The research field of “Toxicologic Pathology” evaluates potentially toxic chemical exposures and chemically mediated illnesses in humans and experimental animals. Comparative studies of chemical exposures between model organisms and humans are essential for the risk assessment of chemicals and human health. Here we review the development and activities of the Japanese Society of Toxicologic Pathology (JSTP) during its 37-year history. Toxicological pathology studies provide many interesting and valuable findings. Rodent cancer bioassay data demonstrate the importance of dose levels, times, and duration of exposures to chemicals that possibly cause human cancers. Studies of toxic injuries in the nasal cavity demonstrate that specific chemical compounds affect different target cells and tissues. These observations are relevant for current air pollution studies in the preventive medicine field. Future toxicological pathology studies will be enhanced by applying molecular pathology with advanced observation techniques. In addition to the nasal cavity, another sense organ such as the tongue should be a potential next program of our mission for risk assessment of inhaled and ingested chemicals. As a message to the younger members of the JSTP, interdisciplinary and global cooperation should be emphasized. Elucidating the mechanisms of toxicologic pathology with a combination of advanced expertise in genetics and molecular biology offers promise for future advances by JSTP members.

The constitutive androstane receptor (CAR)-mediated mode of action (MOA) for phenobarbital (PB)-induced rodent liver tumor formation has been established, with increased hepatocyte proliferation, which is a key event in tumor formation. Previous studies have demonstrated that PB and other CAR-activators stimulate proliferation in cultured rodent hepatocytes, but not in cultured human hepatocytes. However, in the genetically humanized CAR and pregnane X receptor (PXR) mouse (hCAR/hPXR mouse, downstream genes are still mouse), PB increased hepatocyte proliferation and tumor production in vivo. In contrast to the hCAR/hPXR mouse, studies with chimeric mice with human hepatocytes (PXB-mouse, both receptor and downstream genes are human) demonstrated that PB did not increase human hepatocyte proliferation in vivo. PB increased hepatocyte proliferation in a chimeric mouse model with rat hepatocytes, indicating that the lack of human hepatocyte proliferation is not due to any functional defect in the chimeric mouse liver environment. Gene expression analysis demonstrated that the downstream genes of CAR/PXR activation were similar in hCAR/hPXR and CD-1 mice, but differed from those observed in chimeric mice with human hepatocytes. These findings strongly support the conclusion that the MOA for CAR-mediated rodent liver tumor formation is qualitatively implausible for humans. Indeed, epidemiological studies have found no causal link between PB and human liver tumors. There are many similarities with respect to hepatic effects and species differences between rodent CAR and peroxisome proliferator-activated receptor α activators. Based on our research, the chimeric mouse with human hepatocytes (PXB-mouse) is reliable for human cancer risk assessment of test chemicals.

5-Fluorouracil (5-Fu) is a DNA-damaging agent and teratogenic in rodents. This study aimed to investigate its influence on neural progenitor cells (NPCs) in the developing fetal rat brain. Dams were intraperitoneally injected with 5-Fu (50 mg/kg b.w.) on gestation day 13 and its effects on fetal NPCs were observed from 3 to 72 hours after treatment (HAT), via periodic examination at six intervals. In NPCs of the fetal brain, the p53-labeling index (LI%) was markedly elevated at 3 HAT. Pyknosis and cleaved caspase-3-LI% also increased at 3 HAT, reaching peak values at 9 and 12 HAT. These parallel changes suggested the induction of apoptosis through a p53-mediated pathway. Pyknotic NPCs were distributed across the ventricular zone (VZ) of the telencephalic wall until 12 HAT, and became localized in the medial and dorsal layers at 12 and 48 HAT. Significant decreases in the numbers of mitotic NPCs and BrdU-LI% were noted from 3 HAT and 24 HAT, respectively. BrdU-positive NPCs were located in the ventral and middle layer at 24 and 48 HAT. p21-positive cells were detected at 12 and 24 HAT. The present results demonstrated that p53-mediated apoptosis was induced in all phases of the cell cycle of the NPCs in the early stage after 5-FU treatment. Furthermore, apoptosis of NPCs and suppression of cell proliferative activity are the events that take place in parallel leading to prominent reduction in the width of the telencephalic wall.

