Lobular distribution of enhanced expression levels of heat shock proteins using in-situ hybridization in the mouse liver treated with a single administration of CCl4.

This study was conducted to visualize the lobular distribution of enhanced mRNA expression levels of heat shock proteins (HSPs) in liver samples from carbon tetra chloride (CCl4)-treated mice using in-situ hybridization (ISH). Male BALB/c mice given a single oral administration of CCl4 were euthanized 6 hours or 1 day after the administration (6 h or 1 day). Paraffin-embedded liver samples were obtained, ISH for HSPs was conducted, as well as hematoxylin-eosin staining and immunohistochemistry (IHC). At 6 h, centrilobular hepatocellular vacuolization was observed, and increased signals for Hspa1a, Hspa1b, and Grp78, which are HSPs, were noted in the centrilobular area using ISH. At 1 day, zonal hepatocellular necrosis was observed in the centrilobular area, but mRNA signal increases for HSPs were no longer observed there. Some discrepancies between ISH and IHC for HSPs were observed, and they might be partly caused by post-transcriptional gene regulation, including the ribosome quality control mechanisms. It is known that CCl4 damages centrilobular hepatocytes through metabolization by cytochrome P450, mainly located in the centrilobular region, and HSPs are induced under cellular stress. Therefore, our ISH results visualized increased mRNA expression levels of HSPs in the centrilobular hepatocytes of mice 6 hours after a single administration of CCl4 as a response to cellular stress, and it disappeared 1 day after the treatment when remarkable necrosis was observed there.

No obvious toxicological influences of 50 µL microsampling from rats administered phenacetin as a drug with hematological toxicity.

According to ICH S3A Q&A focusing on microsampling, its application should be avoided in main study animals for test drugs that could exacerbate hematological parameters with frequent blood sampling. However, no study has reported the effects of microsampling on toxicity parameters of drugs known to induce hematological toxicity. Therefore, we assessed the toxicological effects of serial microsampling on rats treated with phenacetin as a model drug. In a common 28-day study, 50 µL of microsampling was performed at 6-time points on days 1 to 2 and 7-time points on days 27 to 28 from the jugular vein of Sprague Dawley rats. The study was performed independently by two organizations. The toxicological influence of microsampling was evaluated on body weight, food consumption, hematology, blood clinical chemistry, urine parameters, organ weights, and tissue pathology. Phenacetin treatments induced significant changes of various hematological parameters (including hemoglobin and reticulocytes), some organ weights (including liver and spleen), and some hematology-related pathological parameters in the liver, spleen and bone marrow. Meanwhile, serial microsampling exhibited minimal influence on the assessed parameters, although 20 parameters showed statistical differences mostly at one organization. The current results support the notion that serial 50 µL microsampling from the jugular vein had minimal impacts on overall toxicological profiles even in rats treated with a drug inducing hematological toxicity, but the potential adverse effect on certain parameters could not be fully excluded. Accordingly, this microsampling technique has possibility to be employed even for non-clinical rat toxicity studies using drugs with potentially hematological toxicity.

Scientific and Regulatory Policy Committee Best Practices: Documentation of Sexual Maturity by Microscopic Evaluation in Nonclinical Safety Studies.

The sexual maturity status of animals in nonclinical safety studies can have a significant impact on the microscopic assessment of the reproductive system, the interpretation of potential test article-related findings, and ultimately the assessment of potential risk to humans. However, the assessment and documentation of sexual maturity for animals in nonclinical safety studies is not conducted in a consistent manner across the pharmaceutical and chemical industries. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology convened an international working group of pathologists and nonclinical safety scientists with expertise in the reproductive system, pathology nomenclature, and Standard for Exchange of Nonclinical Data requirements. This article describes the best practices for documentation of the light microscopic assessment of sexual maturity in males and females for both rodent and nonrodent nonclinical safety studies. In addition, a review of the microscopic features of the immature, peripubertal, and mature male and female reproductive system and general considerations for study types and reporting are provided to aid the study pathologist tasked with documentation of sexual maturity.

Histopathological significance of microRNA-210 expression in acute peripheral ischemia in a murine femoral artery ligation model.

