Cell proliferation analysis is a reliable predictor of lack of carcinogenicity: Case study using the pyrethroid imiprothrin on lung tumorigenesis in mice.

In the mouse carcinogenicity study, an apparent increase in lung adenocarcinoma was observed in male mice at 7000 ppm. Based on the overall evaluation of toxicology, oncology, pathology and statistics, we concluded that the apparent increase in lung tumors is not relevant for evaluation of carcinogenicity of imiprothrin (Regul Toxicol Pharmacol, 105, 1-14, 2019). To investigate whether imiprothrin has any mitogenic effect on mouse Club cells, the present study examined its effects on replicative DNA synthesis of Club cells and lung histopathology in male mice treated with imiprothrin for 7 days at 3500 and 7000 ppm in the diet. Isoniazid, a known mouse lung mitogen and tumor inducer, was also examined at 1000 ppm in the diet as a positive control of Club cell mitogenesis and morphological changes. Neither imiprothrin nor isoniazid caused any necrotic changes in lung by light or electron microscopy. There were no increases observed in the bromodeoxyuridine (BrdU) labeling index in the imiprothrin groups, while there was a statistically significant increase in the BrdU labeling index in the isoniazid group. These findings demonstrate that imiprothrin does not induce mouse Club cell proliferation or morphologic changes, supporting our previous conclusion described above. Thus, imiprothrin should not be classified as a carcinogen. Furthermore, this study indicates that short-term studies focusing on cell proliferation can be reliable for predicting a lack of carcinogenic potential of test chemicals.

Correction: Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

The HTML version of this Article contained errors in Supplementary Figure S2 "Flowchart of the lyso-Gb3 screening and gene analysis in female patients", which have been detailed in the associated Correction.

Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

PURPOSE: Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. METHODS: Between 1 July 2014 and 31 December 2015, we screened 2,359 patients (1,324 males) referred from 168 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. RESULTS: Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml-1) and low α-Gal A activity (≤4.0 nmol h-1 ml-1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 14 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 7 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). CONCLUSION: Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.

Correction: Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

In the above article, we noticed that one female patient in the positive group (plasma lyso-Gb3 7.6 ng/ml, α-galactosidase A activity 4.9 nmol/h/ml) who presented at the neurology clinic was already diagnosed with Fabry disease before the current study. We excluded patients with a confirmed diagnosis of Fabry disease and those with relatives known to have Fabry disease. To accurately describe the information in the current study, we must exclude this patient from the analysis. We have accurately revised this information as follows.

Correction: Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

The PDF and HTML versions of the article have been updated to include the Creative Commons Attribution 4.0 International License information.

Toxicological evaluation of carcinogenicity of the pyrethroid imiprothrin in rats and mice.

The carcinogenic potential of a non-genotoxic pyrethroid imiprothrin was examined in rats and mice. There was no carcinogenicity in rats up to a maximum dose of 5000 ppm of the diet. There was a higher (p = 0.03) incidence of lung adenocarcinomas at 7000 ppm in males, and females showed an increasing (p < 0.01) trend in the incidence of lung adenomas and combined lung adenoma/adenocarcinomas. Additional step sections of lung demonstrated no significant increases in any tumor at p < 0.05, although an increasing trend with dose was observed among females. We argue that, the 7000 ppm dose exceeded the Maximum Tolerated Dose (MTD) for both sexes, based on systemic toxicity as evidenced by body weight gain reduction (both sexes) and high mortality (females). If the 7000 ppm dose is therefore removed from consideration, there are not significant (p < 0.05) increases in tumor formation. Moreover, a consideration of multiple comparisons reveals that the lung tumor increases observed are totally consistent with what would be expected by chance alone. Based on high susceptibility of this mouse strain for the appearance of lung tumors and the lack of a statistically significant increase in tumors by appropriate analysis, the mouse study does not indicate a carcinogenic effect of imiprothrin, and thus no classification for carcinogenicity is warranted.

Segmental aplasia of a uterine horn in two RccHan:WIST rats.

