Effects of the dietary phytoestrogens daidzein and genistein on the incidence of vulvar carcinomas in 129/J mice.

The objective of this study was to determine the effect of dietary phytoestrogens on the incidence of spontaneous vulvar carcinomas in 129/J mice using three natural ingredient diets and two purified diets containing predetermined levels of daidzein and genistein. Eighty weanling female mice without clinical evidence of vulvar carcinomas were randomly assigned 16 per diet to each of 5 test diets. Mice were clinically examined for vulvar masses weekly for 3 months and at monthly intervals thereafter. Vulvar carcinomas in representative groups of mice were confirmed using routine histological procedures. The incidence of vulvar carcinomas increased sharply in mice on all test diets during the first 2 months with minor changes during the remainder of the study. Within one month, the incidence of vulvar carcinomas in mice fed the AIN-76A modified soy protein diet was significantly (P < .05) increased over those of mice fed the AIN-76A modified casein diet, the #5K96, or the # 5058 diet. At three months, the incidence of vulvar carcinomas in mice fed the soy protein diet was significantly (P < .05) increased over those of mice fed the NIH-31 diet or the PMI #5K96 diet. There was a marginally significant (P < .10) correlation between the total daidzein and genistein levels in the five test diets and the incidence of vulvar carcinomas in mice as determined by clinical examination. We concluded that dietary levels of daidzein and genistein were associated with an increase in the incidence of vulvar carcinomas in mice and that the 129/J mouse may provide an animal model for studying the development of vulvar carcinomas.

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.

Neuropathologic findings associated with seizures in FVB mice.

The FVB mouse is used extensively in transgenic research because of its defined inbred background, superior reproductive performance, and prominent pronuclei, which facilitate microinjection of genomic material. Seizures associated with a known mutation and seizure-susceptible inbred strains are well documented in mice; however, to the authors' knowledge, seizures in the FVB strain have not been evaluated. Affected nonmanipulated FVB/N (n = 5) and transgenic FVB/N mice generated, using eight unrelated transgenic constructs (n = 63), were submitted for pathologic examination. Most cases were detected during routine observations in animal rooms; however, seizure induction by tail tattooing, fur clipping, and fire alarms has been observed. The majority of mice were female (62 of 68), with mean age of 5.8 months (range, 2 to 16 months). Observations made during seizure presentation in 12 of 68 mice included facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that frequently progressed to tonic convulsions and death. Four mice were dead at presentation, with matting of the fur of the neck and forelimbs. The remainder of the mice had nonspecific signs of disease, such as lethargy, moribundity, or matting of the fur. Vendor and in-house animal health surveillance reports indicated that mice were seronegative to all murine pathogens. Results of gross pathologic examination were unremarkable. Microscopic findings were limited to the brain and liver. In all mice, neuronal necrosis was present in the cerebral cortex, hippocampus, and thalamus. Concurrent astrocyte hypertrophy, as evidenced by an increase in glial fibrillary acidic protein staining, was detected. Acute coagulative necrosis of centrilobular hepatocytes was present in the liver of some cases (19 of 68). Infective agents were not detected in selected brain specimens submitted for electron microscopy or in brain and liver specimens evaluated by use of special stains. Cytopathologic effect was not observed in 3T3, Vero, and BHK-21 cell lines inoculated with brain and liver specimens. The ischemic neuronal necrosis observed in these mice is consistent with lesions associated with status epilepticus in humans. The hepatocellular changes are interpreted to be agonal and associated with terminal hypoxia in seizuring animals. These results provide evidence of a previously unrecognized, often lethal epileptic syndrome in FVB mice that may have a major impact on transgenic research and other disciplines using this mouse strain.

Pica behavior associated with buprenorphine administration in the rat.