Under hypoxic conditions, microRNA-210 is upregulated and plays multiple physiological roles including in cell growth arrest, stem cell survival, repression of mitochondrial respiration, angiogenesis, and arrest of DNA repair. In this study, we investigated the histopathological expression of microRNA-210 under hypoxic conditions using a femoral artery ligation model established in C57BL/6J mice to determine the pathological significance of microRNA-210. Following femoral artery ligation, ischemia was represented by decreased blood flow compared to the control, in which a sham operation was performed. On histopathology, degeneration/necrosis of the muscle fibers, inflammatory cell infiltration, and regeneration of the muscle fibers were sequentially observed from 3 h to 3 d after ligation of the artery. The degree of these effects was more severe in the area in which type I muscular fibers are dominant. The histological expression of hypoxia-inducible factor 1α, a well-known biomarker of hypoxia, and microRNA-210 was observed in a few necrotic muscle fibers, macrophages, and myoblasts, a distribution consistent with the histopathological lesions, and their signal increased over time. The expression of microRNA-210 in macrophages and myoblasts under ischemia might be indicative of a significant role in the recovery from ischemic lesions. In addition, the in situ hybridization of microRNA-210 could potentially be used for the detection of hypoxia as a histological marker in addition to the immunohistochemistry of hypoxia-inducible factor 1α.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs) Part III: Discovery of 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2f as a clinical candidate.

We previously reported that 4-(pyrrolidin-1-yl)benzonitrile derivative 1b was a selective androgen receptor modulator (SARM) that exhibited anabolic effects on organs such as muscles and the central nervous system (CNS), but neutral effects on the prostate. From further modification, we identified that 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2a showed strong AR binding affinity with improved metabolic stabilities. Based on these results, we tried to enhance the AR agonistic activities by modifying the substituents of the 5-oxopyrrolidine ring. As a consequence, we found that 4-[(2S,3S)-2-ethyl-3-hydroxy-5-oxopyrrolidin-1-yl]-2-(trifluoromethyl)benzonitrile (2f) had ideal SARM profiles in Hershberger assay and sexual behavior induction assay. Furthermore, 2f showed good pharmacokinetic profiles in rats, dogs, monkeys, excellent nuclear selectivity and acceptable toxicological profiles. We also determined its binding mode by obtaining the co-crystal structures with AR.

Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.

The molecular mechanism responsible that determines cell fate after mitotic slippage is unclear. Here we investigate the post-mitotic effects of different mitotic aberrations--misaligned chromosomes produced by CENP-E inhibition and monopolar spindles resulting from Eg5 inhibition. Eg5 inhibition in cells with an impaired spindle assembly checkpoint (SAC) induces polyploidy through cytokinesis failure without a strong anti-proliferative effect. In contrast, CENP-E inhibition causes p53-mediated post-mitotic apoptosis triggered by chromosome missegregation. Pharmacological studies reveal that aneuploidy caused by the CENP-E inhibitor, Compound-A, in SAC-attenuated cells causes substantial proteotoxic stress and DNA damage. Polyploidy caused by the Eg5 inhibitor does not produce this effect. Furthermore, p53-mediated post-mitotic apoptosis is accompanied by aneuploidy-associated DNA damage response and unfolded protein response activation. Because Compound-A causes p53 accumulation and antitumour activity in an SAC-impaired xenograft model, CENP-E inhibitors could be potential anticancer drugs effective against SAC-impaired tumours.

Expression of steroidogenic factor 1 and pituitary specific transcription factor 1 in rat pituitary adenomas.

The protein expressions of steroidogenic factor l (SF-l) and pituitary-specific transcription factor 1 (Pit-1) were investigated immunohistochemically for 53 spontaneous pituitary adenomas of the pars distalis from male Crl:CD(SD) rats. Luteinizing hormone (LH)-positive/prolactin (PRL)-negative and LH-negative/PRL-positive adenomas showed that the expression of SF-1 and Pit-1 was exclusively related to the immunoreactivity of LH and PRL, respectively. All double-positive adenomas (positive for both LH and PRL) were positive for Pit-1 and were supposed to be derived from PRL cells, although some of them also showed SF-1 immunoreactivity. In addition, all null cell adenomas (negative for all anterior pituitary hormones) were positive for SF-1 and negative for Pit-1, indicating that they originated from the gonadotroph cell lineage. This is the first report focusing on the application of transcription factors for the classification of rat pituitary adenomas.

