Yumi Wako

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

Histopathology of Incidental Findings in Beagles Used in Toxicity Studies

The purpose of our publication is to widely communicate the pictures of spontaneous findings occurring in beagles. Spontaneous arteritis occurs commonly in beagles. Frequent sites of arteritis are the heart, spleen, pancreas, epididymis and spinal cord. Morphological similarities between spontaneous and drug-induced arterial lesions may cause confusion when evaluating vascular toxicity of chemicals such as vasodilating agents. Focal and minimal inflammatory lesions are occasionally seen in the lung and may be associated with aspiration of food particles or of unknown causes. A cystic change with copious mucin production occurs occasionally in the mucosal epithelium of the gall bladder. Nesidioblastosis is seen rarely in the pancreas of beagles. C-cell complex and lymphocytic thyroiditis are common thyroid lesions. Spontaneous focal hypospermatogenesis and lobular Sertoli-cell-only seminiferous tubules occurring frequently in beagles must be distinguished from drug-induced damage of the seminiferous tubules in toxicity studies. The morphological differences of the female genital system in each cycle need to be understood; therefore, we present the normal features of the cyclic changes of the female genital organs. Further, we provide more information on spontaneous findings in beagles for exact diagnoses in toxicity studies.

Histopathology of Incidental Findings in Cynomolgus Monkeys (Macaca Fascicularis) Used in Toxicity Studies

The purpose of our publication is to widely communicate pictures of spontaneous findings occurring in cynomolgus monkeys. Focal lymphoplasmacytic infiltration is commonly seen in the general organs. The frequency and severity of these lesions may be influenced by the administration of drugs with an effect on the immune system. Lymphoplasmacytic infiltration in the lamina propria of the stomach is also frequently seen in cynomolgus monkeys, and it is caused mainly by a Helicobacter pylori infection. Various degrees of brown pigments are observed in various organs, and it is possible to distinguish the material of the pigments by its morphological features and site. A focal/segmental glomerular lesion is occasionally seen in a section of the kidney, and the minimal lesion has no influence on the urinalysis. We showed the common glomerular lesions in HE-stained sections, as well as in PAM- or PAS-stained sections, for understanding the details. Young and pubertal monkeys are usually used in toxicity studies; therefore, understanding various maturation stages of the genital system is important. In particular, the female genital system needs to be understood in the morphology, because their cyclic changes are different from other laboratory animals. Thus, we present the normal features of the cyclic changes of the female genital organs. Furthermore, we provide more information on spontaneous findings in cynomolgus monkeys for exact diagnoses in toxicity studies.

Elevated susceptibility of newborn as compared with young rats to 2‐tert‐butylphenol and 2,4‐di‐tert‐butylphenol toxicity

ABSTRACT  In order to determine the susceptibility of newborn rats to 2‐tert‐butylphenol (2TBP) and 2,4‐di‐tert‐butylphenol (DTBP) toxicity, studies were conducted with oral administration from postnatal days (PND) 4 to 21 and the findings were compared with results for young rats exposed from 5 or 6 weeks of age for 28 days. In the newborn rats, specific effects on physical and sexual development and reflex ontogeny were not observed. While there were no clear differences in toxicological profiles between newborn and young rats, the no‐observed‐adverse‐effect levels (NOAELs) differed markedly. For 2TBP, clinical signs such as ataxic gait, decrease in locomotor activity and effects on liver, such as increase in organ weight, were observed and the NOAELs were concluded to be 20 and 100 mg/kg/day in newborn and young rats, respectively. Based on hepatic and renal toxicity (histopathological changes and increase in organ weight with blood biochemical changes), the respective NOAELs for DTBP were concluded to be 5 and 20 mg/kg/day. Therefore, the susceptibility of newborn rats to 2TBP and DTBP was found to be 4–5 times higher than that of young rats.

Repeated-dose liver micronucleus assay of mitomycin C in young adult rats.

