Kazutoshi Shinoda

Doxorubicin induces male germ cell apoptosis in rats

Abstract To clarify whether apoptosis is involved in doxorubicin (DXR)-induced testicular toxicity and to identify the target germ cell type, adult Sprague-Dawley rats were treated with a single intravenous dose of DXR (8 or 12 mg/kg) and euthanized at 3, 6, 12, 24, and 48 h subsequently. Histologically, germ cell degeneration was first found 6 h after dosing in meiotically dividing spermatocytes and early round spermatids of seminiferous tubules at stage I, and subsequently observed in spermatogonia at stages I–VI showing ultrastructural characteristics of apoptosis. Coincident with the appearance of morphological changes, degenerating germ cells were shown to be undergoing apoptosis as revealed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The frequency of TUNEL-labeled germ cells increased in a stage- and cell type-specific manner, the peak of frequency gradually progressing from stage I of seminiferous tubules to later stages with time after dosing, suggesting that the damaged germ cells, especially spermatogonia, gradually underwent the processes leading to apoptosis. DNA laddering on gel electrophoresis was apparent 24 and 48 h after dosing. The results demonstrate that apoptosis plays an important role in the induction of testicular toxicity caused by DXR with meiotically dividing spermatocytes and type A and intermediate spermatogonia as highly vulnerable target cells.

Involvement of apoptosis in the rat germ cell degeneration induced by nitrobenzene

Abstract Nitrobenezene (NB) produces germ cell degeneration, especially of spermatocytes in rats. To examine the possible involvement of apoptosis in this process, the extent and nature of nuclear DNA fragmentation after NB dosing were assessed using both terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and DNA gel electrophoresis, in addition to conventional histological and electron microscopic procedures. Adult Sprague Dawley rats were treated with a single oral dose of NB (250 mg/kg) and euthanized subsequently at 6, 12, and 24 h and 2, 3, 5, and 7 days. The earliest morphological signs of germ cell degeneration in testes were found in pachytene spermatocytes 24 h after dosing. Electron micrographs of degenerating spermatocytes showed marked nuclear chromatin condensation at the nuclear periphery and crowding of cytoplasmic constituents, which are characteristic of apoptosis. Coincident with the appearance of such morphological changes, degenerating spermatocytes contained fragmented DNA as revealed by TUNEL. The presence of DNA laddering, a hallmark of apoptosis on gel electrophoresis, was first apparent and most prominent at 24 h, gradually becoming less detectable. No such changes were observed up to 12 h after dosing or in control animals. These results demonstrated unequivocal involvement of apoptosis in the induction of germ cell degeneration caused by NB.

Induction and inhibition of testicular germ cell apoptosis by fluoroacetate in rats

Abstract Fluoroacetate (FA), an inhibitor of aconitase, is known to lower the intracellular level of adenosine triphosphate (ATP), which recently has been suggested to be a possible determinant of the form of cell death, apoptosis or necrosis. To investigate which form of germ cell death occurs in FA-induced testicular toxicity, adult Sprague Dawley rats were given a single oral dose of FA (0.5 or 1.0 mg/kg) and euthanized at 3, 6, 12, 24, 48, and 72 h thereafter. Germ cell degeneration was histologically first found in early round spermatids at stage I and in spermatogonia at stages II-IV of seminiferous tubules 6 and 12 h, respectively, after dosing. Degenerating spermatogonia exhibited characteristic features of apoptosis as demonstrated by both electron microscopy and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), whereas spermatids did not. At the 24 and 48 h time points, degenerating spermatids were continually present and subsequently formed multinucleated giant cells, while the number of degenerating spermatogonia and TUNEL-labeled spermatogonia was drastically and/or significantly decreased compared to those from the control group, indicating that spontaneous male germ cell apoptosis is inhibited. Coincident with these morphological changes, DNA laddering on gel electrophoresis was apparent only 12 h after dosing. The results demonstrate that FA induces either apoptosis or necrosis of male germ cells in the early stage after dosing and subsequently inhibits spontaneous apoptosis.