Proliferative and nonproliferative lesions of the rat and mouse male reproductive system.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and differential diagnosis for classifying microscopic lesions observed in the male reproductive system of laboratory rats and mice, with color microphotographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available for society members electronically on the Internet (http://goreni.org). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions of the male reproductive system in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Suppressive effects of acid-forming diet against the tumorigenic potential of pioglitazone hydrochloride in the urinary bladder of male rats.

Pioglitazone hydrochloride (PIO), a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, was administered orally for 85 weeks at 16 mg/kg/day to male rats fed either a diet containing 1.5% ammonium chloride (acid-forming diet) or a control diet to investigate the effects of urinary acidification induced by the acid-forming diet on the tumorigenic potential of PIO in the urinary bladder. The surviving animals at the end of the administration period were followed to the end of the 2-year study period without changes in the diet and were subjected to terminal necropsy on Week 104. The number of urinary microcrystals, evaluated by manual counting with light microscopy and by an objective method with a laser diffraction particle size analyzer, was increased by PIO on Weeks 12 and 25 and the increases were markedly suppressed by urinary acidification. Urinary citrate was decreased by PIO throughout the study period, but no changes were seen in urinary oxalate at any timepoint. The incidences of PIO-treated males bearing at least one of the advanced proliferative changes consisting of papillary hyperplasia, nodular hyperplasia, papilloma or carcinoma were significantly decreased from 11 of 82 males fed the control diet to 2 of 80 males fed the acid-forming diet. The acid-forming diet did not show any effects on the toxicokinetic parameters of PIO and its metabolites. Microcrystalluria appears to be involved in the development of the advanced stage proliferative lesions in bladder tumorigenesis induced by PIO in male rats.

Salivary mucocele in a laboratory beagle.

The histologic characteristics of a salivary mucocele in a beagle used in a toxicity study are described in this report. A pale yellowish cyst under the mandibular skin containing frothy mucus was observed at necropsy. Microscopically, numerous villous projections arose from the internal surface of the cyst and were lined by stratified epithelial-like macrophages, which were immunopositive for macrophage scavenger receptor A. A ruptured sublingual interlobar duct connected to the lumen was observed near the cyst. Luminal amorphous material showed a positive reaction with Alcian blue and periodic acid-Schiff staining as did mucin in the sublingual gland. Ultrastructurally, the epithelial-like macrophages had numerous vacuoles containing electron-lucent material, which was presumed to be lysosomal in origin, and had pseudopods on their cell surfaces interdigitating with those on the adjacent cells. This case report helps to understand the diversity of the background findings in beagles used in toxicity studies.

High mobility group box associated with cell proliferation appears to play an important role in hepatocellular carcinogenesis in rats and humans.

To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.

Influence of atrazine administration and reduction of calorie intake on prostate carcinogenesis in probasin/SV40 T antigen transgenic rats.

Atrazine, which has been used worldwide as a pesticide, is now known to exert endocrine disrupting (antiandrogenic) effects in mammals. In this study, modifying effects of dietary feeding of 500 and 1000 p.p.m. atrazine on the development of androgen-dependent prostate cancer were investigated using male probasin/SV40 T antigen transgenic (TG) rats. As administration of atrazine has now been identified as causing a decrease in bodyweight, a dietary-restricted TG rat group was also included in order to elucidate the influence of reduction of calorie intake per se on the development of prostate cancer. At week 13, almost the entire lobes of the prostate were occupied with tumor lesions, with no clear intergroup differences in the incidences and multiplicities. Therefore, morphometrical assessment ratios of the prostate epithelial area to the whole prostate tissue area were evaluated. The ratio in the lateral lobe of the 1000 p.p.m. atrazine-treated group was significantly decreased, and there was a tendency to decrease in the ratios in the dorsal lobe of the atrazine-treated groups. However, dietary restriction itself without atrazine treatment caused the same reduction to a similar or greater extent. Testosterone levels were not affected by atrazine administration or dietary restriction. Our results indicate that the observed atrazine-related suppression of prostate carcinogenesis was probably caused by the decrease in calorie intake, rather than by atrazine-related endocrine disruption.