Aplasia of the uterine horn caused by developmental defects has been reported in several species but has not been reported in RccHan:WIST rats. We encountered spontaneous segmental aplasia of a right uterine horn in two RccHan:WIST rats and detailed its pathological characteristics. The right uterine horn of both rats had similar gross and histological appearances. At necropsy, there was segmental loss of the tissues corresponding to normal right uterine horn, which consisted of a fibrous band connected to the uterine cervix. A cystic structure with clear and colorless fluid was observed in the cranial segment of the right uterine horn close to the right oviduct. The cystic structure was thought to be a partially developed tissue to the right uterine horn. The cystic structure seemed to be derived from the right uterine horn. Histologically, a single layer of cuboidal epithelium lined the luminal surface of the cystic structure, the endometrium was thin, and no uterine glands were observed. The fibrous band was composed of α-SMA positive smooth muscle cells, connective tissue, and blood vessels, but cytokeratin AE1/AE3 positive epithelium and uterine endometrium were absent. Based on these gross findings and histological features, segmental aplasia of a uterine horn was diagnosed. To our knowledge, these cases of segmental aplasia of a uterine horn are the first ones described in RccHan:WIST rats.

Alteration of microRNA expressions in the pons and medulla in rats after 3,3'-iminodipropionitrile administration.

Although 3,3'-iminodipropionitrile (IDPN) is widely used as a neurotoxicant to cause axonopathy due to accumulation of neurofilaments in several rodent models, its mechanism of neurotoxicity has not been fully understood. In particular, no information regarding microRNA (miRNA) alteration associated with IDPN is available. This study was conducted to reveal miRNA alteration related to IDPN-induced neurotoxicity. Rats were administered IDPN (20, 50, or 125 mg/kg/day) orally for 3, 7, and 14 days. Histopathological features were investigated using immunohistochemistry for neurofilaments and glial cells, and miRNA alterations were analyzed by microarray and reverse transcription polymerase chain reaction. Nervous symptoms such as ataxic gait and head bobbing were observed from Day 9 at 125 mg/kg. Axonal swelling due to accumulation of neurofilaments was observed especially in the pons, medulla, and spinal cord on Day 7 at 125 mg/kg and on Day 14 at 50 and 125 mg/kg. Furthermore, significant upregulation of miR-547* was observed in the pons and medulla in treated animals only on Day 14 at 125 mg/kg. This is the first report indicating that miR-547* is associated with IDPN-induced neurotoxicity, especially in an advanced stage of axonopathy.

Circulating miR-9* and miR-384-5p as potential indicators for trimethyltin-induced neurotoxicity.

Circulating microRNAs (miRNAs) show promise as biomarkers due to their tissue-specific expression and high stability. This study was conducted to investigate whether nervous system-enriched miR-9* and hippocampus-enriched miR-384-5p could be indicators of neurotoxicity in serum. Rats were given a single administration of trimethyltin (TMT) chloride at 6, 9, or 12 mg/kg by gavage, and brain and serum were collected 1, 4, and 7 days after administration. MiR-9* and miR-384-5p levels in serum and hippocampus were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), and their neurotoxicity detection sensitivities were compared with nervous symptoms, auditory response, and histopathology. TMT caused tremor, hypersensitivity, and decreased auditory response at 12 mg/kg on day 1 and at 9 mg/kg on day 4. Histopathologically, neural cell death and glial reaction were observed in brain (mainly hippocampus) at 12 mg/kg on day 1, 4, and 7 and at 6 and 9 mg/kg on day 4 and 7. MiR-9* and miR-384-5p levels were elevated in serum at 9 and 12 mg/kg on days 4 and 7 (at 9 mg/kg on day 7, miR-9* only) but were not changed in hippocampus. These miRNAs were considered to be elevated with the evolution of neural cell death and were thus considered possible novel indicators of neurotoxicity.

Effect of collaborative intervention by medical and dental professionals on adherence to smoking abstinence.