Marked gastric distention was-observed in rats 20 h after they underwent partial hepatectomy under isoflurane anesthesia and received buprenorphine (0.3 mg/kg of body weight) after surgery. Hardwood bedding comprised the bulk of the gastric contents. A study was undertaken to determine the cause of the pica behavior (consumption of non-nutritive substances) and resultant gastric distention. Ten-week-old male Sprague Dawley rats were randomly assigned to one of six groups. Group-1 rats (n = 11) underwent laparotomy under isoflurane anesthesia, with buprenorphine (0.3 mg/kg) administered after surgery. Group-2 rats (n = 12) underwent laparotomy under isoflurane anesthesia with buprenorphine (0.05 mg/kg) administered after surgery. Group-3 rats (n = 24) underwent laparotomy under isoflurane anesthesia, with saline administered after surgery. Isoflurane was administered at the same rate, concentration, and duration for all groups that underwent laparotomy (groups 1 to 3). Buprenorphine or saline was administered subcutaneously as a single injection when anesthesia was discontinued (groups 1 to 3). Group-4 rats (n = 6) received buprenorphine (0.3 mg/kg) only. Group-5 rats (n = 6) received buprenorphine (0.05 mg/kg) only. Group-6 rats (n = 12) received saline only. Rats not undergoing laparotomy (groups 4 to 6) received buprenorphine or saline 18 to 20 h before euthanasia. Rats were housed individually in filter-topped polycarbonate cages containing hardwood bedding. A purified, pelleted diet and water were offered ad libitum. Food and water consumption were measured over the posttreatment period. Eighteen to 20 h after treatment, rats were euthanized, each stomach and its contents were weighed, contents were examined grossly, and wet and dry gastric content weights were recorded. All weights were significantly (P < 0.05) increased in rats receiving buprenorphine administered after surgery (groups 1 and 2), compared with rats of the control group (group 3). Weights of the stomach and contents, wet gastric contents, and dry gastric contents were significantly (P < 0.05) increased in rats receiving 0.3 mg of buprenorphine/kg only (group 4), compared with values for their controls (group 6). Hardwood bedding comprised the bulk of the gastric contents in all groups receiving buprenorphine. Stomachs of rats not receiving buprenorphine contained the purified diet with little or no hardwood bedding. These results indicate that a single injection of buprenorphine at a dosage of 0.05 or 0.3 mg/kg resulted in rats ingesting hardwood bedding, leading to gastric distention. It was concluded that pica behavior associated with administration of buprenorphine should be considered when evaluating experimental data from rats housed on contact bedding.

Randomly amplified polymorphic DNA polymerase chain reaction assay for molecular epidemiologic investigation of Pasteurella pneumotropica in laboratory rodent colonies.

After an episode of clinical Pasteurella pneumotropica infection was diagnosed in a C57BL/6N mouse, a randomly amplified polymorphic DNA polymerase chain reaction assay (RAPD-PCR) was developed and used to genetically characterize and differentiate 52 field isolates and laboratory reference strains of P. pneumotropica and related bacteria. A survey of rodents in the facility recovered 36 isolates of P. pneumotropica from 30 mice, six isolates from hamsters, and three isolates from rats during the follow-up investigation. Antibiograms and routine bacteriologic evaluations for morphologic and biochemical characteristics on selective media did not substantively aid in the differentiation of these isolates, but the RAPD-PCR revealed four strains of P. pneumotropica in the colony, two of which were confined to rats and hamsters. The RAPD-PCR unambiguously differentiated Heyl and Jawetz biotypes of P. pneumotropica recovered from mice, identified two additional genetic groups for rat and hamster isolates, and clearly distinguished P. pneumotropica from related bacteria. Most field isolates were genetically consistent with the Jawetz biotype of P. pneumotropica. The RAPD-PCR is a fast, sensitive, and efficient method for identifying genetic differences between strains of the P. pneumotropica complex and can contribute substantially in addressing the epidemiology, pathogenesis, and taxonomic classification of this common opportunistic pathogen.

Efficacy of various therapeutic regimens in eliminating Pasteurella pneumotropica from the mouse.

