal cytosol stimulated Ca2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca2+-binding protein. 30 ng/ml 1,25(OH)2D-3 stimulated Ca2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 greater than 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca2+ concentration from 3 . 10(-6) to 1 . 10(-5) M increased Ca2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)2D-3 in vitro increased Ca2+ accumulation less than 2-fold, we conclude that 1,25(OH)2D-3 influences mitochondrial accumulation of Ca2+ in vivo primarily by altering cytosol concentrations of free Ca2+.

Immunoperoxidase localization of vitamin D dependent calcium binding protein.

Calcium binding protein (CaBP) was localized by the indirect peroxidase-labeled antibody method in chick duodenum 72 hr after administering 32.5 nmol of cholecalciferol to vitamin D-deficient chicks. CaBP was observed in cytoplasm and nuclei of absorptive cells but was absent from goblet cells. Our results are consistent with the suggested functional role for CaBP in the prevention of intracellular accumulation of calcium by preventing mitochondrial accumulation of calcium, enhancing removal of calcium from absorptive cells, and/or preventing the "leaking" of calcium into cells through the lateral borders. They are not consistent with an extracellular functional role for CaBP.

Action of vitamin D on intestinal calcium transport.

The means by which 1,25(OH)2D3 regulates calcium transport across the intestine still remains an enigma. The movement of calcium across the brush border membrane into the cell occurs down a steep concentration gradient. Stimulation of this process by 1,25(OH)2D3 occurs within 2h and does not require new protein synthesis. Although the mitochondria may be involved in moving the calcium across the cell under rather high concentrations of cytosol calcium (10(-5) M Ca), a different organelle (lysosome-like vesicle) may assume primary responsibility for intracellular transport at lower calcium concentrations. This change from mitochondrial to vesicular calcium movement requires time (within 18 h) and may involve new protein synthesis. In the absence of this changeover, calcium transport can still occur but only at the cost of higher intracellular calcium concentrations. Movement of calcium out of the cell across the basolateral membrane occurrs against a steep concentration gradient. The difficulty in preparing pure basolateral membranes with homogeneous orientation of the inner and outer surfaces has limited progress in our understanding of calcium movement across this membrane. However, the need for an energy-driven calcium pump at this membrane that is responsive to micromolar calcium concentrations is apparent. Whether 1,25(OH)2D3 induces the synthesis of such a pump or leads to its activation by factors such as CaBP, cAMP, and calmodulin remains to be determined.

Thirteen-week oral toxicity study of difluoromethylornithine in combination with tamoxifen citrate in female dogs.

PURPOSE: Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM), a nonsteroidal antiestrogen, is approved for use in the treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of TAM with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. METHODS: The toxicity of DFMO in combination with TAM was evaluated in female Beagle dogs following 13 weeks of daily oral administration by capsule. Dose levels in milligrams per kilogram body weight per day were: 0 (vehicle control), 100 DFMO, 0.1 TAM, 1.0 TAM, 0.1 TAM + 100 DFMO and 1.0 TAM + 100 DFMO. RESULTS: No mortalities occurred. Diarrhea was produced by TAM and vaginal discharge, due to reproductive tract lesions, was produced by both DFMO and TAM, either alone or in combination. DFMO decreased reticulocyte counts and TAM increased counts of mature neutrophils. DFMO alone resulted in lesions to the intestines and ovaries, and cornified epithelium of vagina and cervix. TAM produced cornified epithelium of vagina and cervix, and numerous lesions in the ovaries, fallopian tube, uterus, cervix and vagina which were likely due to an estrogen agonist effect. Coadministration of DFMO increased the incidence and/or severity of these reproductive tract lesions. Each compound alone produced ovarian atrophy, and antral follicles and corpora lutea were completely absent in the 1.0 TAM + 100 DFMO group. CONCLUSIONS: Coadministration of DFMO and TAM resulted in additive toxicity involving the female reproductive system.

Difluoromethylornithine in combination with tamoxifen in female rats: 13-week oral toxicity study.