The need for smoking cessation care is widely recognized. It is, however, difficult to achieve continued smoking abstinence, even when cessation has initially been achieved. The aim of this study was to determine the effectiveness of a collaborative smoking cessation program involving both medical and dental professionals on smoking abstinence. A total of 10 patients visiting our Smoking Cessation Outpatient Clinic were followed up and monitored for smoking abstinence. All received smoking cessation care consisting mainly of counseling by dental and medical professionals and pharmacotherapy. They also concurrently received an oral examination, instruction on oral hygiene, and professional tooth cleaning. The 4-week smoking abstinence rate was 90.0% on completion of the program. One patient failed to complete the program. At one month after the program, 8 out of 9 patients remained abstinent (4-month abstinence; 88.9%). At 3 months after the program, 7 patients remained abstinent (6-month abstinence; 77.8%). Follow-up was impossible in one patient. Within the limitations of the present study, it is suggested that such collaborative intervention including subsequent dental care has the potential to promote short-term adherence to smoking abstinence.

Prediction of efficacy to pegylated interferon-α-2b plus ribavirin in patients with genotype 2 hepatitis C virus using viral response within 2 weeks.

AIM: Rapid virological response (RVR), defined as serum hepatitis C virus (HCV) RNA negativity at 4 weeks, is the most useful predictor of sustained virological response (SVR) to standard pegylated interferon (PEG IFN) plus ribavirin therapy for patients infected with genotype 2 HCV. The aim of the present study was to predict SVR using viral response within 2 weeks of therapy initiation. METHODS: Of 64 HCV genotype 2 patients with a high viral load treated with standard PEG IFN-α-2b plus weight-based ribavirin for 24 weeks, 58 patients whose adherence was more than 67% were analyzed. RNA and core antigen levels were measured at four time points: the day of therapy initiation, the following day, and at 1 and 2 weeks. RESULTS: SVR was achieved in 73% (47/64) of patients. Univariate analysis of SVR contributing factors showed significant differences with age, bodyweight, white blood cell count, platelet count, fibrosis marker levels, baseline core antigen level and viral response. The area under the receiver-operator curve (AUC) of the core antigen level at 1 week (AUC, 0.940) was the highest among the significant SVR predicting factors. Setting 100 fmol/L as the cut-off value for core antigen level at 1 week, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy for predicting SVR were 100%, 86%, 96%, 100% and 97%, respectively, and for predicting RVR were 66%, 93%, 97%, 46% and 72%, respectively. CONCLUSION: The HCV core antigen level at 1 week after therapy initiation is the most useful predictor for SVR.

Robot therapy aids mental health in patients with hematological malignancy during hematopoietic stem cell transplantation in a protective isolation unit.

Patients with hematological malignancy experience physical and psychological pain, such as a sense of isolation and confinement due to intensive chemotherapy in a protective isolation unit (PIU). We examined whether the intervention of a robotic puppy, aibo (manufactured by Sony), could improve patients' mental health as an alternative therapy for pet therapy, which is not feasible in PIU. This study included 21 patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) (n = 16) or autologous HSCT (n = 5). The patients were randomly divided into the aibo and control groups. Psychological effects were regularly assessed by measuring the levels of salivary stress hormone chromogranin A (CgA), serum oxytocin, and serum cortisol and the quick Inventory of Depressive Symptomatology Self-Report (QIDS-SR) scores. The aibo group demonstrated a significant decrease in CgA level, while the control group showed the opposite trend. In addition, changes in serum oxytocin and cortisol levels indicated that aibo helped reduce stress. There was no significant difference in the QIDS-SR scores between the two groups; however, the psychomotor activity in the aibo group improved significantly. These findings suggest that aibo intervention during a stay in a PIU can improve the mental health of patients with hematological malignancies who have undergone HSCT.

Effect of Ala replacement with Aib in amphipathic cell-penetrating peptide on oligonucleotide delivery into cells.

A number of cell-penetrating peptides (CPPs) have been characterized and their usefulness as delivery tools has been clarified. As one of the CPPs, model amphipathic peptide (MAP) was developed by integrating both hydrophobic and hydrophilic amino acids in its sequence. In our previous work, we designed MAP(Aib) by replacing five alanine (Ala) residues on the hydrophobic face of the helix in the MAP sequence with α-aminoisobutyric acid (Aib) residues, and the replacement resulted in higher helix propensity, stronger resistance to protease, and higher cell membrane permeability than MAP. As a next step, we examined the efficiency of oligonucleotide (ODN) delivery into cells by MAP(Aib) in comparison with that by MAP. The electrostatically formed MAP(Aib)/ODN complex was more easily taken up by cells than the MAP/ODN complex, and the ODN delivery by MAP(Aib) was via an endocytic pathway. We demonstrated that the incorporation of Aib residues into CPPs enhances the delivery of hydrophilic molecules, such as ODN, into cells.

