SPF Mice Research Articles

The importance of glyoxylate and other glycine precursors in the hepatic and renal conjugation of benzoate in normal and hyperammonemic mice.

Benzoate conjugation, represented by hippurate synthesis, was measured in hepatocytes isolated from normal and sparse-fur (spf) mutant mice, with X-linked ornithine transcarbamylase deficiency, to compare the effects of glyoxylate and piridoxylate (a hemiacetal of glyoxylate and pyridoxine), substituted for glycine. Various amino acid precursors of glycine described in the literature, including serine, threonine, glutamine, and glutamate, were studied in a similar manner. The role of glyoxylate and piridoxylate was also assessed in the renal cortex, in comparison with liver homogenates from normal and hyperammonemic mice. The results indicate the importance of glyoxylate and piridoxylate to completely substitute for glycine (96-115%) in isolated hepatocytes of spf/Y mice, as compared with 53-69% (p less than 0.05) in normal +/Y controls. The mean value of amino acid precursors to substitute for glycine in spf mice was serine 51%, threonine 29% (p less than 0.05), and glutamine 9%. In normal mice, only serine (21%) (p less than 0.01) partly substituted for glycine, whereas threonine, glutamine and glutamate gave negative values of net hippurate synthesis. The specific activity of renal cortex for hippurate synthesis from glycine, glyoxylate and piridoxylate was 3-4 times that of liver homogenates (p less than 0.01 - less than 0.001). A scheme for the transamination of glyoxylate by alanine is presented. Besides alanine, the excess of glycine, serine, and threonine is readily deaminated in the body to take part in gluconeogenic reactions, thus contributing to hyperammonemia. The cumulative effect of benzoate conjugation to drain these ammoniagenic precursors through glycine may be the basis of its therapeutic effect in hyperammonemia.

Chronic toxicity of nivalenol in female mice: A 2-year feeding study with Fusarium nivale Fn 2B-moulded rice

Groups of 42 7-wk-old female C57BL/6CrSlc SPF mice were fed diets containing 0, 6, 12 and 30 ppm nivalenol (NIV) for 2 years. Body-weight gain was reduced in all treated groups of animals and feed efficiency was reduced, significantly so, in the high-dose group. The absolute weights of the liver in the 30-ppm group, and of the kidneys in the 12- and 30-ppm groups were significantly reduced, compared with those of the controls. When expressed relative to brain weight there was a reduction in the kidney weight of the 12-ppm NIV group only. Some leucopenia was seen in the treated mice, particularly in the 30-ppm group, although this was not statistically significant, and there were dose-dependent increases in the serum concentrations of alkaline phosphatase and non-esterified fatty acids. No tumours attributable to NIV were found in any of the experimental groups. Naturally occurring tumours, mostly lymphomas, were of similar incidence in all groups, but developed later and appeared to grow more slowly in the mice of the 30-ppm group than in those of other groups. The incidence of amyloidosis, particularly in the small intestine, was low in the two higher dose groups compared with that in the control group. The mortality rate of the 30-ppm NIV group was lower than that of the control group and this may be partly due to the lower tumour incidence in the earlier period and partly due to the lower incidence of amyloidosis.

免疫抑制状態に発症する敗血症の発症機序と予防

Nosocomial infections due to aerobic gram-negative bacilli are life-threatening problems for immunocompromised patients. Most of these infections might arise from the patient's own flora, especially from their faecal flora. The purpose of this study was to investigate the bacterial portal of entry in experimental mice with bacteremia which were treated by cyclophosphamide (CY). It was clarified that almost all of the isolates from blood of CY and antibiotics treated mice were identical with their own faecal flora, and that organisms were isolated in some cases only from the portal vein but not from heart blood. From these results it was concluded that some of the bacteria entered from the intestinal tract through the portal vein to the systemic circulation in the immunosuppressed state. The time course of bacterial translocation were examined with SPF mice which were given Pseudomonas aeruginosa orally prior to CY treatment. Twelve strains of Escherichia coli were isolated, 10 of which were isolated only from mesenteric lymph nodes (MLN). Though six strains of P. aeruginosa were isolated from the heart blood, 4 were not isolated from MLN. It was suggested that translocations of E. coli and P. aeruginosa occurred by lymphogenous and hematogenous routes respectively. And translocation of viable E. coli was observed only after ABPC treatment but before CY inoculation, on the other hand translocation of viable P. aeruginosa was observed after CY inoculation. So only disruption of the normal flora ecology was necessary for translocation of E. coli, but immunosuppression was additionally necessary for translocation of P. aeruginosa.

