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In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.

Human epigenetic studies suggest that consumption of seaweed prevents mammary cancer, which possibly is explained by iodine daily intake. In this study, we evaluated the efficacy of dietary intake of iodine-enriched eggs on mammary tumor incidence caused by the expression of activated type ErbB2. Female transgenic mice were divided into three groups, and fed a basic diet, a diet supplemented with ordinary eggs, or with iodine-enriched eggs. The number of mammary tumors greater than 5 mm in diameter was recorded in mice at 6 months of age. We report that the average number of mammary tumors per mouse was significantly lower in the iodine-enriched egg-added diet group than in either the basic diet or ordinary egg diet groups. These results indicate that iodine intake through livestock-derived products can reduce the incidence of mammary cancers caused by the expression of activated type ErbB2.

In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.

We encountered a patient on long-term oral corticosteroids for systemic lupus erythematosus, who did not complain of pain during caries treatment. Since the patient had no subjective sense of dental pain, all her remaining teeth became stumps, and total tooth extraction was conducted 25 years after presentation. We examined her premolar stumps histopathologically and obtained the following findings: 1. the dental pulp was narrowed in nearly all samples; 2. odontoblasts were not observable; 3. regions with dentinal tubules forming lamellae and regions with fibrotic dentinal tubules were observed. 4. fibrosis of the entire dentin was observed in some regions. 5. nerve fibers stained by anti-neurofilament protein antibody were observed in the dental pulp cavity, but the nerve fibers appeared degenerate and no nerve fiber endings were observed.

Some mutagens are inactivated rapidly by components included in culture media, especially by serum. Long incubation periods may not be appropriate for the comet assay because DNA lesions may be repaired during the time that mutagens are inactivated, leading to false negative results. We questioned how the exposure period of Chinese hamster ovary cells to 8 unstable mutagens affected outcome of the assay.Although the longest biological half-life of the test mutagens was 1.98 h, four were positive following 0.5—24 h incubations while other four were positive only when the incubation period was ≤ 4 h, suggesting that the DNA damage was repaired and the mutagens were inactivated. The rapid inactivation of mutagens in the medium did not affect whether the outcome of the comet assay was positive or negative when cells were exposed for 1—4 h. Based on these results, we concluded that long exposure should not be employed for the compounds that are unstable in culture media, and appropriate incubation time should be determined for them individually.

Hereditary renal carcinomas (RCs) develop in Tsc2 gene mutant (Eker) rats around the age of 1 year. We previously reported that Tsc2 mutations were detected in chemically (N-ethyl-N-hydroxyethylnitrosamine (EHEN) and diethylnitrosamine)-induced non-Eker rat RCs, suggesting an involvement of Tsc2 alteration in rat RC development. In this study, we evaluated the effect of extra copies of the Tsc2 gene on renal and hepatocarcinogenesis that was induced by EHEN in vivo. The incidence of RCs in non-transgenic rats (2/17) is slightly higher than in transgenic rats (0/32), although it is statistically not significant. These results suggest the presence of other target RC gene(s) in chemically (EHEN)-induced renal carcinogenesis. We observed no difference in the numbers and areas of the hepatic glutathione S-transferase placental type positive foci.

Summary 1,4-Dioxane is a nongenotoxic hepatocarcinogen but in our previous replicative DNA synthesis (RDS) studies with the [ 3 H]thymidine (TdR)-technique, it failed to increase hepatocyte RDS values when given by gavage to male F344 rats as a single 2000 mg/kg body weight dose. However, in a current series of trials with TdR, it showed equivocal responses 24 or 48 hr following treatment with 2000 mg/kg in time-course experiments, and positive responses 24 hr following 1000, 1500 and 2000 mg/kg in dose-response experiments. An increased RDS incidence was also observed at the dose of 2000 mg/kg with data for 5-bromo-2′-deoxyuridine (BrdU)-incorporation. These present findings thus support the hypothesis that a capacity to induce cell proliferation may play a key role in 1,4-dioxane hepatocarcinogenesis.

