Genome editing of Nf1, Pten, and Trp53 in neonatal mice induces glioblastomas positive for oligodendrocyte lineage transcription factor 2.

To generate a mouse glioblastoma model by genome editing, we introduced Cas9 protein and guide RNAs specific for Nf1, Pten, and Trp53 into the neonatal mouse forebrain by electroporation. We found a high incidence (approximately 90%) of glial tumor development, including glioblastomas, 15 weeks later. The histological features of the tumors were similar to those of diffuse gliomas and, in some cases, similar to human glioblastomas, with microvascular proliferation (glomeruloid structure). In addition, unlike glial fibrillary acidic protein (GFAP)-positive glioblastomas generated using a similar method in a previous model, the majority of tumor cells were positive for oligodendrocyte lineage transcription factor 2, but negative for GFAP and neurofilaments. One base pair insertions identical to those seen in a previous model were found around the target sequences in Nf1, Pten, and Trp53, and additional deletions were found only in Pten. Considering that the histological characteristics were different from those seen in the previous model, our new model provides an additional research tool to investigate the early stages of glioblastoma development.

Long-Term Chronic Toxicity and Mesothelial Cell Reactions Induced by Potassium Octatitanate Fibers (TISMO) in the Left Thoracic Cavity in A/J Female Mice.

The present study was conducted to examine the chronic effects of potassium octatitanate fibers (trade name TISMO; chemical formula K2O·6TiO2) on the mouse lung and thoracic cavity. This method of infusion was employed to examine the direct effects of the fibers to the pleura. In the present study, 52- and 65-week experiments were employed to examine the long-term chronic effects after infusion of fiber-shaped TISMO into the thoracic cavities of A/J mice. Following this infusion, TISMO fibers were observed in the alveoli, indicating penetration through the visceral pleura. The additional histopathological detection of TISMO fibers in the liver, spleen, kidneys, ovary, heart, bone marrow, and brain of TISMO-infused mice indicated migration of the fibers out from the thoracic cavity. Atypical mesothelial cells with severe pleural proliferation were observed, but malignant mesotheliomas were not detected. This study demonstrated that intrathoracic infusion of TISMO fiber did not cause malignant mesothelioma but did cause severe chronic inflammation and proliferation of pleural mesothelial cells.

Immunohistochemical characteristics of surfactant proteins a, B, C and d in inflammatory and tumorigenic lung lesions of f344 rats.

Surfactant proteins (SPs), originally known as human lung surfactants, are essential to respiratory structure and function. There are 4 subtypes, SP-A, SP-B, SP-C and SP-D, with SP-A and SP-D having immunological functions, and SP-B and SP-C having physicochemical properties that reduce the surface tension at biological interfaces. In this experiment, the expressions of SP-A, SP-B, SP-C and SP-D in lung neoplastic lesions induced by N-bis (2-hydroxypropyl) nitrosamine (DHPN) and inflammatory lesions due to quartz instillation were examined and compared immunohistochemically. Formalin fixed paraffin embedded (FFPE) lung samples featuring inflammation were obtained with a rat quartz instillation model, and neoplastic lesions, hyperplasias and adenomas, were obtained with the rat DHPN-induced lung carcinogenesis model. In the rat quartz instillation model, male 10-week old F344 rats were exposed by intratracheal instillation (IT) to quartz at a dose of 2 mg/rat suspended in saline (0.2 ml) on day 0, and sacrificed on day 28. Lung tumorigenesis in F344 male rats was initiated by DHPN in drinking water for 2 weeks, and the animals were then sacrificed in week 30. Lung proliferative lesions, hyperplasias and adenomas, were observed with DHPN, and inflammation was observed with quartz. The expressions of SP-A, SP-B, SP-C and SP-D were examined immunohistochemically. SP-B and SP-C showed strong expression in lung hyperplasias and adenomas, while SP-A and SP-D were observed in mucus or exudates in inflammatory alveoli. These results suggest the possibility that SP-B and SP-C are related to lung tumorigenesis.

Rat strain differences in levels and effects of chronic inflammation due to intratracheal instillation of quartz on lung tumorigenesis induced by DHPN.