The repeated-dose liver micronucleus (RDLMN) assay has been previously reported to be effective for the detection of hepatocarcinogens and suitable for general toxicology studies. A collaborative study was conducted to evaluate whether this RDLMN assay using young adult rats without collagenase perfusion of the liver can be used to detect genotoxic carcinogens. In this study, we performed the RDLMN assay in young adult rats that received intraperitoneal injections of 0.25, 0.5 or 1.0mg/kg/day of mitomycin C (MMC) for 14 and 28 days. The micronucleus induction in the bone marrow was concurrently measured, and a histopathological examination of the liver was conducted. The results revealed that the frequency of micronucleated hepatocytes (MNHEPs) was significantly increased in all of the treatment groups. However, the highest occurrence of MNHEPs was observed in the low-dose treatment group in both the 14- and the 28-day study periods. In addition, histopathological changes indicating hepatotoxicity were not observed even in the group that received the highest dose of MMC. There was no change in the frequency of metaphase hepatocytes in any of the treatment groups compared with our facilitys background data. However, the frequency of proliferating hepatocytes, as assessed by Ki-67 positivity, was decreased at the highest dose, as was the frequency of MNHEPs. Therefore, the decreased induction of MNHEPs in the high-dose groups might be explained by suppression of hepatocyte cell division. In contrast, the frequency of micronucleated immature erythrocytes in the bone marrow significantly increased in a dose-dependent manner in all of the treatment groups in both study periods. Repeated treatment of MMC induced micronuclei in the liver. These results suggest that the novel RDLMN assay can be used to detect MMC genotoxicity in the liver.

Morphological Study of Progressive Glomerulonephropathy in Common Marmosets (Callithrix jacchus)

Spontaneous progressive glomerulonephropathy often occurs in common marmosets. However, there are few detailed reports concerning the age-related progressive process of glomerular changes. We discuss the glomerular changes in the early stage and the progressive changes in the advanced stage of nephropathy. We investigated the kidneys of common marmosets (2–11 years old; 9 males and 12 females) using hematoxylin and eosin, periodic acid–Schiff, periodic acid–methenamine-silver, and Masson’s trichrome (MT) stains and a transmission electron microscope. There was no remarkable change in urine cytology, hematology, or blood chemistry. In the early stage of nephropathy, effacement of podocyte foot processes was observed ultrastructurally even though there were no marked glomerular lesions in the light microscopy. Subsequently, mesangial proliferation occurred from the hilar to peripheral side along the tuft. In the middle stage, red deposits were visible at the glomerular basement membrane (GBM) and the mesangial region directly under the GBM (paramesangial area) with the MT stain. Electron dense deposits were seen at the same area. In the advanced stage, the irregularity became prominent with or without dense deposits. It is necessary to investigate in detail whether the change of podocyte in the early stage was immuno-mediated or due to podocyte failure.

Activity of 2′, 3′-Cyclic Nucleotide 3′-Phosphodiesterase (CNPase) in Spinal Cord with Spongy Change Induced by a Single Oral Dose of Aniline in Rats

A spongy change in the spinal cord white matter was observed in four-week-old rats treated with aniline. Although this change was found to be a result of the myelin sheath splitting at the ultrastructural level, the mechanism is unknown. This study was conducted to identify the mechanism of the spongy change in aniline-treated rats. The spongy change in the spinal cord white matter was first detected on day 5 in the histopathologic examination. The incidence and severity of the lesions, especially in the lateral and ventral funiculi of the thoracic spinal cord white matter, increased prominently from day 8 to day 10. In all rats, immunohistochemical staining by anti-2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) occurred along the cytoplasmic boundaries of the normal oligodendroglia. However, mild to moderate anti-CNPase staining extended to the swollen cytoplasm of the oligodendroglia in the aniline-treated rats from day 2 to day 4. In the electron microscopic examination, free ribosomes and rough endoplasmic reticula in the cytoplasm of the oligodendroglia increased on days 3 and 4. These changes were considered to be related to CNPase expression. However, CNPase expression decreased, whereas the spongy changes were detected from day 5. The reduction in CNPase expression may contribute to the changes in the myelin morphology observed in aniline intoxication.