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice.

Transforming growth factor-ß (TGF-ß) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-ß/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-ß-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.

Flumioxazin, a PPO inhibitor: A weight-of-evidence consideration of its mode of action as a developmental toxicant in the rat and its relevance to humans.

Flumioxazin, is a herbicide that has inhibitory activity on protoporphyrinogen oxidase (PPO), a key enzyme in the biosynthetic pathway for heme. Flumioxazin induces anemia and developmental toxicity in rats, including ventricular septal defect and embryofetal death. Studies to elucidate the mode of action (MOA) of flumioxazin as a developmental toxicant and to evaluate its relevance to humans have been undertaken. The MOA in the rat has now been elucidated. The first key event is PPO inhibition, which results in reduced heme synthesis in embryonic erythroblasts. The critical window for this effect is gestational day 12 when almost all erythroblasts are at the polychromatophilic stage, synthesizing heme very actively. Embryonic anemia/hypoxemia is induced and the heart pumps more strongly as a compensatory action during organogenesis, leading to thinning of the ventricular walls and failure of the interventricular septum to build completely and close. Investigations showed that this MOA is specific to rats and has no relevancy to humans. Flumioxazin inhibited PPO in rat hepatocyte mitochondria more strongly than in human. A 3-dimensional molecular simulation revealed that species differences in binding affinity of flumioxazin to PPO, observed previously in vitro, were due to differences in binding free energy. In vitro studies using several types of rat and human cells (erythroblasts derived from erythroleukemia cell lines, cord blood, or pluripotent stem cells), showed that flumioxazin decreased heme synthesis in rat cells but not in human cells, demonstrating a clear, qualitative species difference. Considering all available information, including data from PBPK modelling in rat and human, as well as the fact that anemia is not a symptom in patients with variegate porphyria, a congenital hereditary PPO defect, shows that the sequence of events leading to adverse effects in the rat embryo and fetus are very unlikely to occur in humans.

Development of a new, fully automated system for electron backscatter diffraction (EBSD)-based large volume three-dimensional microstructure mapping using serial sectioning by mechanical polishing, and its application to the analysis of special boundaries in 316L stainless steel.

We report the development of a fully automatic large-volume 3D electron backscatter diffraction (EBSD) system (ELAVO 3D), consisting of a scanning electron microscope (ZEISS crossbeam XB 1540) with a dedicated sample holder, an adapted polishing automaton (Saphir X-change, QATM), a collaborative robotic arm (Universal Robots UR5), and several in-house built devices. The whole system is orchestrated by an in-house designed software, which is also able to track the process and report errors. Except for the case of error, the system runs without any user interference. For the measurement of removal thickness, the samples are featured with markers put on the perpendicular lateral surface, cut by plasma focused ion beam (PFIB) milling. The individual effects of both 1 µm diamond suspension and oxide polishing suspension polishing were studied in detail. Coherent twin grain boundaries (GBs) were used as an internal standard to check the removal rates measured by the side markers. The two methods for Z-spacing measurements disagreed by about 10%, and the inaccurate calibration of the PFIB system was found to be the most probable reason for this discrepancy. The angular accuracy of the system was determined to be ∼2.5°, which can be significantly improved with more accurate Z-spacing measurements. When reconstructed grain boundary meshes are sufficiently smoothed, an angular resolution of ±4° is achieved. In a 3D EBSD dataset of a size of 587 × 476 × 72 µm3, we focused on the investigation of coincidence site lattice ∑9 GBs. While bearing predominantly a pure tilt character, ∑9 GBs can be categorized into three groups based on correlative 3D morphologies and crystallography.

Well-differentiated teratoma in a mouse uterus.