The pasteurization effect of far-infrared (FIR) radiation on Escherichia coli suspended in physiological phosphate buffered saline in the exponential growth phase (exponential phase) was studied. FIR irradiation decreased the number of viable cells in their exponential phase more rapidly than that in their stationary growth phase (stationary phase). By using four kinds of antibiotics as selective reagents to examine the injuries of bacteria, the damaged parts induced by FIR irradiation were specified on the basis of the changes in sensitivities to selective reagents. FIR irradiation damaged mainly RNA ploymerase of E. coli the exponential phase. Under the condition that the transient behavior of the bulk temperature of bacterial suspension irradiated by FIR was the same as that heated by thermal conduction, the pasteurization effect by FIR irradiation on the exponential phase cells was much greater than that by thermal conduction. However, both FIR irradiation and thermal conductive heating induced similar changes in the sensitivities to selective reagents for the exponential phase cells of E. coli. Under the same irradiation conditions, the changes in sensitivities to the selective reagents for the exponential phase cells were different from those for the stationary phase cells. The injuries of ribosome in the exponential phase cells were not as serious as those in the stationary phase cells. It was suggested that the mechanisms of death of the exponential phase cells and the stationary phase cells were different from each other.

The hypothesis has been proposed that cell proliferation, or replicative DNA synthesis (RDS) in S-phase cells, is a nongenotoxic (Ames-negative) mechanism involved in tumorigenesis, providing a very useful conceptual basis for carcinogen testing. In this present study, hepatocyte RDS experiments were conducted using 5-bromo-2'-deoxyuridine (BrdU) labeling in combination with histopathological observation, comparing our results with earlier findings for in situ [3H]thymidine (TdR) labeling. The present BrdU data proved to be consistent with the previous TdR data in all but one case. Hepatocyte RDS induction was observed for some chemicals without hepatotoxicity. BrdU labeling in combination with histopathological observation is therefore a reliable approach to assessment of test compound effects in vivo.

The present study was undertaken to establish the experimental animal model by histological staining methods for minerals. After intraperitoneal injections of minerals, precipitates deposited on the surface of the liver. Liver tissues were fixed in paraformaldehyde, embedded in paraffin and cut into thin sections which were used as minerals containing standard section. Several reagents for histological stains and spectrophotometry for minerals were applied in both test-tube experiments and stainings of tissue sections to test for minerals. Hematoxylin-lake was found of capable of staining minerals in tissue. A simple technique used was described for light microscopic detection of minerals.

Summary Background and aims Large-scale epidemiological studies have shown that drinking more than two cups of coffee per day reduces the risks of hepatitis and liver cancer. However, the heterogeneity of the human genome requires studies of experimental animal models with defined genetic backgrounds to evaluate the coffee effects on liver diseases. We evaluated the efficacy of coffee consumption with one of experimental animal models for human disease. Method We used the Long Evans Cinnamon (LEC) rat, which onsets severe hepatitis and high incidence of liver cancer, due to the accumulation of copper and iron in livers caused by the genetic mutation in Atp7B gene, and leading to the continuous oxidative stress. We determined the expression of inflammation related genes, and amounts of copper and iron in livers, and incidence of the pre-neoplastic foci in the liver tissue of LEC rats. Results Coffee administration for 25 weeks delayed the occurrence of hepatitis by two weeks, significantly improved survival, reduced the expression of inflammatory cytokines, and reduced the incidence of small pre-neoplastic liver foci in LEC rats. There was no significant difference in the accumulation of copper and iron in livers, indicating that coffee administration does not affect to the metabolism of these metals. These findings indicate that drinking coffee potentially prevents hepatitis and liver carcinogenesis through its anti-inflammatory effects. Conclusion This study showed the efficacy of coffee in the prevention of hepatitis and liver carcinogenesis in the LEC model.

Saposin D-deficient (Sap-D−/−) mice develop polydipsia/polyuria and die prematurely due to renal failure with robust hydronephrosis. Such symptoms emerged when they were around 3 mo of age. To investigate the pathogenesis of their water mishandling, we attempted to limit water supply and followed sequential changes of physiological and biochemical parameters. We also analyzed renal histological changes at several time points. At 3 mo old just before water restriction challenge was started, their baseline arginine vasopressin level was comparable to the wild-type (WT) level. Twenty-four-hour water deprivation and desamino d-arginine vasopressin administration improved polydipsia and polyuria to certain degrees. However, creatinine concentrations in Sap-D−/− mice were significantly higher than those in WT mice, suggesting that some renal impairment already emerged in the affected mice at this age. Renal histological analyses revealed that renal tubules and collecting ducts were expanded after 3 mo old. After 6 mo old, vacuolar formation was observed, many inflammatory cells migrated around the ducts, and epithelial monolayer cells of tubular origin were replaced by plentiful cysts of various sizes. At 10∼12 mo old, severe cystic deformity appeared. On the other hand, 8-mo-long water restriction started at 4 mo old dramatically improved tubular damage and restored once-dampened amount of tubular aquaporin2 protein to the WT level. Furthermore, 10-mo-long water restriction ameliorated their renal function. Remarkably, by continuing water restriction thereafter, overall survival period became comparable with that of the WT. Together, polyuria, devastating renal tubular lesions, and renal failure were ameliorated by the mere 10-mo-long water restriction, which would trigger lethal dehydration if the disease were to be caused by any processes other than primary polydipsia. Our study demonstrates that long-term water restriction surely improved renal histopathological changes leading to prevention of premature death in Sap-D−/− mice.