2-(l-Menthoxy)ethanol has been frequently employed as a flavoring agent; however, data regarding 2-(l-menthoxy)ethanol toxicity remain limited. We performed a 13-week subchronic toxicity study of 2-(l-menthoxy)ethanol in male and female F344 rats, with doses of 0, 15, 60, or 250 mg/kg body weight (BW)/day orally administered by gavage using corn oil as the vehicle. No significant toxicological changes in general condition, body weight, or food intake were observed in any groups. The hematological assessment showed decreased hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin and increased platelet count in the male 250 mg/kg group. Serum biochemistry revealed elevated total cholesterol in the 250 mg/kg group of male and female rats, reduced triglyceride in the female 250 mg/kg group, and increased total protein in the male 250 mg/kg group, indicating effects on lipid metabolism and protein synthesis. For organ weights, absolute and relative weights of the liver and adrenal glands were increased in the 250 mg/kg group of both sexes and the male 250 mg/kg group, respectively. Histopathological analysis showed chronic nephropathy in the male 15 mg/kg or higher groups, with increased absolute and relative kidney weights, as well as elevated serum creatinine, in the male 60 and 250 mg/kg groups. However, eosinophilic granules containing α_2u-globulin were identified in proximal tubules, suggesting α_2u-globulin nephropathy specific to male rats and without toxicological significance. These results indicated that the no-observed-adverse-effect level of 2-(l-menthoxy)ethanol was 60 mg/kg BW/day for both sexes.

Bleeding during surgical procedures is a common complication. Therefore, hemostatic agents have been developed to control bleeding, and fibrin sealants have several benefits. sFilm-FS is a novel fibrin sealant that comprises a biodegradable co-polymeric film embedded with human fibrinogen and thrombin. Herein, the safety and efficacy of sFilm-FS were compared using a liver and spleen puncture model of Göttingen minipigs with those of the standard hemostatic techniques (control animals) and EVARREST^®, a reference fibrin sealant. Hemostasis and reduced blood loss were more effectively achieved with sFilm-FS than with the standard techniques in the control animals and comparable to those achieved with EVARREST^®. No treatment-related adverse effects were observed in any of the groups. Histopathological evaluation indicated that sFilm-FS was slightly and moderately reactive at the liver puncture site and spleen, respectively, compared with the standard techniques in the control animals. These changes are expected degradation reactions of the co-polymeric film and are not considered as adverse events. No treatment-related abnormalities were noted in the other evaluated organs. Additionally, no evidence of local or systemic thromboses was noted. These results support the use of sFilm-FS for hemostasis in humans.

Testicular histopathology is considered the most sensitive and reliable method to detect the effects of chemicals on sperm production. To carry out a sensitive examination of testicular histopathology and interpret the changes require knowledge of spermatogenic stages. Spermatogenic staging based on acrosome development during spermiogenesis is conventionally performed in animal species routinely used for research and toxicity testing. In contrast, small ruminants, such as sheep and goats, are rarely used as animal models to evaluate toxicity in male reproductive organs. To the best of our knowledge, a comparable spermatogenic staging system in rams has not yet been fully characterised. Hence, this study aimed to adapt the existing spermatogenic staging based on acrosome development in bull testes to fit the seminiferous epithelium cycle of ram testes. The results show that spermatogenic staging based on acrosome development in bull testes can, with slight modifications, be efficiently used for the staging of ram testes.

A 130-week-old male Royal College of Surgeons rat kept as a non-treated animal in a long-term animal study presented with a mass in the hepatic portal region that adhered to a dilated common bile duct and the duodenum. Histopathologically, the solitary mass showed expansive growth with no apparent compression and continued to dilate the common bile duct, which had a hyperplastic epithelium with intestinal metaplasia. The mass mainly consisted of small to large dilated and/or tortuous ducts with abundant dense connective tissue and many inflammatory cells. The single-layer lining epithelium of the duct changed from cuboidal to columnar. Immunohistochemically, the lining cells were positive for cytokeratin 7, cytokeratin 19, and OV-6, which are bile duct markers. Based on the pathological characteristics, the rat was diagnosed as spontaneous cholangiofibrosis adjacent to a dilated common bile duct with intestinal metaplasia.