Case report: spontaneous hemangiosarcoma in the pancreas of a Fischer rat.

Spontaneous hemangioma or hemangiosarcoma is sometimes found in the viscera and soft tissue of rats and mice. However, there is no report of the tumor occurring in the pancreas of rats. We report a pancreatic hemangiosarcoma in a 109-wk-old, male Fischer 344 rat, which was used in the control group of a carcinogenicity study. The tumor destroyed and compressed the normal pancreatic tissue and displayed a high density in terms of the numerous capillaries and strands of endothelial tumor cells. A reticulin stain revealed a dense network formation. The frequency of proliferating cell nuclear antigen-positive staining showed active proliferation of the tumor cells. Immunohistochemically, some of the tumor cells stained positive with factor VIII-related antigen, and ultrastructurally, Weibel-Palade bodies were rarely observed in the cytoplasm of the tumor cell. From these findings, the tumor was diagnosed as a hemangiosarcoma that occurred naturally in the pancreas.

Evaluation of a repeated dose liver micronucleus assay in rats treated with two genotoxic hepatocarcinogens, dimethylnitrosamine and 2-acetylaminofluorene: the possibility of integrating micronucleus tests with multiple tissues into a repeated dose general toxicity study.

As part of a collaborative study by the Collaborative Study Group for Micronucleus Test (CSGMT) of the Mammalian Mutagenicity Study Group (MMS) in the Japanese Environmental Mutagen Society (JEMS), the present study evaluated the effectiveness of the repeated dose liver micronucleus (RDLMN) assay. Two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF), were administered orally to male rats (6 weeks old at the initial dosing) once daily for 14 and 28 days to evaluate the micronucleus (MN) inducibility in the liver. In addition, these chemicals were evaluated for MN inducibility in the bone marrow (BM) and gastrointestinal (GI) tract, i.e. glandular stomach and colon of the same animals used in the RDLMN assay. As a result, both chemicals produced positive results in the liver, although a weak positive response was given by 2-AAF. DMN gave negative results in the tissues other than the liver. 2-AAF produced positive responses in the BM and glandular stomach, and a prominent response was particularly observed in the glandular stomach, which is directly exposed to the test chemicals by gavage. The present results suggest that the RDLMN assay is a useful method for detecting genotoxic hepatocarcinogens, and that it is especially effective for evaluating test chemicals, such as DMN, undetectable by the BM and GI tract MN assay. Moreover, the results in this investigation indicate that the use of multiple tissues in the study integrating the MN tests is more effective than using a single tissue, for detection of the MN induction produced by chemical exposure to rats, and helps to determine the characteristics of the test chemicals.

New Findings Concerning Eosinophilic Substance Deposition in Mouse Nasal Septum: Sex Difference and No Increase in Seniles

An eosinophilic substance (ES) is usually observed in the mouse nasal septum. In contrast to textbooks and one report describing ES as amyloid, a previous study by the authors revealed that ES is not amyloid but consists of collagen and an amorphous material. Furthermore, it was suggested that the amorphous material was produced by clear HE-stained nasal gland epithelial cells present at the dorsal portion directly above the vomeronasal organ. In this histological examination, ES deposition showed sex difference (more intense in males than in females). ES increased with age but not in seniles, suggesting that the increase has a limit. In the detailed examination using subserial HE-stained nasal sections, it was revealed that the clear HE-stained nasal glands continued to the vomeronasal glands, which communicated with the lumen of the vomeronasal organ, and the vomeronasal gland epithelial cells contained strongly periodic acid-Schiff (PAS) positive granules, similar to the clear HE-stained nasal gland epithelial cells. ES also deposited in the interstitium of the vomeronasal glands. The results suggested a possibility that ES deposition may be related to vomeronasal organ.