Traditionally, safety evaluation at the early stage of drug discovery research has been done using in silico, in vitro, and in vivo systems in this order because of limitations on the amount of compounds available and the throughput ability of the assay systems. While these in vitro assays are very effective tools for detecting particular tissue-specific toxicity phenotypes, it is difficult to detect toxicity based on complex mechanisms involving multiple organs and tissues. Therefore, the development of novel high throughput in vivo evaluation systems has been expected for a long time. The zebrafish (Danio rerio) is a vertebrate with many attractive characteristics for use in drug discovery, such as a small size, transparency, gene and protein similarity with mammals (80% or more), and ease of genetic modification to establish human disease models. Actually, in recent years, the zebrafish has attracted interest as a novel experimental animal. In this article, the author summarized the features of zebrafish that make it a suitable laboratory animal, and introduced and discussed the applications of zebrafish to preclinical toxicity testing, including evaluations of teratogenicity, hepatotoxicity, and nephrotoxicity based on morphological findings, evaluation of cardiotoxicity using functional endpoints, and assessment of seizure and drug abuse liability.
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.
We performed a medaka bioassay for the carcinogenicity of methylazoxymethaol acetate (MAM-Ac) to examine the sequential histological changes in the liver from 3 days after exposure until tumor development. The medaka were exposed to MAM-Ac at a concentration of 2 ppm for 24 hours, and were necropsied at 3, 7, 10, 14, 21, 28, 35, 42, 49, 60, and 91 days after exposure. MAM-Ac induced four cases of hepatocellular adenoma and one case of hepatocellular carcinoma in 8 fish after 60 or 91 days of exposure. Histological changes in the liver until tumor development were divided into three phases. In the cytotoxic phase (1–10 days), MAM-Ac-exposed hepatocytes showed vacuolar degeneration and underwent necrosis and apoptosis, resulting in multiple foci of hepatocyte loss. In the repopulation phase (14–35 days), the areas of hepatocyte loss were filled with hepatic cysts and the remaining hepatocytes were surrounded by hepatic stellate-like cells (or spindle cells) and gradually disappeared. In the proliferation phase (42–91 days), the original hepatic parenchyma was regenerated and progressively replaced by regenerative hyperplastic nodules and/or liver neoplasms. The medaka retained a strong hepatocyte regenerative ability in response to liver injury. It is considered that this ability promotes the proliferation of initiated hepatocytes in multistep carcinogenesis and influences the development of liver tumor over a short period in medaka.
Duchenne muscular dystrophy (DMD) is a progressive muscular disorder caused by X-chromosomal DMD gene mutations. Recently, a new CRISPR/Cas9-mediated DMD rat model (cDMDR) was established and is expected to show cardiac lesions similar to those in humans. We therefore investigated the pathological and pathophysiological features of the cardiac lesions and their progression in cDMDR. For our cDMDR, Dmd-mutated rats (W-Dmdem1Kykn) were obtained. Dmd heterozygous-deficient females and wild-type (WT) males were mated, and male offspring including WT as controls were used. (1) Hearts were collected at 3, 5, and 10 months of age, and HE- and Masson’s trichrome-stained specimens were observed. (2) Electrocardiogram (ECG) recordings were made and analyzed at 3, 5, and 8 months of age. (3) Echocardiography was performed at 9 months of age. In cDMDR rats, (1) degeneration/necrosis of cardiomyocytes and myocardial fibrosis prominent in the right ventricular wall and the outer layer of the left ventricular wall were observed. Fibrosis became more prominent with aging. (2) Lower P wave amplitudes and greater R wave amplitudes were detected. PR intervals tended to be shorter. QT intervals were longer at 3 months but tended to be shorter at 8 months. Sinus irregularity and premature ventricular contraction were observed at 8 months. (3) Echocardiography indicated myocardial sclerosis and a tendency of systolic dysfunction. Pathological and pathophysiological changes occurred in cDMDR rat hearts and progressed with aging, which is, to some extent, similar to what occurs in humans. Thus, cDMDR could be a valuable model for studying cardiology of human DMD.
Resident and infiltrative macrophages play important roles in the development of pathological lesions. M1/M2 macrophage polarization with respective CD68 and CD163 expression remains unclear in chemically induced liver injury. This study was aimed at investigating the influence of macrophages on normal and chemically induced liver injury. For this, dexamethasone (DX), an immunosuppressive drug, was administered in normal rats and thioacetamide (TAA)-treated rats. Liver samples were collected and analyzed with immunohistochemical methods. Repeated injections of DX (0.5 or 1.0 mg/kg BW) for 3, 7 and 11 days reduced the number of CD163 positive hepatic resident macrophages (Kupffer cells) in normal livers, while increasing AST and ALT levels. In TAA (300 mg/kg BW)-treated rats injected with DX (0.5 mg/kg BW) pretreatment, the number of M1 and M2 macrophages showed a significant decrease compared with that of TAA-treated rats without DX treatment. Additionally, reparative fibrosis resulting from hepatocyte injury induced by TAA injection was suppressed by DX pretreatment. Our data suggested that macrophages could influence not only normal hepatic homeostasis (reflected by AST and ALT levels) but also chemically induced hepatic lesion development (reduced reparative fibrosis).