Subchronic feeding studies with nivalenol in C57BL/6 mice

Groups of ten C57BL/6CrSlc SPF mice of each sex were fed diets containing 0, 6, 12 or 30 ppm nivalenol for 4 or 12 wk. Body-weight gains of males and females were depressed, dose-dependently in the case of males. Feed consumption was also depressed. Treatment-related changes in liver, kidney, spleen and thymus weights were seen in some groups but showed no clear trends. No gross or histopathological lesions were seen in the organs examined but treated groups had considerably less fatty tissue at autopsy than did controls. There was a dose-dependent increase in serum alkaline phosphatase activity. Other serum parameters showed scattered significantly altered value but no clear trends, except for serum GOT values for males fed 12 and 30 ppm for 12 wk; these showed a statistically significant dose-related increase, but were within the normal range and were not considered to indicate hepatotoxicity.

The acute and chronic toxicities of nivalenol in mice

In an attempt to ascertain precisely the toxic effects of nivalenol (NIV), we conducted the determination of LD50 values, and interim kills during the carcinogenic study in mice. LD50 values (mg/kg) of NIV in 6-week-old male ddY mice were determined as 38.9 (po), 7.4 (ip), 7.2 (sc), and 7.3 (iv). Seven-week-old female C57BL/6CrSlc SPF mice were fed diets containing 0, 6, 12, and 30 ppm (mg/kg) NIV over 1 year, and were assessed for effects on body weight gain, feed efficiency, terminal organ weights, hematology, and histopathology. The rates of body weight gain and feed efficiency showed a good dose-dependent correlation in all experimental periods. Gross and histopathological evaluation of the liver, thymus, spleen, kidneys, stomach, adrenal glands, pituitary gland, ovaries, sternum, bone marrow, lymph node, brain, and small intestines with or without Peyer's patch portion from control and all NIV-exposed mice reveated that these tissues were normal in appearance and in histopathological structure. Also, no changes were observed in the ultrastructural studies on the bone marrow. Dietary NIV did, however, cause dose-dependent decreases of absolute organ weights (mg) and increases of relative organ weights (mg/g body weight) in the terminal organ weights recorded. A significant leukopenia was observed in the 30 ppm group at 6 months and in all NIV-treated groups at 1 year. No marked changes were observed in the other hematological parameters. These results indicated that 6 ppm or more of dietary NIV for 1 year showed a characteristic toxic effect of trichothecene mycotoxins in mice.

Kinetic arguments for the existence of a single form of intestinal ornithine decarboxylase during the postnatal development of normal and sparse-fur mutant mice and after EGF treatment.

The Km for ornithine is remarkably constant during the course of postnatal development in both normal and spf mutant mice even if a large but transient increase in ornithine decarboxylase (ODC) activity is noted. Four hours after EGF injection (4 micrograms/g b.wt) to 17-day-old normal and spf mice, a marked stimulation of ODC activity is observed but Km remains unaffected. These data argue against the existence of multiple forms of ODC in the intestinal mucosa of mice.

Enzyme-linked immunosorbent assay for detection of serum antibody to CAR bacillus.

