Histopathological studies on cases of chronic mouse hepatitis by natural Helicobacter infections.

It is known that Helicobacter hepaticus or Helicobacter bilis infection causes chronic inflammation of the colon and liver. Chronic active hepatitis was found in radiation exposure experiments using male C3H/HeNrs mice at our institute. Histopathologically, 103 cases among 978 mice (64-91 weeks of age at autopsy) had hepatic lesions regardless of irradiation exposure. Mild lesions showed only focal necrosis and focal inflammation in the liver. Severe cases were accompanied by hepatocytomegaly, bile duct hyperplasia, hypertrophy and activation of Kupffer cells, cholangitis, pleomorphic hepatocytes and/or tumor. Helical-shaped bacteria were detected between hepatocytes by Warthin-Starry silver stain and immunohistochemistry (IHC) with an antibody against Helicobacter pylori. It was suggested that these cases of chronic hepatitis were caused by Helicobacter spp. Although chronic hepatitis occurred frequently in mice exposed high-dose irradiation compared with nonirradiated mice in one lot, it was not concluded that radiation might influence the incidence or degree of hepatitis. Our report suggested that natural Helicobacter spp. infection in mice can occur in an experimental animal facility. Therefore, it is suggested that monitoring of Helicobacter infection is very important for quality control of animal experiments.

Functional analysis of lysosomes during mouse preimplantation embryo development.

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

A novel Kit gene mutation in CF1 mice involved in the extracellular domain of the KIT protein.

We screened for natural mutations in Crl:CF1 closed colony mice using an ordinary backcrossing system. Five of 30 CF1 males carried novel genes that caused white spots on colored coats. Their backcross progenies showed a white spot phenotype. The white spot gene was mapped to approximately 39 cM on chromosome 5, where the Kit gene is known to reside. Allelism testing between this spot gene and the Kit gene was performed using two already known Kit alleles, Kit(W), and Kit(W-v). We demonstrated that the spot mutation was semidominant and a novel allele of the Kit gene, which was tentatively named Kit(W-Ham). No infertility or anemia was observed in Kit(W-Ham) homozygotes. However, a reduced number of germ cells and mast cells was observed in Kit(W-Ham)/Kit(W) and Kit(W-Ham)/Kit(W-v) transheterozygotes. Sequencing of the 21 exons of the Kit gene in the Kit(W-Ham) mutants revealed that a unique guanine-to-adenine (G-A) transition at nucleotide position 545 (c.545G>A) of exon 3 changes arginine (R) to glutamine (Q) at position 182 in the extracellular domain of the KIT protein (p.R182Q). This extracellular KIT domain is a binding site for stem cell factors (SCF). It was concluded that the Kit(W-Ham) mutant may serve as a new model of human piebaldism.

Current status of memorial services for laboratory animals in Japan: a questionnaire survey.

In this study, we found that almost all institutions conducting animal experiments, such as universities, corporations, and research laboratories, also conducted memorial services for the animals sacrificed during animal experimentation. A questionnaire survey was conducted among 120 institutions. A total of 83 (69.1%) valid responses were obtained from the participating institutions. Memorial services were held at 79 institutions (95.1%). Memorial services for laboratory animals have been mainly conducted to show appreciation, comfort the spirit, and console the souls.

Neurobehavioral changes in mice exposed to fast neutrons in utero.

Epidemiological studies have revealed that radiation causes brain development abnormalities in atomic bomb survivors exposed in utero. Rat and mouse studies have also shown that prenatal exposure to low-linear energy transfer radiation induces developmental brain anomalies. Because the effects of prenatal irradiation on adult behavior patterns remain largely unknown, the present study investigated the effects of neutron exposure in utero on postnatal behavior patterns in mice. [C57BL/6J × C3H/He] hybrid (B6C3F1) mice were exposed to cyclotron-derived fast neutrons with peak energy of 10 MeV (0.02-0.2 Gy) or Cs-137 gamma-rays (0.2-1.5 Gy) on embryonic day 13.5. At 5.5-8 months of age, the neurobehavior of male offspring was examined by Rota-rod treadmill and locomotor activity. The accumulation of radio-labeled drug at muscarinic acetylcholine and serotonin receptors in mice from control and neutron-irradiated groups was determined by the tracer method. Locomotor activity during the dark period increased in the 0.02 Gy neutron-irradiated group. Furthermore, at 5.5 months of age, tracer binding in vivo to the muscarinic acetylcholine increased and to the serotonin receptors decreased in the 0.02 Gy neutron-irradiated group. In conclusion, the present study reveals that a certain "low-dose window" may exist for radiation-induced changes in neurobehavior and binding to neurotransmitter receptors, because there was correlation in neurobehavior and binding to neurotransmitter receptors in the 0.02 Gy neutron-irradiated group though there was not correlation in the neutron-irradiated groups more than 0.05 Gy.

