Download 2 Rats: A Steamy Visual Novel with Multiple Endings
Six weeks old male albinos Wistar rats were selected for obesity induction. They were randomly divided into two groups, normal control (NC, submitted to the standard diet) and high-fat diet (HFD) group (Table 1), with free access to water . After four (4) months under the respective diets, Lee index and body weight gain allowed us to select obese rats . They were then subjected to oral glucose and insulin tolerance tests to select glucose intolerant and insulin resistant ones.
Multi-walled carbon nanotubes (MWCNTs) constitute one of the most promising types of nanomaterials in industry today. With their increasing use, the potential toxicity and carcinogenicity of MWCNT needs to be evaluated in bioassay studies using rodents. Since humans are mainly exposed to MWCNT by inhalation, we performed a 104-week carcinogenicity study using whole-body inhalation exposure chambers with a fibrous straight type of MWCNT at concentrations of 0, 0.02, 0.2, and 2 mg/m3 using male and female F344 rats.
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Lung carcinomas, mainly bronchiolo-alveolar carcinoma, and combined carcinomas and adenomas were significantly increased in males exposed to 0.2 and 2 mg/m3 MWNT-7 and in females exposed to 2 mg/m3 MWNT-7 compared to the clean air control group. However, no development of pleural mesothelioma was observed. Concentration-dependent toxic effects in the lung such as epithelial hyperplasia, granulomatous change, localized fibrosis, and alteration in BALF parameters were found in MWNT-7 treatment groups of both sexes. There were no MWNT-7-specific macroscopic findings in the other organs, including the pleura and peritoneum. Absolute and relative lung weights were significantly elevated in male rats exposed to 0.2 and 2 mg/m3 MWNT-7 and in all exposed female groups. The lung burdens of MWNT-7 were clearly increased in a concentration-dependent as well as a duration-dependent manner.
Taken together, the results of all of the studies cited above led to the hypothesis that inhalation of MWNT-7 aerosol may result in tumor development in the lung and possibly the pleura. Therefore, in order to clarify the carcinogenicity of MWNT-7, we conducted a 2-year carcinogenicity study of MWNT-7 by whole body inhalation based on a widely accepted protocol . In this study, F344 male and female rats, 6-weeks-old at the commencement of the study, were exposed to MWNT-7 aerosol for 6 h/day, 5 days/week for 104 weeks at concentrations of 0, 0.02, 0.2, and 2 mg/m3 using our dry aerosol generation and exposure system.
a, b Survival rates of rats exposed to MWNT-7 or clean air for 104 weeks. The survival rates of the rats are illustrated in panels A (male) and B (female). The survival rate exceeded 72 % in males and 68 % in females at the end of the 104 weeks experimental period. There were no differences in survival rates between the exposed and control groups. c, d Growth curves of rats exposed to MWNT-7 or clean air for 104 weeks. The growth curves of the rats are illustrated in panels c (male) and d (female). Body weights were measured once a week for the first 14 weeks and every 4 weeks thereafter. There was no growth retardation in any of the groups
Urinary, hematological, and blood biochemical analyses revealed no toxicological changes in either male or female rats exposed to 0.02, 0.2, or 2 mg/m3 compared with their respective controls (data not shown).
At the terminal necropsy, multiple grayish, white areas and nodules were found in the lungs of a large number of male and female rats exposed to 2 mg/m3 MWNT-7. The color tone of the lung surface was darkened in line with the exposure concentration (Fig. 3a and b). There were no MWNT-7-specific macroscopic findings in the other organs, including the pleura and peritoneum of the animals that survived to the end of the experiment or the animals that died or were sacrificed before the end of the experiment.
Absolute and relative lung weights were significantly elevated in male rats exposed to 0.2 and 2 mg/m3 MWNT-7, and they were significantly increased in all exposed female groups (Table 2). No MWNT-7 exposure-related increases were found in the other organs.
The results of histopathological examination of the lungs, peritoneum and pleura are shown in Table 3. The incidences of bronchiolo-alveolar carcinomas, total carcinomas (bronchiolo-alveolar carcinomas, adenosquamous carcinoma, adenocarcinoma and squamous cell carcinoma), and total carcinomas and/or adenomas in males exposed to 0.2 and 2 mg/m3 MWNT-7 and females exposed to 2 mg/m3 MWNT-7 were significantly increased compared with their respective control groups. One adenosquamous carcinoma was found in each of the 2 mg/m3-exposed male and female groups, and one poorly differentiated adenocarcinoma and one squamous cell carcinoma were found in the 2 mg/m3-exposed female group; these malignant tumors very rarely arise spontaneously: no such tumors were observed in 599 control males or 600 control females in previous 104-week inhalation carcinogenicity studies in rats conducted by our institute during the last 10 years. Most bronchiolo-alveolar tumors were single tumors, while adenosquamous carcinoma, poorly differentiated adenocarcinoma, and squamous cell carcinoma tended to be multiple tumors. Tumor cells of the bronchiolo-alveolar tumors destroyed the alveolar structure and invaded the walls of bronchioles and blood vessels (Fig. 3c). It was noteworthy that the MWNT-7-induced bronchiolo-alveolar carcinomas were often accompanied by proliferative fibrous connective tissue, which was not present in the spontaneous lung carcinoma that developed in the control male rat (Fig. 3d).