We previously reported that exposure to α-glycosyl isoquercitrin (AGIQ) from the fetal stage to adulthood facilitated fear extinction learning in rats. The present study investigated the specific AGIQ exposure period sufficient for inducing this behavioral effect. Rats were dietarily exposed to 0.5% AGIQ from the postweaning stage to adulthood (PW-AGIQ), the fetal stage to postweaning stage (DEV-AGIQ), or the fetal stage to adulthood (WP-AGIQ). Fear memory, anxiety-like behavior, and object recognition memory were assessed during adulthood. Fear extinction learning was exclusively facilitated in the WP-AGIQ rats. Synaptic plasticity-related genes showed a similar pattern of constitutive expression changes in the hippocampal dentate gyrus and prelimbic medial prefrontal cortex (mPFC) between the DEV-AGIQ and WP-AGIQ rats. However, WP-AGIQ rats revealed more genes constitutively upregulated in the infralimbic mPFC and amygdala than DEV-AGIQ rats, as well as FOS-immunoreactive(+) neurons constitutively increased in the infralimbic cortex. Ninety minutes after the last fear extinction trial, many synaptic plasticity-related genes (encoding Ephs/Ephrins, glutamate receptors/transporters, and immediate-early gene proteins and their regulator, extracellular signal-regulated kinase 2 [ERK2]) were upregulated in the dentate gyrus and amygdala in WP-AGIQ rats. Additionally, WP-AGIQ rats exhibited increased phosphorylated ERK1/2+ neurons in both the prelimbic and infralimbic cortices. These results suggest that AGIQ exposure from the fetal stage to adulthood is necessary for facilitating fear extinction learning. Furthermore, constitutive and learning-dependent upregulation of synaptic plasticity-related genes/molecules may be differentially involved in brain regions that regulate fear memory. Thus, new learning-related neural circuits for facilitating fear extinction can be established in the mPFC.
The use of polyhexamethylene guanidine hydrochloride (PHMG·HCl) as a humidifier disinfectant caused an outbreak of pulmonary disease, leading to the deaths of pregnant women and children in South Korea. However, limited information is available on the inhalation toxicity of PHMG·HCl. Therefore, this study aimed to characterize the subacute inhalation toxicity of PHMG·HCl by whole-body exposure in rats. F344 rats were exposed to 0 mg/m3, 1 mg/m3, 5 mg/m3, or 25 mg/m3 of PHMG·HCl for 6 h/day, 5 days/week for two weeks via whole-body inhalation. Emaciation and rale were observed in rats in the 25 mg/m3 PHMG·HCl group. Significant changes in body weight, hematology, serum chemistry and organ weight were observed in all PHMG·HCl-exposed groups. Gross lesions showed ballooning or red focus in the lungs of rats in the PHMG·HCl-exposed groups. In histopathological examination, most of histological lesions (including degeneration, atrophy, ulcer, inflammatory cell infiltration, inflammation, and fibrosis in nasal cavity, larynx, trachea, and lungs) indicated tissue damage by PHMG·HCl in all PHMG·HCl-exposed groups. Additionally, atrophy of the spleen, thymus, and reproductive organs; immaturity of the testes; and cell debris in the epididymides were affected by the reduction in body weight in PHMG·HCl-exposed groups. In conclusion, two-week repeated whole-body inhalation exposure of rats to PHMG·HCl reveled toxic effects on the respiratory system and secondary effects on other organs. The results of this study indicate that the no observable adverse effect level (NOAEL) for PHMG·HCl is below 1 mg/m3.
Phosphorylation of histone H2AX at serine 139 (γ-H2AX) is known to be induced by direct DNA damage or cellular metabolic imbalances and malfunctions. Previous studies have reported that γ-H2AX is a useful biomarker for early detection of genotoxic bladder carcinogens in rats. The purpose of the present study was to determine the role of γ-H2AX as a biomarker for detection of non-genotoxic bladder carcinogens in rats. Six-week-old male F344 rats were treated with 15 different chemicals for 4 weeks. Immunohistochemical analyses revealed that all three genotoxic bladder carcinogens and six out of seven non-genotoxic bladder carcinogens significantly increased γ-H2AX formation in the bladder urothelium of rats. In addition, four out of five rat bladder noncarcinogens did not increase γ-H2AX formation in the bladder urothelium regardless of genotoxicity. These results suggest that γ-H2AX is a useful biomarker for detection of both genotoxic and non-genotoxic bladder carcinogens in rats.