An enzyme-linked immunosorbent assay (ELISA) for detection of CAR bacillus antibody in rat sera was developed by Ganaway et al., in 1985 although the ELISA method was not described in detail. We investigated antigen preparation and test procedures of the ELISA using two strains of CAR bacillus which we isolated from a mouse (CB-M) and a rat (CB-R). Allantoic fluids containing 2.4 X 10(8)/ml of CB-M and 2.0 X 10(8)/ml of CB-R were washed with sterile phosphate buffered saline (PBS), resuspended in a 1/5 volume of sterile carbonate buffer (pH 9.8) and sonicated. Then 1/40 and 1/80 dilutions of CB-M and CB-R lysates in PBS, respectively, were used for antigen solutions of ELISA. Briefly, antibodies in sera are reacted with antigens coated on the surface of microtiter plates. The amount of horse radish peroxidase labeled protein-A or anti-rat IgG bound to the antigen-antibody complexes is measured on the spectro photometer at wave length of 492 nm. A total of 180 mouse and 205 rat sera were tested against both antigens. The optical density (OD) values of 140 mouse and 161 rat sera obtained from SPF mice and rats free from CAR bacillus infection were on the average 0.005 and 0.019, respectively. On the other hand, OD values of the sera collected from CB-M or CB-R infected animals ranged from 0.20 to 1.52. According to these results, the cut-off OD value for positive reaction was set at 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)

The Acute and Chronic Toxicities of Nivalenol in Mice

The Acute and Chronic Toxicities of Nivalenol in Mice. Ryu, J.-C, Ohtsubo, K., Izumiy-ama, N., Nakamura, JL, Tanaka, T., Yamamura, H., and Ueno, Y. (1988). Fundam. Appl Toxicol. 11, 38–47. In an attempt to ascertain precisely the toxic effects of nivalenol (N1V), we conducted the determination of LD50 values, and interim kills during the carcinogenic study in mice. LD50 values (mg/kg) of NIV in 6-week-old male ddY mice were determined as 38.9 (po), 7.4 (ip), 7.2 (sc), and 7.3 (iv). Seven-week-old female C57BL/6CrSlc SPF mice were fed diets containing 0, 6, 12, and 30 ppm (mg/kg) NIV over 1 year, and were assessed for effects on body weight gain, feed efficiency, terminai organ weights, hematology, and histopathology. The rates of body weight gain and feed efficiency showed a good dose-dependent correlation in all experimental periods. Gross and histopathological evaluation of the liver, thymus, spleen, kidneys, stomach, adrenal glands, pituitary gland, ovaries, sternum, bone marrow, lymph node, brain, and small intestines with or without Peyer's patch portion from control and all NIV-exposed mice revealed that these tissues were normal in appearance and in histopathological structure. Also, no changes were observed in the ultrastructural studies on the bone marrow. Dietary NIV did, however, cause dose-dependent decreases of absolute organ weights (mg) and increases of relative organ weights (mg/g body weight) in the terminal organ weights recorded. A significant leukopenia was observed in the 30 ppm group at 6 months and in all NIV-treated groups at 1 year. No marked changes were observed in the other hematological parameters. These results indicated that 6 ppm or more of dietary NIV for 1 year showed a characteristic toxic effect of trichothecene mycotoxins in mice.

Accumulation of large neutral amino acids in the brain of sparse-fur mice at hyperammonemic state.

Sparse-fur mice which are deficient in ornithine transcarbamylase, the second-step enzyme in the urea cycle, were examined for hyperammonemia and its relationship with encephalopathy. We compared amino acid concentrations in the serum and brain of spf mice with those of control mice. Unlike hepatic encephalopathy we could not find marked amino acid changes in the serum of spf mice besides low levels of citrulline and arginine. But in the brain of spf mice, glutamine was increased strikingly during hyperammonemia, and a concomitant accumulation of large neutral amino acids such as tyrosine, phenylalanine, methionine, and histidine was observed. The accumulation of these large neutral amino acids in the brain was not influenced by 24-hr fasting which caused increases in branched chain amino acids in the serum. From these results, we conclude that the accumulation of the large neutral amino acid in the brain of hyperammonemic state is caused by uptake of ammonia in the brain and the subsequent accumulation of glutamine, but is not influenced by a decreased ratio of branched chain amino acids to aromatic amino acids in the serum.

Biochemistry and physiology of brain ammonia.

Biochemistry and physiology of brain ammonia.