Pasteurella pneumotropica, a gram-negative opportunistic pathogen, can be isolated from the oropharynx, intestinal tract, and reproductive tract of clinically normal mice and has been associated with various clinical syndromes, including conjunctivitis, infections of the reproductive tract, otitis, and subcutaneous abscess formation. Enrofloxacin, a fluoroquinolone bactericidal antimicrobial, has been shown to be effective in eliminating P. multocida from rabbits. We sought to determine whether enrofloxacin would eliminate P. pneumotropica from mice known to be asymptomatically infected with the agent. Pasteurella pneumotropica-positive (culture and immunofluorescence assay) male (n = 55) and female (n = 55) C57BL/6N mice were randomly assigned to one of seven treatment groups or to a control group. These groups were designed to evaluate the efficacy of enrofloxacin administered orally via the drinking water or parenterally at three dosages (8.5, 25.5, and 85.0 mg/kg of body weight per day) over a 14-day treatment period. A tetracycline-treated group (60 mg/kg per day) and an untreated control group were included for comparisons. Repeated oropharyngeal swab and fecal specimens were obtained for culture through posttreatment day 30, and specimens from numerous enteric and reproductive organs collected during necropsy were used to evaluate group differences. Enrofloxacin eliminated evidence of P. pneumotropica from all sites when administered at 25.5 or 85 mg/kg but not at 8.5 mg/kg by either route for at least 30 days after treatment. Tetracycline-treated and control groups remained consistently culture-positive throughout the study. We concluded that the oral route may be a more practical method for treating large numbers of mice. Enrofloxacin may be a practical and inexpensive alternative to cesarian rederivation or embryo transfer for the elimination of P. pneumotropica in mice.

A standard procedure for measuring pellet hardness of rodent diets.

A Chatillon Model TCM-200 test stand with exchangeable flat horizontal or concave receptacle bases and a DFI-200 gauge load cell with multiple types of upper exchangeable test jaws (large round-flat, medium round-flat, chisel, bullet, and cone-shaped) were compared by using preautoclaved and autoclaved NIH-31 rodent diet pellets to determine which type of hardness testing system would give the most accurate and reproducible results for measuring pellet hardness. The type and size of the contact area of the upper jaws significantly affected the force required to break the pellets. Significant differences were observed between the flat-horizontal and concave receptacle bases in the force required to break the pellets when using the two round-flat upper jaws. In contrast, similar results were obtained with both bases when the bullet, chisel, or cone-shaped upper jaws were used. Autoclaved pellets were 69.4% (range, 49 to 94%) harder than preautoclaved pellets. These results suggest that different testing systems can be used for measuring pellet hardness and that a standard procedure must be used in order to compare pellet hardness results between different testing laboratories. It was concluded that the flat-horizontal base and the larger round-flat end upper jaw gave the most reproducible results for measuring pellet hardness.

The use of dirty bedding for detection of murine pathogens in sentinel mice.

Sentinel Swiss (CD-1) mice, housed without filter bonnets, were seronegative for mouse hepatitis virus (MHV) for 8 consecutive months in an experimental colony of CD-1 mice. MHV titers had been detected sporadically in sentinel mice housed in this colony during a 2 year period. In an effort to determine whether MHV was still present in the colony, two methods of exposing sentinel mice to an animal room environment were compared under routine husbandry practices. Eight cages (12 mice per cage; 2 cages per rack) of experimental virus antibody free sentinel mice, housed without filter bonnets, were placed on the bottom shelf of 4 of 12 racks in the room. Twice each week, four cages of sentinel mice received a composite sample of dirty bedding (bedding used previously by mice in the room). The remaining four cages of experimental sentinels received fresh non-used bedding. Sentinel mice were bled at monthly intervals for MHV serology. After 4 months, mice from two cages which received dirty bedding seroconverted to MHV and mice from one cage were positive for Myobia musculi (mites). Three weeks later, all four cages of mice which received dirty bedding were positive for MHV and three were positive for mites. In contrast, only two of four cages of mice which received fresh bedding were positive for MHV and all were negative for mites. These findings indicate the importance of exposing sentinel mice to dirty bedding and that MHV and mites may go undetected for several months in a mouse colony when the incidence levels are low where standard sanitation procedures are used.