In this study, we report the features of an adenocarcinoma with giant cell formation spontaneously occurring in the accessory sex glands of a male 10-month-old Sprague-Dawley rat. A milky white mass was found in the region corresponding to the left seminal vesicle and the left coagulating gland. Histologically, tumor cells exhibited diverse growth patterns, including glandular/trabecular, cystic, and sheet-like growth areas. The tumor cells were pleomorphic, with round- or oval-shaped nuclei and abundant eosinophilic cytoplasm. Mitotic figures were occasionally observed. Giant cells were also prominent in the sheet-like growth area, with intracytoplasmic vacuoles containing eosinophilic material. The stroma was rich in collagen fibers and fibroblasts. Numerous inflammatory cells were observed in the glandular and cystic lumina and stroma. Immunohistochemically, the tumor cells were positive for cytokeratin AE1/AE3 and proliferating cell nuclear antigen. In the sheet-like growth area, some of the tumor cells and giant cells were positive for vimentin in the cytoplasm adjacent to the nucleus. Electron microscopy revealed that the tumor cells contained a small number of mitochondria and rough endoplasmic reticulum, and had no basement membrane or desmosome. The giant cells occasionally contained variably sized intracytoplasmic lumina and globular filamentous bodies, probably corresponding to vimentin. Considering these morphological features, the tumor was diagnosed as an adenocarcinoma with the formation of giant tumor cells originating from the male accessory sex glands.

Zymbal’s gland neoplasms are induced in rats through the administration of various carcinogens, but spontaneous neoplasia is rare. This report describes a spontaneous Zymbal’s gland carcinoma with lung metastasis found in an aged male Fischer 344 rat. Macroscopically, the dome-like tumor nodule, approximately 30 mm in diameter with ulceration, was located near the ear canal of the rat. No healthy tissue or structure of Zymbal’s gland was identified on the corresponding side, while the normal salivary glands and a lacrimal gland were observed. Histologically, a large part of the tumor mass was occupied by poorly differentiated neoplastic cells, the shapes of which were oval to polygonal or fusiform. Additionally, clusters of sebaceous-like foamy cells and squamous metaplasia with prominent keratinization were observed. Tumor cells were found to metastasize to the lung; these cells displayed histological similarities, including a sebaceous gland-like pattern, to those in the primary site. The tumor cells were immunohistochemically positive for cytokeratin AE1/AE3 or vimentin but negative for CD68, S100, α-smooth muscle actin, von Willebrand factor, and desmin. Our results indicate that the tumor was a poorly differentiated Zymbal’s gland carcinoma with lung metastasis.

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.

Antisense oligonucleotide (ASO) therapies have been identified as a new treatment modality for intractable diseases. In kidneys treated with ASOs, vacuoles, in addition to basophilic granules, are often observed in the proximal tubules. Some reports have described that these vacuoles are likely to be a secondary phenomenon resulting from the extraction of ASOs during tissue processing. In this study, we compared renal morphology after fixation with Karnovsky’s fixative or 4% paraformaldehyde phosphate buffer (PFA) with that of 10% neutral-buffered formaldehyde solution (NBF). Female Sprague-Dawley rats, intravenously treated four times with 50 mg/kg locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) for 1 or 2 weeks, were examined. Microscopically, vacuoles and basophilic granules in the proximal tubules were observed in the kidneys fixed with NBF. Basophilic granules are indicative of the accumulation of ASOs. Moreover, some of the vacuoles also contained faint basophilic granules, suggesting that the vacuoles were relevant to the accumulation of ASOs. Although moderate vacuolation was observed in the proximal tubules, the majority of the vacuolated epithelia were negative for kidney injury molecule-1 on immunohistochemical staining. Vacuoles in the proximal tubules were not observed in samples subjected to Karnovsky’s fixation, although basophilic granules were observed. In samples subjected to PFA fixation, vacuoles and basophilic granules were observed in the proximal tubules, similar to those in samples subjected to NBF fixation. Overall, our findings demonstrated the possibility of overestimation of vacuolation due to artifacts during tissue processing when using conventional NBF fixation. Karnovsky’s fixative is considered a useful alternative for distinguishing artificial vacuoles from true nephrotoxicity.