A novel mutation in Hr causes abnormal hair follicle morphogenesis in hairpoor mouse, an animal model for Marie Unna Hereditary Hypotrichosis.

Hairpoor mice (Hr(Hp)) were derived through N-ethyl-N-nitrosourea (ENU) mutagenesis. These mice display sparse and short hair in the Hr(Hp)/+ heterozygous state and complete baldness in the Hr(Hp)/Hr(Hp) homozygous state. This phenotype was irreversible and was inherited in an autosomal semidominant manner. Hair follicles (HFs) of Hr(Hp)/+ mice underwent normal cycling and appeared normal, although smaller than those of the wild-type mice. In contrast, HFs of Hr(Hp)/Hr(Hp) mice became cyst-like structures by postnatal day (P) 21. The number and length of vibrissae decreased in a dose-dependent manner as the number of mutant alleles increased. A positional candidate gene approach was used to identify the gene responsible for the hairpoor phenotype. Genetic linkage analysis determined that the hairpoor locus is 2 cm from D14Mit34 on chromosome 14. Sequence analysis of the exons of the candidate gene hairless revealed a T-to-A transversion mutation at nucleotide position 403 (exon 2), presumably resulting in abolishment of an upstream open reading frame (uORF). In addition, we also found that the near-naked mouse (Hr(N)), a spontaneously arising mutant, harbors a A402G transition in its genome. Both mutations were in the uATG codon of the second uORF in the 5' UTR and corresponded to the mutations identified in Marie Unna Hereditary Hypotrichosis (MUHH) patients. In the present study we describe the phenotype, histological morphology, and molecular etiology of an animal model of MUHH, the hairpoor mouse.

Recurrent exposure to welding fumes induces insufficient recovery from inflammation.

Previous studies on welding-fume-induced lung fibrosis have indicated that recovery is possible when the degree of exposure is short-term and moderate. However, this study investigated the recovery after recurrent exposure to welding fumes, as welders are invariably re-exposed to welding fumes after recovering from radiographic pneumoconiosis. Thus, to investigate the disease and recovery processes of welding-fume-induced pneumoconiosis in the case of recurrent welding-fume exposure, rats were exposed to manual metal arc-stainless steel (MMA-SS) welding fumes with a total suspended particulate (TSP) concentration of 51.4 +/- 2.8 mg/m(3) (low dose) or 84.6 +/- 2.9 mg/m(3) (high dose) for 2 h/day in an inhalation chamber for 1 mo and then allowed to recover from the inflammation for 1 mo. Thereafter, the rats were exposed again to MMA-SS with a TSP concentration of 44.1 +/- 8.8 mg/m(3) (low dose) or 80.1 +/- 9.8 mg/m(3) (high dose) for another 30 d and then allowed to recover from the inflammation for 1 mo. The recovery from the first exposure was then compared with that from the second exposure. The first and second exposures to MMA-SS welding fumes were found to produce significant increases in the lung weights and inflammatory parameters, including total cell numbers, alveolar macrophages (AMs), polymorphonuclear cells (PMNs), lymphocytes, and lactate dehydrogenase (LDH) in the bronchoalveolar lavage fluid (BALF) when compared with the unexposed controls. Following the first and second recovery, a significant reduction in inflammatory parameters of BALF was observed between the exposure and recovery groups. Histopathological observations showed foamy or pigmented macrophage accumulation, cellular debris, or pigment from burst macrophages after the first or second exposure. Following the first or second recovery, cellular debris or pigment from burst macrophages was cleared away from the lungs and accumulation of foamy or pigmented macrophages was decreased when compared to previous exposure. Reactive hyperplasia was noticed after second exposure or either recovery. However, significant differences were observed between the first and second exposure or the first and second recovery. In particular, the number of PMNs was significantly higher after the second exposure than after the first exposure. Also, all cell types in the BALF were significantly elevated in the high-dose second recovery group than in the first recovery group, indicating an incomplete recovery from second exposure. In conclusion, these results indicated that the lung damage caused by the second welding-fume exposure was more difficult to recover from than the first exposure.

Implications for tooth development on ENU-induced ectodermal dysplasia mice.