Incidences of bronchiolo-alveolar hyperplasia and alveolar hyperplasia were significantly increased in male rats exposed to 0.2 and 2 mg/m3 MWNT-7 and in female rats exposed to 2 mg/m3 MWNT-7. Incidences of bronchiolar hyperplasia and atypical hyperplasia were significantly increased in male and female rats exposed to 2 mg/m3 MWNT-7. Atypical hyperplasia consisted of epithelial cells with atypia and was accompanied by development of proliferative fibrous connective tissue and alveolar macrophages with phagocytosed MWNT-7 (Fig. 3e). Alveolar hyperplasia was composed of localized proliferation of type II alveolar epithelial cells, accompanied by fractured alveolar macrophages in the alveolar space. Bronchiolar hyperplasia was composed mainly of proliferating ciliated columnar epithelial cells in the terminal bronchiole area. Significantly elevated incidences of granulomatous change and focal fibrosis of the alveolar wall were noted in male and female rats exposed to 0.2 and 2 mg/m3 MWNT-7. Focal fibrosis, characterized by thickening and increased collagen fibers, was often associated with the granulomatous changes. Alveolar macrophages, with phagocytosed MWNT-7 s accumulated in the alveolar spaces in male and female rats exposed to 2 mg/m3 MWNT-7.
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In the pleura, incidences of simple mesothelial hyperplasia of the parietal pleura (Fig. 3f) and focal fibrosis of the parietal pleura side of the diaphragm were increased in male rats exposed to 2 mg/m3 MWNT-7. In female rats exposed to 2 mg/m3 MWNT-7, there was a non-significant increase in the incidences of simple mesothelial hyperplasia and focal fibrosis of the parietal pleura. The incidence of focal fibrosis of the ventral pleura was elevated in male and female rats exposed to 2 mg/m3 MWNT-7. Finally, the incidence of inflammation of the mediastinum was elevated in male rats exposed to 2 mg/m3 MWNT-7.
Goblet cell hyperplasia was also observed in the frontal respiratory epithelium of the nasal cavity and nasopharynx of male and female rats exposed to 0.2 and 2 mg/m3 MWNT-7 and in the respiratory epithelium of the nasopharynx of female rats exposed to 0.02 mg/m3 MWNT-7. Incidences of eosinophilic globules, which appeared in the cytoplasm of the respiratory and olfactory epithelia in the nasal cavity, were increased in male and female rats exposed to 0.2 and 2 mg/m3 MWNT-7. Eosinophilic globules could be the normal reaction to mild irritation . No remarkable non-neoplastic changes were found in the other organs.
Microscopically, single or aggregated MWNT-7 s were found in the nasal cavity, larynx, trachea, lungs, lymph nodes, spleen, liver, kidneys, olfactory bulb, and brain of the exposed rats of both sexes. MWNT-7 fibers in the kidney, olfactory bulb, and brain were single and not aggregated. The degree of MWNT-7 deposition in the lungs and mediastinal lymph nodes was concentration-dependent. Little MWNT-7 was observed by light microscopy outside of the lungs and mediastinal lymph nodes.
Figure 5 shows the MWNT-7 lung burden of the rats that were euthanized or died before the end of the 104-week experimental period. The amount of retained MWNT-7 was related to the MWNT-7 concentration and overall exposure time. The results of the lung burden analysis of MWNT-7 at the end of exposure period are presented in Fig. 6. Amounts of MWNT-7 in the lungs increased linearly with concentration in both males and females (Fig. 6a). The relative amounts of MWNT-7 per body weight were similar between the males and females in the different exposure groups (Fig. 6b).
Results of lung burden analysis. MWNT-7 fibers in the lung were measured for rats that died or were sacrificed in a moribund condition before the end of the experiment. The lung burden increased with concentration and time of exposure
Results of lung burden analysis in rats at 104 weeks. The lung burdens of 10 rats from each exposure group were measured. a Total amounts of MWNT-7 (μg) in whole lungs in male and female rats is plotted against the exposure concentration. The amount of MWNT-7 in the lungs increased linearly in a concentration-dependent manner. b The relative amount of MWNT-7 (μg/body weight) in the lungs of male and female rats. The relative amounts of MWNT-7 found in the lungs are the same in males and females