Genomic and gene expression signatures of radiation in medulloblastomas after low-dose irradiation in Ptch1 heterozygous mice.

Accurate cancer risk assessment of low-dose radiation poses many challenges that are partly due to the inability to distinguish radiation-induced tumors from spontaneous ones. To elucidate characteristic features of radiation-induced tumors, we analyzed 163 medulloblastomas that developed either spontaneously or after X-ray irradiation at doses of 0.05-3 Gy in Ptch1 heterozygous mice. All spontaneous tumors showed loss of heterozygosity in broad regions on chromosome 13, with losses at all consecutive markers distal to Ptch1 locus (S-type). In contrast, all tumors that developed after 3 Gy irradiation exhibited interstitial losses around Ptch1 with distal markers retained (R-type). There was a clear dose-dependent increase in the proportion of R-type tumors within the intermediate dose range, indicating that the R-type change is a reliable radiation signature. Importantly, the incidence of R-type tumors increased significantly (P = 0.007) at a dose as low as 50 mGy. Integrated array-comparative genomic hybridization and expression microarray analyses demonstrated that expression levels of many genes around the Ptch1 locus faithfully reflected the signature-associated reduction in genomic copy number. Furthermore, 573 genes on other chromosomes were also expressed differently between S-type and R-type tumors. They include genes whose expression changes during early cerebellar development such as Plagl1 and Tgfb2, suggesting a recapitulation of gene subsets functioning at distinct developmental stages. These findings provide, for the first time, solid experimental evidence for a significant increase in cancer risk by low-dose radiation at diagnostic levels and imply that radiation-induced carcinogenesis accompanies both genomic and gene expression signatures.

Phenotype-based search of natural mutations related to hereditary diseases existing in a closed colony of mice.

We attempted to detect natural mutations existing in the Jcl:ICR closed colony of mice which is maintained by random mating. We used ordinary genetic backcrosses to efficiently detect recessive mutations carried by individual mice in the colony. Crosses of DBA/2 females and ICR males were performed to obtain F(1) mice. Four F(1) females randomly selected from each cross were backcrossed to the male parent. More than thirty backcross progeny were obtained from each F(1) female by several deliveries. Phenotypes of the backcross progeny were observed macroscopically at about one month of age. As a result, 18 (26.1%) of 69 Jcl:ICR males carried 11 recessive mutation(s). Based on the phenotypes, the tentative names were abnormal kidney, aplasia of eyelids/hind limb digits, circling, dwarfism, heterotaxy, hind limb paralysis, hydrocephalus, rigidity (or rigor), testicular hypoplasia, tremor, and wobbling. The genes responsible for aplasia of eyelids/hind limb digits and dwarfism were each carried by two males, the genes responsible for hydrocephalus and testicular hypoplasia were each carried by three males and the gene responsible for wobbling by four males. It was strongly suggested that the genes shared by several males originated from an identical mutated gene. Surprisingly, male No. 43 had the responsible genes of abnormal kidneys and testicular hypoplasia, and No. 79 had those of dwarfism and tremor. The results obtained in this study suggest that breeders need to be aware of the presence of natural mutations in their colonies.

Age dependence of radiation-induced renal cell carcinomas in an Eker rat model.