SODIUM BENZOATE (SB) INCREASES FREE TRYPTOPHAN (TRP) IN BLOOD AND SEROTONIN (5-HT) FLUX IN CORTEX OF OTC DEFICIENT SPF MICE

We have previously shown that both hyperammonemia and SB used to treat hyperammonemia increase central 5-HT flux in rats and mice (Pediat Res 19:309A, 1985; 20:325A, 1986). In this study we injected ip. 1 or 5 mmol/kg SB or sodium acetate (Ac) in hyperammonemic Spf/y and normoammonemic CD-1/y mice (n=5/eondition) with sacrifice after 1 h. Compared to CD-1 nice, Spf (all conditions) had significantly higher cortical levels of the 5-HT precursor Trp (p<.001), of 5-HT (p<.01), and of its metabolite 5-hydroxyindoleacetic acid (HIAA), (p<.001). SB did not change plasma ammonium levels but further increased cortical Trp (p<.005) and HIAA (p<.01) in CD=1 mice. The effect of increasing SB from 1 to 5 mmol/kg was to increase cortical Trp (p<.001) and HIAA (p<.005) in Spf and Trp (p<.05) in CD-1 mice. Plasma benzoate levels ranged from 1.8 to 4.1 oH 1 h post 5 mmol/kg SB. Serum free Trp levels doubled in both Spf (25±.4.2 vs. 13.7±3.9 uM, p<.001) and CD-1 mice (23.9±.2 vs. 11.0±1.8uM, p<.001) following 5 mmol SB. No change occurred following injection of 1 and 5 mmol/kg Ac or sodium phenylacetate, which is also used to treat hyperammonemia. We suggest that SB competes for albumin binding sites with Trp, resulting in increased free Trp in blood, Trp transport across the blood-brain barrier, and increased serotonin flux. It is possible that the clinical symptoms of SB intoxication are related to this alteration in serotonin metabolism.

In situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels

Ornithine transcarbamylase deficiency is a human genetic disease potentially susceptible to gene therapy. A murine model system exists for the disease in the sparse-fur ( spf) mouse. Before gene therapy studies can be performed it is necessary to have practical methods which could detect successful gene transfer. Therefore we have developed an in situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels. Following electrophoretic separation under nondenaturing conditions inorganic phosphate cleaved from carbamyl phosphate in gels as a result of enzymatic activity was precipitated as phosphomolybdic acid and visualized by reduction with ascorbic acid. Results from the procedure correlated with ornithine transcarbamylase activity as measured by solution assay for citrulline, the other product of the reaction. This procedure readily distinguished mutant forms of ornithine transcarbamylase as exemplified by the murine spf mutation and resolved ornithine transcarbamylases of all animals tested into multiple forms. The procedure further distinguished ornithine transcarbamylases of animals of several different genera while yielding virtually identical patterns of the enzyme from species within the same genus. This procedure also suggested that the human enzyme was more labile than murine ornithine transcarbamylase; direct thermolability studies confirmed this finding.

Effects of nivalenol on the bone marrow in mice.

In order to investigate the toxic effects of nivalenol, one of the trichothecene mycotoxins, we performed a short-term feeding trial for 24 days using feed supplemented with rice artificially molded with nivalenol producing fungus, Fusarium nivale Fn 2B, in female C57BL/6CrSlc SPF mice. A significant erythropenia and slight leukopenia were observed in the 30 ppm group, but no marked changes were observed in other hematological parameters, feed consumption, body weight gain, or weights of the liver, spleen, and thymus. Ultrastructural studies also revealed polyribosomal breakdowns of the bone marrow cells in the 30 ppm group.

Experimental cryptosporidiosis in mice, calves and chicken

Experimental infections attributed solely to Cryptosporidium were carried out in newborn SPF mice, calves and chicken in order to study the prepatency, patency and incubation periods, describe the clinical symptoms and find and describe any correlations between the association of Cryptosporidium with the intestinal mucosa and presence of pathological lesions. The paper also gives the clinical and paarsitological parameters of Cryptosporidium infection of calves from a field survey and compares them to the results of experimental study.

Serological surveys of Corynebacterium kutscheri infection in mice and rats.