BACKGROUND: In this study, the mutated phenotypes were produced by treatment of chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed the mutated mice showing the specific phenotype of ectodermal dysplasia (ED) and examined the affected gene. METHODS: Phenotypes, including size, bone formation, and craniofacial morphology of ENU-induced ED mice, were focused. Tooth development and expression of several molecules were analyzed by histologic observations and immunohistochemistry. We carried out genome-wide screening and quantitative real-time PCR to define the affected and related genes. RESULTS: As examined previously in human ectodermal dysplasia, ENU-induced ED mice showed the specific morphologic deformities in tooth, hair, and craniofacial growth. Tooth development in the ENU-induced ED mice ceased at early cap stage. In addition, skeletal staining showed retardation in craniofacial development. Finally, the affected gene, which would be involved in the mechanism of ED, was located between the marker D3Mit14 and D3Mit319 on chromosome 3. CONCLUSIONS: The affected gene in ENU-induced ED mice showed several defects in ectodermal organogenesis and these results indicate that this gene plays an important role in mouse embryogenesis.

Point mutation of Hoxd12 in mice.

PURPOSE: Genes of the HoxD cluster play a major role in vertebrate limb development, and changes that modify the Hoxd12 locus affect other genes also, suggesting that HoxD function is coordinated by a control mechanism involving multiple genes during limb morphogenesis. In this study, mutant phenotypes were produced by treatment of mice with a chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed mutant mice exhibiting the specific microdactyly phenotype and examined the genes affected. MATERIALS AND METHODS: We focused on phenotype characteristics including size, bone formation, and digit morphology of ENU-induced microdactyly mice. The expressions of several molecules were analyzed by genome-wide screening and quantitative real-time PCR to define the affected genes. RESULTS: We report on limb phenotypes of an ENU-induced A-to-C mutation in the Hoxd12 gene, resulting in alanine-to-serine conversion. Microdactyly mice exhibited growth defects in the zeugopod and autopod, shortening of digits, a missing tip of digit I, limb growth affected, and dramatic increases in the expressions of Fgf4 and Lmx1b. However, the expression level of Shh was not changed in Hoxd12 point mutated mice. CONCLUSION: These results suggest that point mutation rather than the entire deletion of Hoxd12, such as in knockout and transgenic mice, causes the abnormal limb phenotype in microdactyly mice. The precise nature of the spectrum of differences requires further investigation.

Standardization of bronchoalveolar lavage method based on suction frequency number and lavage fraction number using rats.

Bronchoalveolar lavage (BAL) is a useful tool in researches and in clinical medicine of lung diseases because the BAL fluid contains biochemical and cytological indicators of the cellular response to infection, drugs, or toxicants. However, the variability among laboratories regarding the technique and the processing of the BAL material limits clinical research. The aim of this study was to determine the suction frequency and lavage fraction number necessary to reduce the variability in lavage using male Sprague-Dawley rats. We compared the total cell number and protein level of each lavage fraction and concluded that more cells and protein can be obtained by repetitive lavage with a suction frequency of 2 or 3 than by lavage with a single suction. On the basis of total cell recovery, approximately 70% of cells were obtained from fractions 1~3. The first lavage fraction should be used for evaluation of protein concentration because fractions 2~5 of lavage fluid were diluted in manifolds. These observations were confirmed in bleomycin-induced inflamed lungs of rats. We further compared the BAL data from the whole lobes with data from the right lobes and concluded that BAL data of the right lobes represented data of the whole lobes. However, this conclusion can only be applied to general lung diseases. At the end, this study provides an insight into the technical or analytical problems of lavage study in vivo.

Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice.

Many studies have reported that bleomycin, anti-cancer drug, induces pulmonary fibrosis as a side effect. However, few investigations have focused on the dose-response effects of bleomycin on pulmonary fibrosis. Therefore, in the present study, we investigated the effects of different doses of bleomycin in male mice. ICR mice were given 3 consecutive doses of bleomycin: 1, 2, or 4 mg/kg in bleomycin-treated (BT) groups and saline only in vehicle control (VC) groups. The animals were sacrificed at 7 and 24 days postinstillation. The severity of pulmonary fibrosis was evaluated according to inflammatory cell count and lactate dehydrogenase (LDH) activity in the broncho alveolar lavage fluid (BALF) , and lung tissues were histologically evaluated after hematoxylin and eosin (H&E) , and Masson's trichrome staining. BT groups exhibited changed cellular profiles in BAL fluid compared to the VC group, which had an increased number of total cells, neutrophils, and lymphocytes and a modest increase in the number of macrophages at 7 days post-bleomycin instillation. Moreover, BT groups showed a dose-dependent increase in LDH levels and inflammatory cell counts. However, at 24 days after treatment, collagen deposition, interstitial thickening, and granulomatous lesions were observed in the alveolar spaces in addition to a decrease in inflammatory cells. These results indicate that pulmonary fibrosis induced by 4 mg/kg bleomycin was more severe than that induced by 1 or 2 mg/kg. These data will be utilized in experimental animal models and as basic data to evaluate therapeutic candidates through non-invasive monitoring using the pulmonary fibrosis mouse model established in this study.