In recent years lanthanum compounds have been widely used in the optical and electronic industries. Although release of lanthanum (La) into the environment and exposure to humans are feared, acute or chronic biologic effects of La remain to be elucidated. The present study was undertaken to establish the experimental animal model for La toxicity and histological staining methods for La. After intraperitoneal injections of lanthanum chloride, white precipitates deposited on the surface of the liver. Existence of La in the precipitates was confirmed by a X-ray fluorescent microanalysis. Liver tissues from La treated rats were fixed in paraformaldehyde, embedded in paraffin and cut into thin sections which were used as a La containing standard section. Several reagents for histological stains and spectrophotometry for metals were applied in both test-tube experiments and stainings of tissue sections to test for La. Alizarin complexone (ALC) was found of capable of staining La in tissue. A simple new technique used was described for light microscopic detection of La.

We experienced a case of focal nodular hyperplasia (FNH), which is a tumor-like lesion of the liver. A 43-year-old woman was admitted to the hospital because of left hypochondralgia. Abdominal echography, CT, and MRI visualized a tumor in the left lateral segment of the hepatic lobe. A possible malignancy could not be denied and excision of the lesion was carried out. From histopathological examination the tumor was diagnosed as FNH of the liver. Recently, attention is called to administration of female hormone preparation as a probable etiologic factor for heaptic FNH. There was a 10-year history of taking an oral contraceptive for the treatment of dysmenorrhea in this patient. It is strongly suggested that hepatic FNH may be related to long-term administration of the oral contraceptive.

Identification and removal/replacement of sources of indoor air pollutants, such as volatile organic compounds (VOCs) and aldehydes, are most effective measures to reduce indoor chemical exposures. For instance, formaldehyde emissions from building materials have been successfully decreased by the restrictions on interior finishing materials under the amended Building Standard Low in Japan. This study was performed to estimate quantitatively influence of household products on indoor air quality. VOC emissions were investigated for 51 products including interior materials, bedclothes, stationeries, toys and printed matters by the small chamber test method (JIS A 1901) under the standard conditions of 28 degrees C, 50% relative humidity and 0.5 times/h ventilation. Total VOC (TVOC) emissions from the tablecloth and gloves, both of which were made of polyvinyl chloride, showed the highest emission rates; over 2000 microg/(m2 x h) after 1 day, and then rapidly decreased to less than 500 microg/(m2 x h) in a week. Among stationeries/toys for schoolchildren and infants, jigsaw puzzle and play mat exhibited higher TVOC emission rates (38 and 24 microg/(m2 x h) after 1 day, respectively). As for VOCs emitted from printed matters, high boiling-point compounds (higher than that of n-tridecane) were typically identified along with toluene, xylenes and ethylbenzene. These results revealed that VOC emissions from household products may influence significantly indoor air quality.

Indoor air quality is a growing health concern because of the increased incidence of the building-related illness, such as sick-building syndrome and multiple chemical sensitivity/idiopathic environmental intolerance. In order to effectively reduce the unnecessary chemical exposure in the indoor environment, it would be important to quantitatively compare the emissions from many types of sources. Besides the chemical emissions from the building materials, daily use of household products may contribute at significant levels to the indoor volatile organic compounds (VOCs). In this study, we investigated the emission rate of VOCs and carbonyl compounds for 30 air fresheners and deodorizers by the standard small chamber test method (JIS A 1901). The total VOC (TVOC) emission rates of these household products ranged from the undetectable level (20 microg/unit/h) to 6,900 microg/unit/h. The mean TVOC emission rate of the air fresheners for indoor use (16 products) was 1,400 microg/unit/ h and that of the deodorizers for indoor use (6 products) was 58 microg/unit/h, indicating that the fragrances in the products account for the major part of the TVOC emissions. Based on the emission rates, the impacts on the indoor TVOC were estimated by the simple model with a volume of 17.4 m3 and a ventilation frequency of 0.5 times/h. The mean of the TVOC increment for the indoor air fresheners was 170 microg/m3, accounting for 40% of the current provisional target value, 400 microg/m3. These results suggest that daily use of household products can significantly influence the indoor air quality.