Hepatic α2u-globulin mRNA levels and diethylstilbestrol-associated testicular atrophy in rats

Abstract Biosynthesis of α 2u -Globulin (α 2u -g) is under multihormonal regulation. In this study, we investigated histopathologic changes in the testis and hepatic α 2u -g messenger ribonucleic acid (mRNA) levels in male rats after administration of the potent estrogen diethylstilbestrol (DES) at 0.01, 0.1, or 1 mg/kg/day by gavage for 14 days. DES treatment decreased hepatic α 2u -g mRNA levels in a dose-dependent manner accompanied by atrophic histopathologic changes in the testis. In addition, α 2u -g mRNA levels were lowest in animals with the most marked testicular changes. Hepatic α 2u -g mRNA may be a useful biomarker for the evaluation of endocrine disruption in male rats.

Changes in serum alpha2u-globulin levels in male rats given diethylstilbestrol and applicability to a screening test for endocrine-disrupting chemicals.

Abstractα2u-Globulin (AUG) is a major rat urinary protein, which has a molecular weight of 16 kDa (kidney type) or 19 kDa (native type). The biosynthesis of this protein is under multi-hormonal regulation. In this study, we investigated changes in serum AUG level and their association with changes in the reproductive organs of male rats after the administration of the estrogenic chemical, diethylstilbestrol (DES) at doses ranging from 0.01 mg/kg per day to 100 mg/kg per day by gavage for 14 days. Our aim was to establish basic data for the development of a new screening method for endocrine disrupting chemicals based on serum AUG levels. DES treatment decreased the weight of testes in a dose-dependent manner; and was accompanied by atrophic histopathological changes in testes. Testis weights were significantly decreased by the group given 1 mg/kg per day DES; however, histopathological abnormalities were found in the group given 0.1 mg/kg per day DES. In four of five animals in the group given 1 mg/kg per day there was no significant decrease in testis weight and only a slight or moderate degeneration of the pachytene spermatocytes. Despite these findings, serum AUG levels in this group decreased markedly, while the serum AUG level markedly decreased even in the animals with no histopathological change in the 1 mg/kg per day or 0.1 mg/kg per day groups with no histopathological change also showed decreased serum AUG level. These results suggest that the serum AUG level may be a sensitive parameter for detecting the activity of estrogenic chemicals in intact male rats. Although a uterotropic assay has been proposed for immature female or ovariectomized female rats and is currently undergoing validation studies internationally, there is no screening method for estrogenic chemicals in intact male animals. More data on AUG changes by treatment with other estrogenic chemicals are needed in order to determine the sensitivity and specificity of this response to estrogens. Nonetheless, an AUG-based screening test for estrogenic chemicals may be useful owing to its applicability to conventional toxicity studies and an apparently higher sensitivity of this parameter compared to organ weight change or histology of testis in intact male rats and applicability to conventional toxicity studies.

Note on Regulatory Toxicology Requirements for Adjuvants and Vaccines; in View of the Newly Established WHO Guidelines

1 Review Division, Pharmaceuticals and Medical Devices Agency (PMDA), Kasumigaseki 3-3-2, Chiyoda-ku, Tokyo 100-0013, Japan 2 Research and Development Department, POC Clinical Research Inc. Taishidou 4-11, Setagaya-ku, Tokyo 154-0004, Japan 3 Pathology Department, The Chemo-SeroTherapeutic Research Institute, Kyokushi Kawabe 1314-1, Kikuchi-shi, Kumamoto 869-1298, Japan 4 Vaccine Research Laboratories, Kitasato Daiichi Sankyo Vaccine Co. Ltd., Kita-Kasai 1-16-13, Edogawa-ku, Tokyo 134-8630, Japan 5 Research Division, Chugai Pharmaceutical Co. Ltd., Komakado, 1-135, Gotemba, Shizuoka 412-8513, Japan