LC50 Determination of tert-Butyl Acetate using a Nose Only Inhalation Exposure in Rats.

tert-Butyl acetate (TBAc) is an organic solvent, which is commonly used in architectural coatings and industrial solvents. It has recently been exempted from the definition of a volatile organic compound (VOC) by the Air Resources Board (ARB) . Since the use of TBAc as a substitute for other VOCs has increased, thus its potential risk in humans has also increased. However, its inhalation toxicity data in the literature are very limited. Hence, inhalation exposure to TBAc was carried out to investigate its toxic effects in this study. Adult male rats were exposed to TBAc for 4 h for 1 day by using a nose-only inhalation exposure chamber (low dose, 2370 mg/m(3) (500 ppm) ; high dose, 9482 mg/m(3) (2000 ppm) ) . Shamtreated control rats were exposed to clean air in the inhalation chamber for the same period. The animals were killed at 2, 7, and 15 days after exposure. At each time point, body weight measurement, bronchoalveolar lavage fluid (BALF) analysis, histopathological examination, and biochemical assay were performed. No treatment-related abnormal effects were observed in any group according to time course. Based on those findings, the median lethal concentration (LC50) of TBAc was over 9482 mg/m(3) in this study. According to the MSDS, the 4 h LC50 for TBAc for rats is over 2230 mg/m(3). We suggested that this value is changed and these findings may be applied in the risk assessment of TBAc which could be beneficial in a sub-acute study.

Basic Principles of the Validation for Good Laboratory Practice Institutes.

Validation specifies and coordinates all relevant activities to ensure compliance with good laboratory practices (GLP) according to suitable international standards. This includes validation activities of past, present and future for the best possible actions to ensure the integrity of non-clinical laboratory data. Recently, validation has become increasingly important, not only in good manufacturing practice (GMP) institutions but also in GLP facilities. In accordance with the guideline for GLP regulations, all equipments used to generate, measure, or assess data should undergo validation to ensure that this equipment is of appropriate design and capacity and that it will consistently function as intended. Therefore, the implantation of validation processes is considered to be an essential step in a global institution. This review describes the procedures and documentations required for validation of GLP. It introduces basic elements such as the validation master plan, risk assessment, gap analysis, design qualification, installation qualification, operational qualification, performance qualification, calibration, traceability, and revalidation.

Characteristics of ethylnitrosourea-induced cataracts.

PURPOSE: This study analyzed genes associated with the morphology and regulation of ethylnitrosourea (ENU)-induced cataract mouse. MATERIALS AND METHODS: Immunohistochemistry analysis using anti-crystallins and PCNA antibody revealed that the localization pattern of these specific markers differed between the cataractous and wild-type lens epithelium. Two-dimensional electrophoresis and microarray techniques were used to identify the proteins and genes related to ENU-induced cataract. RESULTS: A novel ENU-induced mutation in the mouse led to nuclear and cortical opacity of the eye lens at 5 weeks postnatal. This cataract phenotype was similar to that of the zonular-pulverulent type of human cataract. Crystallin proteins and gap-junction genes have relations to the formation of cataract. CONCLUSIONS: Together, the results suggest that various proteins affect the formation and specific phenotypes of ENU-induced cataract mouse.

Studies on N-Ethyl-N-nitrosourea Mutagenesis in BALB/c Mice.

N-ethyl-N-nitrosoures (ENU) is effective in inducing hypermorphic mutation as well as hypomorphic and antimorphic mutations. Therefore, this mutagen is used to the production of mutant in the mice. In order to perform an effective ENU mutagenesis using BALB/cAnN mice, determination of optimal dosage and dosage regimen of ENU is necessary. And this study tried to develop a suitable screening method and searched for novel and various mutants as model animals in phenotypedriven ENU mutagenesis. We have carried out dosage regimen for mutagenizing dose of 200 mg/kg ENU in the BALB/c mice. Total screened mice were 30,133. As the results of Esaki and Cho's Phenotype Screening, we got 2,516 phenotypic and behavior abnormalities in G1, G2 and G3 mice. One hundred thirty five G1 phenodeviants were tested for inheritance and 16 dominant mutants were discovered. Forty two recessive mutants were also found in tested 201 micropedigrees. Early-onset mutant mice included the dysmorphology of face, eye, tail, limb, skin, and foot and abnormal behavior like circling, swimming, head tossing, stiff-walking, high cholesterol level, and tremor etc. In this study we could effectively screen G3 recessive mutants. The frequent and concise early-onset screening before weaning will be available for ENU mutagenesis.