Characteristics of 2,3,7,8-tetrachlorodibenzo-p-dioxin induced endotoxin hypersensitivity: association with hepatotoxicity.

Hepatic clearance is a major route of endotoxin detoxification. In the present study, the potential relationship between TCDD-induced endotoxin hypersensitivity and hepatotoxicity was examined. Acute doses of 50, 100, or 200 micrograms TCDD/kg body weight induced an endotoxin hypersensitive state in B6C3F1 mice as demonstrated by increased mortality 24-48 h following i.v. injection of endotoxin. This hypersensitive state occurred when endotoxin was administered 7 days following TCDD exposure, but not 1 day post-TCDD exposure. TCDD did not affect endogenous serum endotoxin levels. However, clearance of injected endotoxin was significantly inhibited following exposure to TCDD. Six hours post endotoxin treatment serum triglycerides were significantly increased in TCDD/endotoxin-treated mice compared to either treatment alone. Methylprednisolone and uridine were both examined in this model due to their roles in inflammation and RNA synthesis, respectively. Both compounds significantly reversed the mortality associated with the combined exposure. [3H]Uridine incorporation into liver was decreased following TCDD treatment alone, further suggesting impaired RNA synthesis. Studies performed on congenic mice indicate that the observed effects segregate with the Ah locus. The ability of methylprednisolone and uridine to reverse the mortality associated with TCDD/endotoxin treatment is consistent with an inflammatory response and impaired hepatic detoxification mechanisms. Thus, changes in hepatic handling of endotoxin, caused by progressive TCDD-induced liver dysfunction, may be responsible for the endotoxin hypersensitivity.

A standard procedure for measuring rodent bedding particle size and dust content.

Hardwood dust can cause dermatitis, respiratory disease, allergies and nasal cancer in humans. A major concern with animal hardwood bedding is its dust content and its possible effects on animals and animal technicians. Previous reports on the quality control of rodent bedding did not specify sample size or shake time for measuring bedding particle size and dust content. These variables could alter particle size analyses. In an effort to more accurately characterize bedding particle size and dust content, 50g and 100g samples of hardwood bedding were shaken for 0.5, 1, 2, 3, 4, or 5 minutes in a portable sieve shaker containing U.S. standard sieves (Nos. 8, 20, 30 and 50) to determine optimum sample size and shake time. Significant differences (P less than 0.05 or greater) were observed in the percent of bedding retained on a No. 8 sieve when 50g and 100g samples were taken for 30 seconds or for 1 minute. Samples shaken for 2 or more minutes did not show any statistical differences in the percent of bedding which was retained on or passed through the different sieves. Major differences occurred in the percent of bedding which was retained or passed through the different sieves, when the shake time was varied from 0.5 to 5 minutes. These results indicated that 0.5 or 1 minute was definitely not enough time to accurately measure bedding particle size and dust content and that the sample size and shake time must be consistent in order to accurately compare the particle size and dust content of different shipments of bedding or to compare bedding from different vendors.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatal pneumonia with terminal emaciation in nude mice caused by pneumonia virus of mice.

