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.
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 α2u-globulin were identified in proximal tubules, suggesting α2u-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.
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, 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.
It is extremely rare to have multiple spontaneous proliferative lesions in young adult rats. Here, we report the occurrence of different proliferative lesions in multiple tissues of a 7-week-old female rat in a 1-week repeated toxicity study. Grossly, multiple white patches and nodules in the bilateral kidneys, femoral, and subcutaneous masses, and a nodule in the liver were observed. Renal lesions were diagnosed as renal mesenchymal tumors. One of the femoral subcutaneous masses was diagnosed as an adenolipoma consisting of mammary epithelial cells and mature adipocytes. The other femoral and abdominal subcutaneous masses were diagnosed as lipomas consisting of mature adipocytes. The liver nodule was diagnosed as non-regenerative hepatocellular hyperplasia, which was characterized by the proliferation of slightly hypertrophic hepatocytes. In the cauda equina, the growth of enlarged Schwann cells around the axon was observed, and this lesion was diagnosed as a neuroma.
Recently, intratracheal instillation has been focused on as a simple, low-cost alternative to the inhalation method. In this study, intratracheal instillation of sulfuric acid, a typical acidic compound, was performed to compare the acute toxicity of acidic compounds that could cause damage to the respiratory system between intratracheal instillation and inhalation. Sulfuric acid was administered to male rats at doses of 0.7, 2, 7, 20, and 60 mg/kg by dividing the total dose into four doses. General condition and body weight were examined up to 14 days after administration, and macropathological and histopathological examinations were performed. The half-lethal dose was then estimated. All animals administered 20 and 60 mg/kg sulfuric acid and one animal administered 2 mg/kg sulfuric acid died within 4 hours after administration. No abnormalities were observed in other animals. At 20 and 60 mg/kg, multiple red foci or diffuse red areas were macroscopically observed in the lungs. In these lesions, histopathologically, clefts between the mucosal epithelium and basement membrane and necrosis of the alveolar epithelium were observed. Deaths in these groups may have resulted from lung injury. No notable changes were observed in other animals. Therefore, the half-lethal dose of sulfuric acid by intratracheal instillation was estimated as 7–20 mg/kg. The acute toxicity by intratracheal instillation was evaluated with two-fold sensitivity since the exposure at the half-lethal sulfuric acid concentration in inhalation studies was calculated as 43.2 mg/kg.
There are limited data on the gene expression profiles of ion channels in the sinoatrial node (SAN) of dogs and monkeys. In this study, the messenger RNA (mRNA) expression profiles of various ion channels in the SAN of naïve dogs and monkeys were examined using RNAscope® in situ hybridization and compared with those in the surrounding right atrium (RA) of each species. Regional-specific Cav1.3 and HCN4 expression was observed in the SAN of dogs and monkeys. Additionally, HCN1 in dogs was only expressed in the SAN group. The expression profiles of Cav3.1 and Cav3.2 in the SAN and RA were completely different between dogs and monkeys. Dog hearts only expressed Cav3.2; however, Cav3.1 was detected only in monkeys, and the expression score in the SAN was slightly higher than that in the RA. Although Kir3.1 and NCX1 in dogs were equally expressed in both the SAN and RA, the expression scores of these genes in the SAN of monkeys were slightly higher than those in the RA. The Kir3.4 expression score in the SAN of dogs and monkeys was also slightly higher than that in the RA. The mRNA expression scores of Kv11.1/ERG and KvLQT1 were equally observed in both the SAN and RA of dogs and monkeys. HCN2 was not detected in dogs and monkeys. In summary, we used RNAscope to demonstrate the SAN-specific gene expression patterns of ion channels, which may be useful in explaining the effect of pacemaking and/or hemodynamic effects in nonclinical studies.
Unilaterally swollen eyes were histopathologically characterized in four MG-W gerbils. The primary lesions resided in the anterior segment of the eye where neural crest cells play a critical role in embryonic development. They included indistinct filtration angle, unformed canal of Schlemm, hypoplastic iris, and ciliary body. The findings noted in the retina, optic nerve, optic tract, and lateral geniculate nucleus were consistent with the lesions induced following the persistent elevation of intraocular pressure as a result of insufficient drainage of aqueous humor. Thus, the present cases observed in the eyes of MG-W gerbils exemplified the anterior segment dysmorphogenesis associated with inadequate neural crest migration or differentiation, leading to subsequent glaucoma.
Here, we report a case of spontaneous granulocytic leukemia in a 51-week-old male NOD/Shi-scid IL-2Rγnull (NOG) mouse. The mouse showed progressive anemia and rough respiratory movement. Macroscopically, the spleen was discolored and enlarged. Histologically, the bone marrow of the sternum and femur was highly cellular and almost exclusively filled with neoplastic cells. The nuclei of neoplastic cells were large, oval to slightly irregular in shape, and a small number of cells had kidney- or ring-shaped nuclei. Neoplastic cells extensively infiltrated the organs, and the spleen and liver were prominently involved. Immunohistochemically, a large population of neoplastic cells in the red pulp of the spleen and sinusoid of the liver was positive for myeloperoxidase. Based on the histological features, this case was diagnosed with granulocytic leukemia. This novel information on spontaneous tumors may be helpful for the appropriate use of this mouse strain in further research.