Serological surveys of mice and rats naturally infected with Corynebacterium kutscheri were performed by examining serum samples collected from breeder and laboratory colonies between 1981 and 1983. Among 756 mice from 73 conventional colonies, only 4 animals (0.5%) from 3 colonies (4.1%) developed C. kutscheri antibody of 1:40 to 1:2, 560 titers. Three of them suffered from abscess caused by the organism. Regarding a titer of 1:40 or higher as reliably positive, 87 (13.0%) of 669 conventional rats or 20 (32.8%) of 61 colonies were found to be infected with the organism. The antibodies were detected in both types of animals older than 6 months of age. No lesions caused by C. kutscheri were found in almost all the rats examined. Germ-free and SPF mice and rats were all negative for antibody at 1:5 serum dilution.

Detection of lymphocytic choriomeningitis virus antibody in colonies of laboratory animals in Japan.

Indirect fluorescent antibody method was applied for a detection of lymphocytic choriomeningitis virus (LCMV) antibody in colonies of laboratory animals in Japan. The results showed that the antibody exist in SPF mice (3/152, 2.2%) and conventional mice (30/539, 5.6%) with the titers ranging from 1:10 to 1:160. The antibody was also detected in 2.2% (2/89) of Syrian hamsters, and 2.9% (2/68) of Apodemus agrarius, 21.4% (3/14) of Japanese harvest mice which have been maintained as laboratory colony for several years. However, the antibody was not demonstrated in Mongolian gerbils, Suncus murinus, guinea pigs and rats, thus far. These results indicate that LCMV infection is present in laboratory animals in Japan, and pointed out the importance of microbiological monitoring for LCMV.

1244 SIGNIFICANCE OF TRANSPORTED GLYCINE IN THE CONJUGATION OF BENZOATE IN SPARSE-FUR (spf) MUTANT MICE WITH ORNITHINE TRANSCARBAMYLASE DEFICIENCY

Sodium benzoate (SB) therapy in hyperammonemic children is based on the elimination of excess nitrogen as hippurate, but its mechanism is speculatory. We gave 2 % SB in drinking water to normal (+/Y) and spf/Y mice. After acclimatization for 48 h, i.p. 3H-glycine and 14C-serine were given as sources of transported and biosynthesized glycine respectively. 24 h urine was analyzed for total hippurate, free glycine and orotate. Hippurate spots were separated by TLC and counted for radioactivity. Output of hippurate and free glycine increased significantly in SB treated spf/Y and +/Y as compared to untreated mice, while orotate excretion decreased (p < 0.013). Specific activity of 3H and 14C (DPM/μmol hippurate) showed significant increases in treated groups, indicating utilization of transported and biosynthesiged glycine. However, 3H;14C ratio showed predominant use of 3H-glycine. The ratio was higher in treated spf mice as compared to untreated controls (3.1 ± 0.7 Vs 1.5 ± 0.04; p < 0.02); same increase was seen in +/Y (1.9 ± 0.2 Vs 1.0 ± 0.1; p <0.01). The results indicate that transported glycine has a bigger contribution in the formation of hippurate in vivo than glycine synthesized from serine. Since glycine is highly ammoniagenic, we postulate that the conjugation of excess glycine transported from body pools would result in depletion of ammoniagenic potential. This would be supplemented by the effect of glycine biosynthesis through reactions requiring a metabolic input of NH3.

Tryptophan and serotonin turnover rate in the brain of genetically hyperammonemic mice

An investigation was made into the effects of hyperammonemia on the metabolism of brain serotonin (5-HT). The animal model used was the sparse fur ( spf) mouse, which possesses an inborn error of the urea cycle, i.e. an abnormal form of ornithine transcarbamylase. Several indoles were measured in brain and plasma using liquid chromatography with electrochemical detection coupled to an u.v. detection (LCEC-u.v.). In the mutant mice, plasma total tryptophan (TRP) was higher when compared with the controls, while plasma free-TRP portion was unchanged. In these animals, brain TRP was increased whilst the 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were significantly higher in the hypothalamus and midbrain. Experiments with NSD-1015 (100 mg/kg i.p.) indicated that the 5-hydroxytryptophan (5-HTP) synthesis rate was increased in the hyperammonemic mice. Pargyline experiments (100 mg/kg i.p.) confirmed the enhanced brain 5-HT turnover rate in the spf mice. In addition, these experiments led to the conclusion that hyperammonemia does not affect the various rate constants. After administration of NSD-1015, TRP level slightly increased in the spf mouse brains, while it was stationary in those of the controls. This result could indicate an increased activity of hepatic TRP-pyrrolase in the hyperammonemic mice. Valine (VAL) administration (200 mg/kg i.p.) reduced brain TRP content in the two kinds of mice, but its effect was of shorter duration in the spf when compared with the control. Comparison of brain tryptamine level indicated a slight but not significant increase in the mutant mice. The data reported here indicate that hyperammonemia may affect peripheral TRP metabolism with consequences upon brain 5-HT synthesis, which could promote certain neurologic disorders.