Teratomas commonly occur in the testis and ovary, whereas in the uterus they are rare. The authors report findings for a mass detected in the uterus of a 26-week-old mouse in a colony of C57BL/6 bred in their laboratory. The mass was located in the endometrium and protruded into the lumen. Histopathologically, it consisted of abnormal diploblastic or triploblastic tissues. Bone with a growth plate and myeloid cells, as well as cartilage, was mainly observed. It also included melanocytes, exocrine gland-like cells, striated muscle, and neuron-like cells. While these tissues were accompanied by extensive necrosis, all of them were well differentiated and lacked features of malignancy, such as invasion and metastasis. This mouse had experienced parturition, but fetal tissue was not observed in the lesion. Therefore, the lesion was diagnosed as a benign teratoma, which was spontaneously developed in the uterus.

A simple three-dimensional gut model constructed in a restricted ductal microspace induces intestinal epithelial cell integrity and facilitates absorption assays.

The intestine acts as a center for nutrient and water absorption at the epithelium and plays an important role in immunity. Considering the complexity of its function and roles in living systems, a physiologically relevant gut in vitro model is desirable in both basic biology and the analysis of effects of some substances on functions of the gut; these analyses include the screening of drug and food candidates with regard to intestinal disorder at an early stage of medical development. In the present study, we constructed a three-dimensional (3D) gut model using human absorptive enterocytes (CACO-2 cells) by reconstitution of the gut epithelial sheet restricted on a high-reproducible ductal scaffold of collagen gel. Moreover, using the 3D gut model, we evaluated the morphology at the cellular and tissue levels and conducted a phenotypic analysis of the intestinal physiological functions, which involved a permeability assay mimicking barrier disruption inducing inflammation and an absorption assay reflecting ingestive effects. The ductal structure, in vivo-like 3D epithelial structures, epithelial barrier, and effective absorptive function characterized the 3D gut model. The epithelial cells formed a villus-like buckling epithelium, vertical microvilli of increased density on the cell surface, and a crypt-like localized cell proliferating region. The mature shape of the epithelium may contribute to mimicking barrier function and effective absorption compared with that in the 2D gut model. Furthermore, we successfully mimicked the dextran sodium sulfate-induced epithelial barrier dysfunction as a trigger phenomenon of gut inflammation in the 3D gut model. The integrity of the epithelium and phenotypic analysis of the intestinal physiological functions in the simple and reproducible 3D gut model will allow for a drug screening system for assessing the effects on the functions of the gut epithelium from the lumen side.

Ethanol Does Not Promote MeIQx-initiated Rat Colon Carcinogenesis Based on Evidence from Analysis of a Colon Cancer Surrogate Marker.

Epidemiological studies suggest that alcohol consumption increases the risk of developing colorectal cancer. However, the data are confounded by numerous cosegregating variables. To cast further light on the relationships between alcohol intake and colon cancer development, 21-day-old male F344/DuCrj rats were fed 200 ppm 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and doses of 0, 0.1, 0.3, 1, 3, 10 and 20% of ethanol in their drinking water ad libitum for 16 weeks thereafter. The rats were sacrificed after 24 weeks of experiment, and aberrant crypt foci (ACF), surrogate lesions for colon cancer, were examined under a light microscope at low magnification. Ethanol was found not to affect the ACF formation at any dose compared with the initiated-controls. Furthermore, ethanol did not alter colon epithelial cell proliferation. These data, obtained by analysis of a colon cancer surrogate marker lesion, indicate that ethanol lacks promotion activity for MeIQx-initiated rat colon carcinogenesis.

Spontaneous iron accumulation in hepatocytes of a 7-week-old female rat.

Spontaneous iron accumulation in hepatocytes was observed in a 7-week-old female Han Wistar GALAS rat. Very fine yellowish brown pigments, which showed a positive reaction with Berlin Blue stain, were apparent in the cytoplasm close to the bile canaliculi, with a diminishing periportal-to-centrilobular gradient. There were also differences in distribution between and within lobes. Transmission electron microscopy revealed cytosolic ferritin and pericanalicular siderosomes in hepatocytes. No degeneration or necrotic changes were observed, and non-hepatocyte cells did not demonstrate any obvious accumulation of iron. There were no abnormalities in the animal other than this finding in the liver.