Studies on the Small Body Size Mouse Developed by Mutagen N-Ethyl-N-nitrosourea.

Mutant mouse which show dwarfism has been developed by N-ethyl-N-nitrosourea (ENU) mutagenesis using BALB/c mice. The mutant mouse was inherited as autosomal recessive trait and named Small Body Size (SBS) mouse. The phenotype of SBS mouse was not apparent at birth, but it was possible to distinguish mutant phenotype from normal mice 1 week after birth. In this study, we examined body weight changes and bone mineral density (BMD), and we also carried out genetic linkage analysis to map the causative gene(s) of SBS mouse. Body weight changes were observed from birth to 14 weeks of age in both affected (n = 30) and normal mice (n = 24). BMD was examined in each five SBS and normal mice between 3 and 6 weeks of age, respectively. For the linkage analysis, we produced backcross progeny [(SBS × C57BL/6J) F1 × SBS] N2 mice (n = 142), and seventy-four microsatellite markers were used for primary linkage analysis. Body weight of affected mice was consistently lower than that of the normal mice, and was 43.7% less than that of normal mice at 3 weeks of age (P < 0.001). As compared with normal mice at 3 and 6 weeks of age, BMD of the SBS mice was significantly low. The results showed 15.5% and 14.1% lower in total body BMD, 15.3% and 8.7% lower in forearm BMD, and 29.7% and 20.1% lower in femur BMD, respectively. The causative gene was mapped on chromosome 10. The map order and the distance between markers were D10Mit248 - 2.1 cM - D10Mit51 - 4.2 cM - sbs - 0.7 cM - D10Mit283 - 1.4 cM - D10Mit106 - 11.2 cM - D10Mit170.

Noninvasive Monitoring of Bleomycin-induced Lung Injury in Rats Using Pulmonary Function Test.

The single intratracheal instillation (ITI) of bleomycin (BLM) is a widely used method for inducing experimental pulmonary fibrosis in rat model. In the present study, pulmonary function tests (PFTs) of tidal volume (VT), minute volume (VM), and respiratory frequency (FR) have been applied to study their possibility as a tool to monitor the progress of BLM-induced lung injury in rat model. Rats were treated with a single ITI of BLM (2.5 mg/kg) or saline (control). Animals were euthanized at 3, 7, 14, 21, and 28 days post-ITI. Lung toxicity effects were evaluated by inflammatory cell count, lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage fluid (BALF), and light microscopic examination of lung injury. The PFT parameters were measured immediately before the animals were sacrificed. BLM treatment induced significant cellular changes in BALF-increase in number of total cells, neutrophils, and lymphocytes along with sustained increase in number of macrophages compared to the controls at days 3, 7, and 14. BALF LDH level was significantly increased compared to that in the controls up to day 14. On day 3, infiltration of neutrophils was observed in the alveolar spaces. These changes developed into marked peribronchiolar and interstitial infiltration by inflammatory cells, and extensive thickening of the interalveolar septa on day 7. At 14, 21, and 28 days, mild peribronchiolar fibrosis was observed along with inflammatory cell infiltration. The results of PFT show significant consistencies compared to the results of other toxicity tests. These data demonstrate that the most suitable time point for assessing lung fibrosis in this model is 14 days post-ITI of BLM based on the observation of fibrosis at 14, 21, and 28 days. Further, the progress of lung injury can be traced by monitoring the PFT parameters of FR, VT, and VM.

Fine localization of a new cataract locus, Kec, on mouse chromosome 14 and exclusion of candidate genes as the gene that causes cataract in the Kec mouse.

A mouse with cataract, Kec, was generated from N-ethyl-N-nitrosourea (ENU) mutagenesis. Cataract in the Kec mouse was observable at about 5 weeks after birth and this gradually progressed to become completely opaque by 12 weeks. Dissection microscopy revealed that vacuoles with a radial or irregular shape were located primarily in the cortex of the posterior and equatorial regions of the lens. At the late stage, the lens structure was distorted, but not ruptured. This cataract phenotype was inherited in an autosomal recessive manner. We performed a genetic linkage analysis using 133 mutant and 67 normal mice produced by mating Kec mutant (BALB/c) and F1 (C57BL/6 x Kec) mice. The Kec locus was mapped to the 3 cM region encompassed by D14Mit34 and D14Mit69. In addition we excluded coding sequences of 9 genes including Rcbtb2, P2ry5, Itm2b, Med4, Nudt15, Esd, Lcp1, Slc25a30, and 2810032E02Rik as the candidate gene that causes cataract in the Kec mouse.