PURPOSE: Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM) is currently used for treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of tamoxifen with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. METHODS: The toxicity of DFMO in combination with TAM was evaluated in female rats following 13 weeks of daily administration by gavage. Dose groups were vehicle control, DFMO (1000 mg/kg per day), low TAM (0.25 mg/kg per day), high TAM (2.5 mg/kg per day), low combination (1000 + 0.25) and high combination (1000 + 2.5). RESULTS: No mortalities occurred in the study. Clinical signs of toxicity were limited to dermal lesions consisting of scab formation and abrasions produced by DFMO. Administration of either DFMO or TAM resulted in decreased body weight gains, with coadministration having an additive effect. Serum albumin, total protein, cholesterol and triglyceride levels were decreased in all drug-treated dose groups, although histologic evidence of liver lesions were not seen. TAM resulted in increased numbers of red blood cells, whereas DFMO produced a slightly anemic response. DFMO produced lesions in the small intestine consisting of necrosis of crypt epithelium and crypt microabscess, which were enhanced by TAM coadministration. Administration of TAM resulted in histologic changes in the ovaries, fallopian tube, vagina, cervix and uterus, indicating that inhibition of ovulation and reproductive cycle arrest in the proestrus stage had occurred. Coadministration with DFMO did not affect the changes to the reproductive system induced by TAM. CONCLUSIONS: Coadministration of DFMO with tamoxifen did not result in toxicity unique to the combination drug regimen, but rather toxicity resulted from administration of each drug. Under the conditions of the study, the overall toxicity produced by dual administration of DFMO with tamoxifen was additive with respect to the toxicity associated with each agent alone.

Effect of static pressure on the disappearance rate of specific echocardiographic contrast agents.

Contrast echocardiography has been applied to identify cardiac structures, shunts, and perfusion territories. Most recently, quantification of flow has been proposed based on disappearance of contrast intensity. This requires that contrast agents are stable and produce a predictable effect. To assess the possible effect of pressure on their stability, the rates of backscatter decay of four echocardiographic contrast agents (Albunex, Levovist, agitated Angiovist, and agitated saline solution) exposed to constant pressures (0, 50, 100, 150, and 200 mm Hg) were quantitated. Contrast was recorded by echocardiography and measured to construct time-intensity curves. The peak decay rate for each agent at each pressure was determined. For all four agents, contrast intensity (I) decreased over time and could be described by the sigmoid function: I = a [e-lambda(t-ts)/1 + e-lambda(t-ts)] + C. Peak decay rate was significantly affected by pressure (p < 0.005) in a proportionate fashion. At pressures of 0, 100, and 200 mm Hg, the rates increased for each agent in the following fashion: Albunex, 0.144 +/- 0.109 to 0.410 +/- 0.142 to 1.442 +/- 0.309; Levovist, 0.060 +/- 0.023 to 0.162 +/- 0.049 to 0.495 +/- 0.142; Angiovist, 0.089 +/- 0.028 to 0.166 +/- 0.057 to 0.224 +/- 0.027; and saline solution, 0.068 +/- 0.039 to 0.110 +/- 0.036 to 0.154 +/- 0.057. The effect of pressure on the peak rate of contrast disappearance (lambda) was significantly different among agents (p < 0.001). Thus attempts to quantitate blood flow with contrast agents must take into account the influence of pressure.

Automated assessment of ventricular volume and function by echocardiography: validation of automated border detection.

To determine the utility of a new on-line echocardiographic automated border detection (ABD) algorithm in assessing ventricular volume and ejection fraction, an optimal model was studied. This open-chest canine model allowed continuous measurement of actual left ventricular volume. In four dogs, true end-systolic and end-diastolic volume and ejection fraction were compared with those obtained by two-dimensional echocardiography with an automated method calculated from a border detection algorithm to define left ventricular endocardium and the single-plane Simpson method to calculate volume. Left ventricular volumes that used manual, off-line tracings of the left ventricle by two-dimensional echocardiograms and the single-plane Simpson method were compared. The automated echocardiographic volumes correlated with true volumes (y = 0.7x + 8.9; standard error of the estimate = 13.5 cc; r = 0.81). A significant mean underestimation of 11 +/- 15 cc was noted (p < 0.0001). Volumes obtained from the manual tracings of left ventricular endocardial contours also correlated well with true volumes (y = 0.89x + 4; standard error of the estimate = 6.7 cc; r = 0.96). However, the 3 +/- 7 underestimation was significantly lower than the error of the ABD method (p = 0.00005). Both on-line ABD and off-line ejection fractions correlated well with true ejection fractions (r = 0.94 and 0.96, respectively). There was no statistically significant difference between the mean errors of the ABD or manually derived ejection fractions. In the setting of optimal left ventricular imaging, the on-line and rapid features of this automated method make it potentially useful for quickly obtaining left ventricular volumes and ejection fraction.

Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity.