A collaborative study was conducted to evaluate whether a replicative DNA synthesis (RDS) test using the rat liver can detect nongenotoxic (Ames-negative) hepatocarcinogens with three or seven daily administrations at dose-levels effective in long-term bioassays. The assay methods were well-validated by the 14 participants. Of six compounds tested, carbon tetrachloride (50 and 100 mg/kg), clofibrate (125 and 250 mg/kg), diethylstilbestrol (0.125 and 0.25 mg/kg) and urethane (100 mg/kg) gave positive results, methyl carbamate (200 and 400 mg/kg) exerted equivocal effects, and D,L-ethionine (125 mg/kg) failed to elevate RDS. These findings suggest that the RDS test can detect many nongenotoxic rat hepatocarcinogens with short-term administration at dose-levels used in long-term bioassays.

To assess the efficacy of the in vivo-in vitro hepatocyte replicative DNA synthesis (RDS) test as a short-term assay, 41 putative nongenotoxic (Ames-negative) mouse hepatocarcinogens, as well as 31 noncarcinogens, were examined using male 8-week-old B6C3F1 mice and an in vitro [methyl-3]thymidine-incorporation technique. Animals were exposed to the maximum tolerated dose (MTD) and 1/2 MTD of each chemical by gavage and after 24, 39 or 48 h, hepatocytes were prepared with a collagenase-perfusion technique. Assessment of the distribution of spontaneous RDS in a total of 337 control mice gave an average incidence of 0.15 +/- 0.08% within the range of 0 to 0.39% (mean +/- 3 x SE) with a 99.7% probability. Values of 0.4% or more for RDS incidences induced by test samples were therefore judged as indicating a positive response in our RDS test. Under the experimental conditions applied, 32 of 41 putative nongenotoxic hepatocarcinogens gave clear positive responses (positive sensitivity: 78%), and of 31 noncarcinogens 25 samples gave negative responses (negative specificity: 81%), thus giving an overall concordance for the RDS test with long-term findings of 79%.

To evaluate the applicability of an in vivo-in vitro replicative DNA synthesis (RDS) test using rat hepatocytes, we conducted the RDS test with 22 nongenotoxic (Ames-negative) hepatocarcinogens and 25 noncarcinogens under our standardized conditions and judgement criteria. Compared to controls (RDS incidence of under 1.0%), the RDS test gave positive results for 18 hepatocarcinogens (positive sensitivity: 82%), and negative results for 20 noncarcinogens (negative specificity: 80%), and thus the overall concordance was 81%. These findings strongly suggest that the RDS test is an extremely useful method for early detection of nongenotoxic hepatocarcinogens.

In the sera from chickens inoculated with a transplantable Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C, five peaks (I, II, III, IV and V) were separated by chromatofocusing in a gradient of pH 5-6. Peaks I, II, III and IV were not exactly detectable in normal chicken serum. The contents of two peaks (I and II) correlated to tumor size. Subsequently, Peaks I and II were each purified by Cellulofine gel filtration and six monoclonal antibodies to the purified materials were prepared by somatic cell hybridization. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) revealed that Peaks I and II were each a glycoprotein of the same molecular weight, approximately 105, 000 (105K) daltons, with different isoeltctric points. All monoclonal antibodies obtained cross-reacted equally with either Peak I and Peak II by enzyme-linked immunosorbent assay. By immunoblotting analysis using one (P5-13) of these antibodies, when the same samples from normal and MSB1-41C-transplanted chickens and the purified Peaks I and II were each subjected to SDS-PAGE and blotted on a sheet of nitrocellulose, a single band reacted with P5-13 was formed at a position corresponding with 105K dalton in the respective samples and the density of each band correlated to tumor size. These results suggest that the cross-reactive 105K molecules may increase when chickens develop tumors.