Study of a BALB/c Mouse Model for Allergic Asthma.

Allergic asthma is a worldwide public health problem and a major socioeconomic burden disease. It is a chronic inflammatory disease marked by airway eosinophilia and goblet cell hyperplasia with mucus hypersecretion. Mouse models have proven as a valuable tool for studying human asthma. In the present report we describe a comparison of mouse asthma models. The experiments were designed as follows: Group I was injected with ovalbumin (OVA, i.p.) on day 1 and challenged with 1% OVA (aerosol exposure) on days 14~21. Group II was injected on day 1, 14 and aerosol-immunized on days 14~21. Group III was injected on day 1, 14 and immunized by 1% OVA aerosol on days 18~21. We assessed asthma induction by determining the total number of white blood cells (WBC) and eosinophils as well as by measuring cytokine levels in bronchoalveolar lavage fluid (BALF). In addition, we evaluated the histopathological changes of the lungs and determined the concentration of immunoglobulin E (IgE) in serum. Total WBC, eosinophils, Th2 cytokines (IL-4, IL-13) and IgE were significantly increased in group I relative to the other groups. Moreover, histopathological studies show that group I mice show an increase in the infiltration of inflammatory cell-in peribronchial and perivascular areas as well as an overall increase in the number of mucus-containing goblet cells relative to other groups. These data suggest that group I can be a useful model for the study of human asthma pathobiology and the evaluation of existing and novel therapeutic agents.

Pulmonary Toxicity and Recovery from Inhalation of Manual Metal Arc Stainless Steel Welding Fumes in Rats.

The objectives of this study were to examine the lung injury and inflammation caused by manual metal arc stainless steel (MMA-SS) welding fume inhalation and to evaluate the recovery process. Sprague-Dawley rats were exposed to MMA-SS welding fumes for 2 h per day in a whole-body exposure chamber, with a total suspended particulate (TSP) concentration of 51.4 ± 2.8 mg/m3 (low dose) or 84.6 ± 2.9 mg/m3 (high dose) for 30 days. The animals were sacrificed after 30 days of exposure as well as after a 30-day recovery period. To assess the inflammatory or injury responses, cellular and biochemical parameters as well as cytokines were assayed in the bronchoalveolar lavage fluid (BALF). MMA-SS welding fume exposure led to a significant elevation in the number of alveolar macrophages (AM) and polymorphonuclear cells (PMN). Additionary, the values of ß-nacetyl glucosaminidase (ß-NAG) and lactate dehydrogenase (LDH) in the BALF were increased in the exposed group when compared to controls. After 30 days of recovery from exposure, a significant reduction in inflammatory parameters of BALF was observed between the exposed and recovered groups. Slight, but significant elevations were noted in the number of AM and PMN in the recovered groups, and AM that had been ingested fume particles still remain in the lungs. In conclusion, these results indicated that welding fumes induced inflammatory responses and cytotoxicity in the lungs of exposed rats. Fume particles were not fully cleared from lungs even after a 30-day recovery period.

A novel missense mutation in the mouse hairless gene causes irreversible hair loss: genetic and molecular analyses of Hr m1Enu.

A novel autosomal recessive mutant was produced using N-ethyl-N-nitrosourea mutagenesis. The characteristics of the mutant mice included progressive irreversible hair loss within a month of birth, wrinkled skin, and long curved nails. Linkage analysis revealed that the causative gene is linked to D14Mit193 on chromosome 14. Sequence analysis of the complete cDNA of the candidate gene, hairless (Hr), identified a homozygous G-to-T transition at nucleotide 3572, leading to the substitution of glycine by tryptophan, designated Gly960Trp. This missense mutation occurs in the vicinity of repression domain 3 of the hairless protein (HR). This allele was named Hr(m1Enu). The relative amounts of Hr mRNA and HR protein determined by real-time PCR and Western blot analyses, respectively, were slightly elevated in the mutant mice. Quantitative real-time PCR analysis revealed the increased expression of Kc1 and Vdr in the mutant mice, whereas the expression of Nrs1 and Krtap16-6 was decreased. These results suggest that the Gly960Trp substitution in HR protein in Hr(m1Enu) mice may alter the function of HR as a transcriptional corepressor.