Mutouhapu (610 hap) was a calcium polysulfide colloid preparation, which was sold as a bath salt in Japan. Herein, we report on the autopsy of a suicide case as a result of taking 610 hap orally and present histopathological findings with a focus on corrosive changes observed in upper gastrointestinal tract. The subject was a 60-year-old man who was discovered dead 200–300 m from his home. The site smelled of sulfur. Sixty parts per million hydrogen sulfide was detected in the oral cavity of the deceased. He had schizophrenia since junior high school. At autopsy, the oral and nasal cavities had a rotten-egg smell. Adipose tissues had green coloring. There was thickening and sclerosis of the gastric wall, and a green to pale yellow pseudomembrane-like substance was observed adhering to the gastric mucosa. Gastric content was 400 ml of green to pale yellow sludge. The distal portion of the stomach was highly contracted. Qualitative analysis for hydrogen sulfide was positive. Histological changes in the digestive tract were particularly notable in the stomach. Degeneration and necrosis of the mucosa and submucosa, degeneration of the submucosal fat, and dissection-like changes in the blood vessels of the submucosa were discovered. Basophilic lime granules were found on the mucosal surface. There were hypercontracture changes in the proper muscle layer of the pyloric region. The cause of death was hydrogen sulfide poisoning with associated corrosive gastritis.
We report a spontaneous case of nephroblastoma in a 26-week-old female Slc:CD(SD) rat. Macroscopically, there was a yellow mass in the left kidney that included another small yellowish-white mass. Histologically, the mass was located mainly in the cortex of the kidney. The tumor showed two distinct morphologies corresponding to the macroscopic findings: a blastemal cell dominant area (blastemal area) with primitive glomeruli and immature tubules and a columnar epithelial tubule dominant area with blastemal cell cuffing on (epithelial area). The epithelial area was located inside the blastemal area and the two morphologies were characterized by the lack of a transition region. Nephroblastoma is known to be biphasic or triphasic and showing transitional features. To our knowledge, there is no report of such nephroblastoma comprising two histologically distinct areas without transition. Therefore, the two distinct morphologies of this case with no transitional characteristic is a rare feature in nephroblastoma.
Spontaneous nephroblastomas are uncommon tumors of laboratory rats. This report describes a spontaneous nephroblastoma with peritoneal metastasis in an 11-month-old, female Sprague Dawley rat. The rat was part of a breeding program and presented 15 days post parturition with clinical signs including tachypnea, dyspnea and abdominal distension. At necropsy, the right kidney was markedly enlarged by an expansile pale-tan to white multinodular mass with extension into the retroperitoneal space, with multifocal variably sized nodules involving the mesentery, and surface of pancreas, liver, uterus, and ovarian bursa. The rat also had severe bicavitary effusion. Histologically, the renal parenchyma of the affected kidney was replaced by a moderately cellular, poorly-demarcated, non-encapsulated, multilobulated mass that appeared to compress the adjacent renal outer medulla and cortex. Three distinct neoplastic cell populations were identified in this renal tumor: epithelial cells (convoluted and dilated tubules / rare primitive glomeruloid structures), mesenchymal (neoplastic spindle cells in connective tissue), and blastemal cells (primitive neoplastic cells). The extrarenal nodular masses were predominantly composed of neoplastic mesenchymal and pleomorphic blastemal cells. Immunohistochemically, neoplastic epithelial cells in the renal mass were positive for pancytokeratin, and blastemal cells in both renal and extrarenal masses were positive for Wilms’ tumor 1 protein (WT1) and vimentin. Neoplastic mesenchymal elements in both renal and extrarenal masses were positive for vimentin. The neoplasm was negative for chromogranin A and S100. The tumor was classified as an anaplastic nephroblastoma with metastasis to the mesentery and peritoneal organs.
Hyaline glomerulopathy is a type of glomerular lesion that occurs in aging mice. Spontaneous hyaline glomerulopathy is rare in young mice. Here, we report spontaneous hyaline glomerulopathy in a young adult (15-week-old) ICR mouse. Necropsy revealed discoloration and roughness of the kidney surface. Microscopically, diffuse glomerular lesions were prominent. Amorphous, eosinophilic materials were deposited globally in the glomeruli. The mesangial region was expanded; however, the mesangial cells showed no proliferation. Thickening of the Bowman’s capsule with proliferation of parietal epithelial cells was observed. Glomerular deposits were strongly positive for anti-IgM, anti-IgG, and periodic acid-Schiff stain and were stained red by Masson’s trichrome stain. The deposits were negative for anti C3 and stained negatively with Congo red stain. Periodic acid methenamine silver and electron microscopy revealed glomerular deposits limited to intraglomerular capillaries. Based on the histological features, we diagnosed this lesion as hyaline glomerulopathy. This case could improve our understanding of spontaneous lesions in toxicological and pharmacological studies.