Exposure to carcinogens early in life may contribute to cancer development later in life. The amount of radiation exposure children experience during medical procedures has been increasing, so it is important to evaluate the radiation risk of cancer in developing organs. Toward this goal, we assessed the risk of developing renal cell carcinoma using Eker rats as a kidney tumor model. F1 hybrids of male Eker (Tsc2 mutant) and female F344 rats were irradiated with 0.5 or 2 Gy gamma radiation on gestation days 15 and 19, and on postnatal days 5, 20, and 49. At 27 weeks of age, kidneys were examined for proliferative lesions. Preneoplastic lesions such as phenotypically altered tubules increased after postnatal irradiation as a function of age-at-irradiation, and hyperplasia were greatly increased after perinatal and postnatal irradiation. In contrast, development of adenoma and adenocarcinoma were evident in animals irradiated at perinatal ages, being maximal at gestational day 19. The frequency of LOH at the Tsc2 locus was unexpectedly low - 0% (0 of 4) for the unirradiated control, and 17% (6 of 35) for the irradiated group. Irrespective of LOH, the mTOR (mammalian target of rapamycin) pathway, which is negatively regulated by the Tsc1/2 complex, was activated in both benign and malignant lesions, as evidenced by phosphorylation of S6 ribosomal protein and 4E-BP1. This suggests that the wild-type Tsc2 allele may be functionally inactivated. In conclusion, actively growing kidneys in perinatal-aged (F344 x Eker) F1 rats (Tsc2(+/-)) are at risk for radiation-induced malignant transformation of the renal epithelium associated with mTOR activation.

Chromosomal mapping of host resistance loci to Trichinella spiralis nematode infection in rats.

The differences in host response among strains of rats to intestinal nematode parasite Trichinella spiralis infection could provide a powerful benefit for further elucidation of molecular interactions between the host and the parasite. Using several strains of rats, we previously observed that DA strain is a strong responder and F344 strain is a weak responder with respect to expulsion of the adult worm. To identify the host resistance loci, quantitative trait loci (QTLs) analysis in F2 population from crosses between DA and F344 strains was performed. One significant QTL (designated as Tspe) was mapped to the middle region of chromosome 9. In addition, the effect of DA allele at Tspe locus could act recessively and lead to the rejection of more adult worms from the gut. The results from the present study provide more insights on host-parasite interactions, which may be useful in facilitating the development of novel approaches for treatment and control of intestinal parasites in human and domestic livestock.

Establishment of a set of combined immunodeficient DA/Slc-Foxn1(rnu) Lyst(bg) congenic rat strains.

The congenitally athymic nude rat is used for studying cancer and transplantation owing to its hairlessness and T-cell defective function caused by the Foxn1(rnu) gene. However, NK cell activity of the nude rat is markedly increased. It is known that NK cells play a major role in rejection of xenografts and in cytotoxicity against tumor cells. Thus, the athymic nude rat with impaired NK cell activity should be a useful model for extensive studies. The DA-Lyst(bg)/Lyst(bg) rat, a model for human Chediak-Higashi syndrome (CHS) is characterized by diluted-coat color and impairment of NK cell activity. We planned to establish a combined immunodeficient double mutant rat introgressed with the Foxn1(rnu) and Lyst(bg) genes and a set of congenic strains having an identical genetic backgrounds simultaneously. Based on the phenotypic and genetic characteristics of the parental rat strains, the new strains were produced using continuous backcross and diagnosis with molecular genetic techniques. Each disease gene was diagnosed with PCR-RFLP or the long-nested PCR method. Furthermore, we used a marker-assisted congenic strategy based on scanning the genetic backgrounds of the parental rats with 461 rat microsatellite markers. We think that the newly established DA/Slc-Foxn1(rnu)/Foxn1(rnu) Lyst(bg)/Lyst(bg) double mutant will be useful as a severe disease model for human CHS, and the set of DA/Slc-Foxn1(rnu) Lyst(bg) congenic strains which have impaired NK cell activity and/or defective T cell function should be useful for studying in cancer research, xenotransplantation, immune function and other wide-ranging studies.

An allele-specific genotyping method for rat lyst (lysosomal trafficking regulator) gene.

Two spontaneous mutant beige rats, with phenotypes resembling human Chediak- Higashi syndrome (CHS), were found independently in two inbred strains. Both beige mutations were identified to be recessive alleles in the Lyst locus on rat chromosome 17 and the alleles were denoted Lyst(bg) and Lyst(bg-Kyo). As it is almost impossible to discriminate these mutations phenotypically, we developed an allele-specific genotyping method for the Lyst gene. The nested PCR amplification was followed by restriction fragment length polymorphism (RFLP) analysis. By this method, we could discriminate the mutant Lyst(bg), Lyst(bg-Kyo) alleles, and the normal Lyst allele, easily and accurately.

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.