Athymic nude mice used as sentinel animals in a mouse holding room died of pneumonia 17 to 32 weeks after being placed in the room. Lesions in the pulmonary parenchyma consisted of monocytic exudate, epithelial cell necrosis, hemorrhage, fibrin deposition and interstitial fibrosis. Septal edema, septal cell necrosis and septal capillary stasis were common, but there was limited sloughing of bronchial lining epithelium. Indirect fluorescence microscopy (IFA) of lung sections using pneumonia virus of mice (PVM) antibody was positive. The pneumonia and IFA results were reproduced in euthymic mice inoculated experimentally with lung suspension from naturally infected mice or with tissue culture fluid from cultures infected with American Type Culture Collection PVM. The lungs of a naturally infected nude mouse were studied by transmission electron microscopy. Virus growth was found on Type II alveolar epithelium and on poorly differentiated replacement alveolar epithelium. Virus particles appeared as long exophytic filaments containing one to six linearly arranged nucleocapsids. Inclusion bodies and intracellular virus structures were not observed.

The mouse bioassay for the detection of estrogenic activity in rodent diets: I. A standardized method for conducting the mouse bioassay.

A standardized procedure was developed for conducting the mouse bioassay for detecting estrogenic activity in rodent diets. Studies were conducted with CD-1 mice to determine the appropriate weaning age and length of bioassay period. Uterine growth curves were generated from mice weaned at 15 days of age and fed a negative control diet until 28 days of age. These mice showed slow regular increases in uterine weights from 15 22 days of age followed by rapid uterine growth in some mice from 24 to 28 days of age. Estrogenic bioassays using female mice weaned at 15 days of age and fed the positive control diets containing 4 or 6 ppb diethylstilbestrol (DES) demonstrated significant (P less than 0.05) increases in uterine weight and in uterus to body weight (U:BW) ratios over those of mice fed the negative control diet without DES for 5, 7 or 9 days after weaning. In contrast, mice weaned at 17 days of age showed significant (P less than 0.05) increases in uterine weight and in U:BW ratios only at 5 days after weaning. Six ppb DES was required in the positive control diet to produce a 1.5 fold increase in the U:BW ratio over those of mice fed the negative control diet. It was concluded that mice should be weaned at 15 days of age and that the bioassay period should be terminated at 7 days, when the mice are 22 days old, for best reproducible results. The criteria for a valid bioassay should include the demonstration of a significant statistical increase in the U:BW ratios of mice fed the DES positive diet over those of mice fed the negative control diet.

The mouse bioassay for the detection of estrogenic activity in rodent diets: II. Comparative estrogenic activity of purified, certified and standard open and closed formula rodent diets.

A major source of exogenous estrogenic substances, which may affect laboratory animals, comes from the diet. To test the possibility that commercially available rodent diets may significantly influence uterine weights and uterine:body weight (U:BW) ratios, estrogen bioassays were performed using female CD-1 mice weaned at 15 days of age and assigned randomly to a variety of commercial test diets or to a control diet (Purina #5002) containing 0 or 6 ppb added diethylstilbestrol (DES) for comparison. Mice were housed five per cage and given deionized water and feed ad libitum. Uterine:BW ratios from 15 mice per diet were determined after 3, 5 and 7 days of feeding. Mice fed The American Institute of Nutrition purified diet (AIN-76A) or the Purina #5015 natural ingredient breeder diet had significantly (P less than 0.05) increased U:BW ratios at 3, 5 and 7 days post weaning when compared to the control diet without added DES. This increase in U:BW ratios was similar to the U:BW ratios observed in a natural ingredient maintenance diet (Purina #5002), containing 6 ppb of DES. These results show that significant differences exist in the level of substances which can cause increase in uterine weight in some commercial diets. The diet may be important when performing or comparing certain types of studies, especially those relating to estrogenic substances. A standardized diet with minimal estrogenic activity may be desirable for such studies. It is unclear from the present studies what substances might be responsible for the uterine growth promoting activity in the diets examined.

The mouse bioassay for the detection of estrogenic activity in rodent diets: III. Stimulation of uterine weight by dextrose, sucrose and corn starch.