Chronic inflammatory effects of single intratracheal instillation (i.t.) of quartz on rat lung tumorigenesis were examined using 4 different animal models. At first, in order to determine an appropriate dose of quartz i.t. to promote lung tumorigenesis, F344 male rats were administrated single 0, 0.5, 1, 2 or 4 mg quartz/rat after initiation by N-bis(2-hydroxypropyl) nitrosamine (DHPN). Further studies were performed to examine strain differences of the effects of chronic inflammation caused by quartz i.t. in 3 strains of rat, i.e. F344, Wistar-Hannover and SD. Each was instilled with 2mg quartz/rat after DHPN administration and sacrificed in week 24. In addition, strain differences in generation of inflammation were determined at days 1 and 28. Finally, for determination of long-term effects period, F344 and Wistar-Hannover rats were similarly treated, but the experiment was terminated at week 52. In F344 rats, the tumor areas in DHPN treated groups showed a tendency to increase along with the dose of quartz. F344 rats demonstrated the highest and Wistar-Hannover rats the lowest sensitivity to quartz in acute and chronic phases in the 3 strains. In 52 week, in F344 rats, the multiplicity of tumors and the serum concentration of IL-6 in the group treated with DHPN and quartz were significantly increased. The present experiments indicated that chronic inflammation due to quartz instillation exerted promoting effects on lung carcinogenesis in F344, SD and Wistar-Hannover rats. The strain differences in tumor promotion appeared to correlate with inflammatory reactions to quartz and increase of IL-6.

Napsin A is possibly useful marker to predict the tumorigenic potential of lung bronchiolo-alveolar hyperplasia in F344 rats.

There are 2 types of bronchiolo-alveolar hyperplasia found in rat lungs. One is 'inflammatory hyperplasia' with a potential to recover in future with removal of the stimulating insult and the other is 'latent tumorigenic hyperplasia' as an independent preneoplastic lesion for adenocarcinoma. In the present experiment, we focused on rat lung bronchiolo-alveolar hyperplasia induced by 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which decreases with time after induction and reverts to normal, or by N-bis(2-hydroxypropyl)nitrosamine (DHPN), with tumorigenic potential to progress to adenoma and adenocarcinoma. Though NNK is a typical carcinogen inducing lung adenocarcinoma in female A/J mice, the tumorigenic potential by NNK in rats is weak. Differences between hyperplasias induced by DHPN and by NNK were here examined immunohistochemically. Formalin fixed paraffin embedded lung samples with hyperplastic and inflammatory lesions were obtained from rats exposed to DHPN or NNK and from lung inflammation models induced with fine particles like CuO, NiO and quartz. The 19 markers were examined immunohistochemically. Napsin A, in the inflammatory lesions and hyperplasia induced by NNK, was positive for macrophages and secretions in the alveoli spaces but less so in the walls of the alveoli. In the proliferative lesions including hyperplasia induced by DHPN, strong positive staining for napsin A was observed in the walls of the alveoli. Thus high expression was suggested to be possibly useful for detecting tumorigenic potential of rat lung hyperplasia.

Gender-dependent effects of gonadectomy on lung carcinogenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female and male A/J mice.

The present study was conducted to investigate the effects of gonadectomy on lung carcinogenesis in female and male mice, and to determine an association between sex hormone and lung carcinogenesis. Female and male A/J mice were divided into gonadectomized and unoperated control groups and all animals were treated intraperitoneally with 1 or 2 injections of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at the dose of 2 mg/mouse. The mice were sacrificed 18 or 56 weeks after surgery. Serum levels of estradiol in females and testosterone in males were confirmed to be decreased by gonadectomy. Lung white nodules were detected in all mice of all groups. In the control groups of 18- and 56-week studies, the multiplicities of lung nodules in females were significantly greater than in males. In males in the 56-week study, the multiplicity of macroscopical lung nodules, bronchiolo-alveolar hyperplasias, adenomas and tumors (adenomas and adenocarcinomas) showed significant increase with castration. In females in the 18-week study, the multiplicity of adenomas decreased significantly by ovariectomy. Based on the results of the present study, female A/J mice were confirmed to be more susceptible to NNK-induced lung carcinogenesis than males. Furthermore, it was suggested that the process is inhibited by testosterone and accelerated by estradiol. These findings indicate the possibility that sex hormones play important roles in determining sex differences in lung carcinogenesis in the A/J mice initiated by NNK.

Strain differences in pleural mesothelial cell reactions induced by potassium octatitanate fibers (TISMO) infused directly into the thoracic cavity.

Although we have previously reported that the fiber-shaped TISMO, morphologically similar to asbestos, can induce a severe mesothelial reaction in A/J mice, it is important to clarify any strain differences. In the present study, female A/J, C3H/HeN, ICR and C57BL/6 mice were therefore employed as test strains. At the beginning of the experiment, all mice underwent a left thoracotomy and direct administration of 3mg of TISMO particles suspended in 0.2 ml saline into the left thorax. The experiment was terminated after 21 weeks and all groups were sacrificed and the mesothelium and main organs were examined histopathologically. To contribute to mechanistic analysis, iron staining with Berlin blue and Turnbull's blue, and immunostaining for calretinin were also performed. The present experiment demonstrated only minor strain differences in the degree of pleural reaction to TISMO. However, there was clear variation in the iron and lymphocyte accumulation in the pleura and in the liver. This difference in response to TISMO fibers in vivo is important information when considering the development of mesothelioma as an animal model and the extrapolation to human risk from such animal studies.