We encountered a case of spontaneous thymic carcinosarcoma in a young Crl:CD (Sprague Dawley) rat. Grossly, a white multinodular mass replaced the thymus in the thoracic cavity. Histologically, multiple nodules were separated by fibrous stroma, and each nodule included isolated regions that were composed of epithelial or non-epithelial tumor cells. The epithelial tumor cells were relatively large and round to polygonal cells with large nuclei and weakly eosinophilic cytoplasm. These cells were cytokeratin-positive and vimentin-negative. These cells infiltrated the lungs. The non-epithelial tumor cells were poorly differentiated, small, round to spindle-shaped cells with small nuclei and basophilic cytoplasm. These cells were vimentin-positive and mostly cytokeratin-negative. Many islands of cartilage were observed near non-epithelial cells. Based on these findings, the tumor was diagnosed as a primary thymic carcinosarcoma consisting of a malignant thymoma composed of epithelial tumor cells and a mesenchymal chondrosarcoma composed of non-epithelial tumor cells.
The WHO International Programme on Chemical Safety (IPCS) framework for analyzing the relevance of a cancer mode of action (MoA) for humans (IPCS cancer-HRF) is an application to assess human relevance of tumorigenic hazards found through rodent bioassays. The chloroacetanilide herbicides, butachlor and alachlor, induced enterochromaffin-like (ECL) cell tumors in rat stomachs, at the highest doses. This study analyzed the human relevance of this tumor by applying the IPCS cancer-HRF using published data. In a postulated MoA, early key events (KEs) included decreased mucosal thickness in the fundic region, due to reduced parietal cells. The following KEs included increased pH of gastric acid and hypergastrinemia, leading to enhanced cell proliferation and hyperplasia, and resulting in the outcome of an ECL cell tumor. The data showed consistencies in dose-response and temporal concordance with the KEs and specificity in the tumor response, providing strengthened evidence of the KEs. While the early KE was not the same, similar MoAs have already been established for omeprazole and ciprofloxacin. The integrated data indicated that the postulated MoAs were biologically plausible. Alternative MoAs were excluded.. Based on sufficient evidence, an MoA was established in rats. When addressing chemically inducible MoAs of human relevance, KEs of hypergastrinemia and trophic ECL cell hyperplasia were judged to not be qualitatively and quantitatively plausible in humans. The MoA in rats is unlikely to be present in humans; however, the potential effects on parietal cells cannot be excluded. Thus, the IPCS cancer-HRF is very useful for assessing human relevance.
Herbal products have been in use for many years, but they are becoming more and more popular in recent years, and they are currently in widespread use throughout the world. In this review article we describe the histopathologic findings found after exposure to 12 dietary herbals in studies conducted in rodent model systems. Clear or some evidence for carcinogenic activity was seen with 6 herbals, with the liver being the most common organ affected. The intestine was affected by two herbals (aloe vera nondecolorized extract and senna), three had no clear evidence for carcinogenic activity and one was cardiotoxic (Ephedrine and Ephedra in combination with caffeine). Information from these studies can help to better understand potential target organs for further evaluation from exposure to various herbal products.
A 110-week-old male F344 rat from the high-dose group of a 104-week carcinogenicity study, exhibited a spontaneously occurring subcutaneous mass in the left axilla extending to the chest. Histologically, the mass was well-demarcated from the adjacent mammary tissue and slightly encapsulated without evidence of infiltration into the surrounding tissues. The mass contained both epithelial and adipose components. The epithelial component consisted of ductal structures of various sizes lined by a single layer of flattened to cuboidal epithelial cells with relatively clear or vacuolated cytoplasm. These ductal structures were well-intermingled with an adipose component that consisted of a uniform monomorphic cell population of mature adipocytes. Both cell types were well-differentiated and did not exhibit cellular atypia. Within the mass, fibrous connective tissue was found in the stroma with infiltration of numerous mast cells. Based on these findings, the mass was diagnosed as an adenolipoma of the mammary gland.
A 6-month-old female beagle dog, assigned to the low-dose group in a toxicity study, was evaluated for compound toxicity, and spontaneous hyperadrenocorticism was suspected. The animal had an externally apparent distended abdomen on clinical examination upon arrival. Pre-dose clinical pathology showed slightly higher erythroid parameters and stress leukogram on hematology; plasma biochemistry showed higher total protein, gamma-glutamyl transferase, total cholesterol, and triglyceride levels than the reference data. On necropsy, a prominent increase in adipose tissues of the subcutis and abdomen and increased weight of the adrenal gland and liver were observed. Histopathology revealed diffuse hyperplasia of adrenocortical cells in the zona fasciculata and reticularis, cortical atrophy of the thymus, and abundant glycogen accumulation in the hepatocytes. These findings were incidental and not test-substance-related. Electron microscopy of the adrenocortical cells in the zona fasciculata revealed decreased typical translucent lipid droplets, increased electron-dense lipid droplets, and abundant smooth endoplasmic reticulum and lysosomes. Additionally, increased numbers of various sizes and forms of mitochondria with tubular, vesicular, or lamellar cristae compared to that of normal animals were observed. These ultrastructural characteristics of the adrenocortical cells suggested hyperfunction. The pre-dose plasma cortisol levels were slightly higher than those of other females assigned to the toxicity study, while plasma adrenocorticotropic hormone levels were within the normal range. These findings indicate that hyperadrenocorticism is a possible cause of the systemic changes in this case.