We have shown previously that mice fed the American Institute of Nutrition (AIN-76A) purified diet experience a significant increase in uterine:body weight (U:BW) ratios when compared to the U:BW ratios of mice fed a closed formula natural ingredient diet (Certified Rodent Chow #5002) for 7 days. The AIN-76A purified diet contains 5% corn oil and 65% carbohydrates with 50% of the carbohydrates coming from sucrose or dextrose and 15% from corn starch. The objective of this study was to determine whether the fat and carbohydrate content contributed to the unexpected uterine growth promoting activity observed in mice fed the AIN-76A diet. Estrogen bioassays were performed using CD-1 mice weaned at 15 days of age and assigned randomly to the negative control diet (Certified Rodent Chow #5002) or to the positive control diet (#5002) containing 4 or 6 ppb DES for comparison or to the test diets. The test diets were prepared by adding sucrose, dextrose, corn starch, corn oil or soybean oil to the #5002 negative control diet at 10% w/w concentration. Uterine:BW ratios were determined at 7 days post-feeding. The uterine weights and the U:BW ratios of mice fed the test diets containing dextrose, corn starch, or corn oil, were increased significantly (P less than 0.05) over those of mice fed the negative control diet. The uterine weights and U:BW ratios of mice fed the test diets containing sucrose or soybean oil also were increased over those of mice fed the negative control diet. These increases in uterine weights and U:BW ratios were similar to the increases in uterine weights and U:BW ratios of mice fed the positive control diet containing 4 ppb DES. It was concluded that the fats and carbohydrates caused preferential increases in uterine weights and in U:BW ratios and may account for the estrogen-like uterine growth promoting activity observed in mice fed the AIN-76A purified diet.

Toxicity of inhaled methyl isocyanate in F344/N rats and B6C3F1 mice. I. Acute exposure and recovery studies.

Male and female F344/N rats and B6C3F1 mice were exposed to lethal and sublethal concentrations of methyl isocyanate by inhalation. Mortality, clinical signs, body and organ weights, and changes in clinical pathology and hematology were monitored immediately after 2-hr exposures and during the ensuing 3 months. Additional studies investigated the possible involvement of cyanide in the toxicity of methyl isocyanate. During exposures, signs of restlessness, lacrimation, and a reddish discharge from the nose and mouth were evident in rats and mice. Following exposures, rats and mice were dyspneic and weak. Deaths of rats and mice exposed to lethal concentrations (20 to 30 ppm) began within 15-18 hr, with males more prone to early death than females. A second wave of deaths occurred after 8 to 10 days, affecting primarily female rats and mice exposed to 20 to 30 ppm of methyl isocyanate, and male and female rats exposed to 10 ppm. Most deaths occurred during the first month following the exposures and were preceded by periods of severe respiratory distress. Body weights decreased in proportion to dose early, but then weight gain resumed in survivors at control rates. The only organ with a consistent, dose-related weight change was the lung, which was heavier throughout the studies in animals exposed to high concentrations of methyl isocyanate. No significant clinical pathology, or hematologic changes were observed in exposed rats. Blood and brain cholinesterase were not inhibited. Studies attempting to measure cyanide in the blood of methyl isocyanate-exposed rats, and attempting to affect lethality with a cyanide antidote (sodium nitrite and sodium thiosulfate) gave negative results.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth characteristics of large- and small-colony types of Mycoplasma mycoides subsp. mycoides on 5% sheep blood agar.

Mycoplasma mycoides subsp. mycoides of the large-colony (LC) type was isolated in pure culture on 5% sheep blood agar plates inoculated with lung specimens from a 4-month-old Toggenburg goat. The growth characteristics of this isolate, of four known LC types, and of five known small-colony (SC) types of M. mycoides subsp. mycoides were compared on 5% sheep blood agar at 2, 5, and 7 days. The SC types were not visible at 2 days and did not grow larger than 0.1 mm, whereas the LC types were visible in 2 days and increased in diameter over 7 days to between 0.4 and 0.7 mm. These results indicate that growth on 5% sheep blood agar can be used as an additional marker in differentiating LC and SC types of M. mycoides subsp. mycoides.