To characterize the subchronic oral toxicity of resveratrol, CD rats received daily gavage doses of 0, 200, 400, or 1000 mg resveratrol/kg/day, and beagle dogs received daily capsule doses of 0, 200, 600, or 1200 mg resveratrol/kg/day for 90 days. Resveratrol induced only minimal toxicity, consisting of dose-related reductions in body weight gain in female rats and both sexes of dogs, and a statistically significant increase in bilirubin levels in rats at the 1000 mg/kg/day dose. Clinical observations, hematology, ophthalmology, neurotoxicity evaluations (functional observational batteries), organ weights, and gross pathology provided no biologically significant evidence of resveratrol toxicity in either species. In rats, the high dose of resveratrol reduced the incidence of cardiomyopathy; no other microscopic changes were seen. Histopathologic changes in dogs were limited to minimal inflammatory infiltrates in the kidney and urinary bladder, which were not considered toxicologically significant. A cardiovascular safety pharmacology (telemetry) study in dogs revealed no evidence of resveratrol toxicity. Based on body weight effects, the No Observed Adverse Effect Level (NOAEL) for resveratrol was 200mg/kg/day in rats and 600 mg/kg/day in dogs. The apparent cardioprotective activity of resveratrol in rats demonstrates that its potentially beneficial activities may extend beyond efficacy in cancer prevention.

Respiratory infectivity of a recently isolated Egyptian strain of Rift Valley fever virus.

The respiratory infectivity of a strain of Rift Valley fever virus isolated in Egypt (strain ZH-501) was compared with that of one isolate from Uganda (Entebbe strain) and two isolates from South Africa (strains SA-51 and SA-75). Studies were performed with ICR mice which were infected by exposure to infectious aerosols composed of particles with a mass median diameter of 0.96 micrometer. The respiratory median lethal doses for ZH-501, Entebbe, SA-51, and SA-75 were 2.2, 1.9, 2.6, and 1.9 log10 plaque-forming units, respectively. Although these values are statistically different, the biological implications of such differences seem unimportant. In an additional study of pathogenesis, a single group of mice was infected with 3.1 log10 plaque-forming units of ZH-501, and tissues were assayed sequentially through 96 h postinfection. Between 6 and 30 h, demonstration of an increasing virus concentration only in the lungs indicated that initial replication occurred there; however, determination of histopathological changes did not reveal evidence of pneumonia. Virus was isolated from the liver by 48 h, and the ultimate outcome of infection was a fulminating and fatal hepatic necrosis.

Amplified response to phenobarbital promotion of hepatotumorigenesis in obese yellow Avy/A (C3H x VY) F-1 hybrid mice.

Mottled yellow Avy/A and agouti A/a (C3H x VY) F-1 hybrid male mice were fed untreated control diet or diet with a target dose of 500 p.p.m. sodium phenobarbital (PB) for 17-19 months. No differences in prevalence of hepatocellular adenomas or carcinomas were found between untreated yellow and agouti mice. PB treatment increased prevalence of adenomas but decreased prevalence of carcinomas. No difference in enhancement of adenoma formation by PB was observed between yellow and agouti mice bearing single adenomas. However, the proportion of PB-treated yellow mice bearing multiple adenomas (66%) was much greater than the proportion of analogous agouti mice (18%). Fatty changes in the periportal area of the liver and focal cytoplasmic vacuolization were induced to a much greater extent in PB-treated yellow mice than among treated agoutis. PB increased the prevalence and severity of focal areas of chronic inflammation in the liver considerably more in agouti than in yellow mice. The possible relation of this finding to the altered immune responses of obese yellow mice remains to be determined. The results of this study suggest that the use of yellow Avy/A and agouti A/a (C3H x VY) F-1 hybrid mice in carcinogenicity assays make make it possible to differentiate between weak and strong promoters as well as between promoters and complete carcinogens. Weak promoters should induce hepatocellular adenomas in yellow mice even if they fail to do so in agouti mice. Promoting substances which act similarly to PB may be identified in this system by simultaneously increasing adenoma prevalence and decreasing carcinoma prevalence. Complete carcinogens should increase carcinoma prevalence in the yellow mice even at low dose levels.

Susceptible and resistant subgroups in genetically identical populations: response of mouse liver neoplasia and body weight to phenobarbital.

Following 17-19 months of feeding 500 p.p.m. sodium phenobarbital (PB) in the diet to yellow Avy/A and agouti A/a (C3H X VY) F1 hybrid male mice, two subgroups differing in responsiveness to PB with respect to promotion of hepatocellular adenomas and body weight gain were observed within each genotype. In untreated mice of both genotypes, the presence of an adenoma at necropsy was associated with decreased body weight gain during this study. However, PB treatment inverted this association. In treated mice the presence of an adenoma at necropsy was preceded by a greater increase in body weight during the study than when no tumor was present. This increase in average body weight gain was more pronounced among the yellow mice (44%) than among the agouti mice (21%). Among yellow mice PB treatment had no effect on body weight gain unless an adenoma was present at necropsy. However, in those yellow mice in which an adenoma was found, body weight was greater than in untreated yellow controls throughout the study beginning at week 27. The mean body weight curve of treated yellow mice bearing one adenoma was slightly higher than that of treated yellow mice in which no adenoma was found. The mean body weight curve of treated yellow mice bearing multiple adenomas was significantly higher than those of yellow mice with no or only one adenoma.