Spontaneous thymoma in a 10-week-old sprague-dawley rat.

Spontaneous thymoma was found in the left lobe of the thymus of a male 10-week-old Sprague-Dawley (SD) rat. Microscopically, the thymic mass showed a sheet of dark area with multiple pale foci. The dark area mainly consisted of densely compacted small lymphoid cells with sporadic large epithelioid cells and mitotic figures. The epithelioid cells and mitotic figures were more frequent than those of the normal thymic cortex in this animal. The multiple pale foci were similar to the normal thymic medulla and occasionally had Hassall's corpuscles; thus, they were regarded as medullary differentiation areas. Furthermore, some perivascular spaces recognized as characteristics of thymoma were present in the center of the mass. Immunohistochemically, the epithelioid cells in the dark area were positive for cytokeratin. Ultrastructurally, desmosomes and tonofilaments were observed in the epithelioid cells. Thus, this tumor was diagnosed as a thymoma. This is a rare case of thymoma occurring spontaneously in young adult SD rat.

Toxicity of nicotine by repeated intratracheal instillation to f344 rats.

In vivo, nicotine in cigarette smoke induces various effects not only on the respiratory system but also the central and peripheral nerve systems, circulatory organs and digestive organs, and there is a possibility of promotion of lung tumorigenesis. The present experiment was conducted to examine histopathological changes caused by nicotine in the lung with repeated intratracheal instillation (i.t.). Six-week-old male F344 rats were administered nicotine by i.t. at doses of 0.05, 0.1 and 0.2 mg nicotine/rat every 3 weeks beginning at week 4, for up to a total of 9 times and were then sacrificed at week 30. The total number of administrations, total dose of nicotine and effective number of rats were 9 times, 0.45 mg and 5 rats and 4 times, 0.20 mg and 5 rats for the 0.05 mg nicotine/rat group; 3 times, 0.30 mg and 5 rats and 4 times, 0.40 mg and 3 rats for the 0.1 mg group; and 3 times, 0.60 mg and 3 rats for the 0.2 mg group, respectively. As a control group, 5 rats were administered 0.2 ml saline/rat 9 times. Some rats administered 0.1 and 0.2 mg nicotine suffered convulsions just after administration. Histopathologically, though proliferative changes were not observed, neutrophil infiltration, edema and fibrosis in the lung were induced by nicotine. In conclusion, repeated treatment of nicotine promoted neurologic symptoms in the acute phase, and strong inflammation in the lungs in the chronic phase, even at a low dose. Toxicity of nicotine is suggested to depend not on total dose of nicotine in the experiment but rather on repeated injury with consecutive administration.

An intratracheal instillation bioassay system for detection of lung toxicity due to fine particles in f344 rats.

It is an urgent priority to establish in vivo bioassays for detection of hazards related to fine particles, which can be inhaled into deep lung tissue by humans. In order to establish an appropriate bioassay for detection of lung damage after particle inhalation, several experiments were performed in rats using quartz as a typical lung toxic particle. The results of pilot experiments suggest that Days 1 and 28 after intratracheal instillation of 2 mg of fine test particles in vehicle are most appropriate for detection of acute and subacute inflammatory changes, respectively. Furthermore, the BrdU incorporation on Day 1 and the iNOS level on Day 28 proved to be suitable end-point markers for this purpose. An examination of the toxicity of a series of particles was performed with the developed bioassay. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, a ranking order could be clarified. The bioassay thus appears suitable for rapid hazard identification with a possible ranking of the toxicity of various particles at single concentrations.

One-year chronic toxicity study of Aloe arborescens Miller var. natalensis Berger in Wistar Hannover rats. A pilot study.

This study was conducted to evaluate the chronic toxicity of Aloe arborescens Miller var. natalensis Berger (ALOE) in the diet at doses of 4.0%, 0.8% or 0.16% to groups of male and female Wistar Hannover rats. No deaths occurred at any dose level throughout the treatment period. Both sexes receiving 4.0% showed diarrhea, with a reduced body weight gain. Increase of WBCs in the male 4.0% group, decrease of Hb in the female 4.0% and 0.8% groups, decrease of IP in the male 4.0% and 0.8% groups and female 4.0% group, and decrease of Ca and ALT in the female 4.0% group were observed. Relative kidney weight showed increase in the female 4.0% group and relative heart and brain weights were decreased in the female 4.0% and 0.8% groups. Histopathologically, both sexes receiving 4.0% showed severe sinus dilatation of ileocecal lymph nodes, and yellowish pigmentation of ileocecal lymph nodes and renal tubules. In conclusion, the no observed adverse effect level (NOAEL) for ALOE was the 0.16% in diet, which is equivalent to 87.7 and 109.7 mg/kg/day in males and females, respectively.