Cell-free synthesis and transport of precursors of mutant ornithine carbamoyltransferases into mitochondria

Synthesis, mitochondrial transport and processing of ornithine carbamoyltrasferase (EC 2.1.3.3) were studied in mutant mice strains (sparse-fur, spf, and sparse-fur with abnormal skin and hair, spf-ash) which exhibit a deficiency in this enzyme. Spf mice have an increased amount (about 150% of control) of the enzyme with abnormal kinetic properties, whereas spf-ash mice have a decreased amount (about 10% of control) of the enzyme with apparently normal kinetic properties. Precursors of the mutant enzymes were synthesized in a reticulocyte lysate cell-free system. The hepatic level of translatable mRNA coding for the enzyme and the rate of the enzyme synthesis in liver slices of spf mice were 58 and 60% of the controls, respectively. In the case of spf-ash mice the activity of translatable mRNA for the enzyme was 10% of the controls. These results indicate that the decreased amount of ornithine carbamoyltransferase protein in spf-ash mice is due mainly to a decreased level of translatable mRNA for the enzyme, whereas the increase in the enzyme amount in spf mice is presumably the result of a decreased rate of enzyme degradation. The subunit molecular weight of the spf enzyme precursor was practically the same as that of the normal enzyme precursor ( M r 40 000). Both precursors synthetized in vitro could be taken up and processed similary to an apparently mature form ( M r 37 000). In the case of spf-ash enzyme, two discrete in vitro products were observed on sodium dodecyl sulfate polyacrylamide gel; one comigrated with the normal enzyme precursor and the other moved slightly slower. Both products appeared to be taken up and processed to the mature form of the enzyme.

Anti-mu induces lymphoma in germfree congenitally athymic (nude) but not in heterozygous (nu/+) mice.

To further our understanding of the role of host immunity in the development of lymphoid neoplasia, groups of germfree BALB/c nude and nu/+ mice were either followed unmanipulated or were treated, beginning at birth, with anti-mu, normal goat IgG or with lipopolysaccharide (LPS). The survival and development of neoplasia of all groups of animals were monitored up to 2 yr. Nude mice, under germfree and specific pathogen-free (spf) conditions, had a higher incidence of lymphoid neoplasia and reduced survival when compared to nu/+ littermates. The incidence of lymphoid tumors in nude mice under spf or germfree conditions was 7.2 and 8.7%, respectively, in comparison to 0% in nu/+ animals. Treatment of germfree nude mice with anti-mu, but not with goat IgG, increased the incidence of lymphoid tumors to 39%. Anti-mu did not significantly change the incidence of lymphoid neoplasia in nu/+ animals. Treatment of nu/nu and nu/+ mice with LPS, however, led to a several-fold increase in the appearance of neoplasia, to values of 25.4% in nude and 10% in nu/+ mice. Lymphoid neoplasia found in either unmanipulated, anti-mu, or IgG-treated germfree or spf mice included Thy-1.2+, surface IgM+, and IgG+ tumors. In contrast, all the lymphomas found in LPS-treated mice were surface IgM+. Thus, whereas LPS may have generated a relatively homogeneous group of tumors, anti-mu may have randomly increased the normal incidence of spontaneous tumors. Moreover, although there was significant variation in the histologic appearance of tumors, both within treatment groups as well as in different areas of the same animal, only LPS-treated mice were regularly noted to have distant nonlymphoid involvement, with lesions found in liver, lung, and kidney. In contrast, the incidence of nonlymphoid neoplasia was similar and was less than 2.5% in all groups.