A step-by-step approach for assessing acute oral toxicity without animal testing for additives of quasi-drugs and cosmetic ingredients.

Animal testing of cosmetic ingredients and products has been banned in the European Union since 2013. However, in Japan, the application of new quasi-drugs requires the generation of data on acute oral toxicity through animal testing. A weight of evidence approach for assessing oral toxicity was challenged. This approach used a combination of safety data, including a neutral red uptake cytotoxicity assay using BALB/c3T3 cells (3T3-NRU cytotoxicity assay), which can assess the acute oral toxicity of quasi-drugs or cosmetic ingredients. We conclude that the step-by-step approach can be used to assess test substances that cause low acute oral toxicity, such as the median lethal dose (LD 50) > 2000 mg/kg, thereby avoiding animal testing.

Successful treatment of G-CSF-related aortitis with prednisolone during preoperative chemotherapy for breast cancer: a case report.

BACKGROUND: With the introduction of dose-dense therapy, the use of primary pegfilgrastim (PEG-G) has been increasing in breast cancer treatment. A rare side effect of PEG-G is aortitis. We describe a case of PEG-G-induced aortitis. CASE PRESENTATION: The patient was a 43-year-old woman with stage IIA breast cancer. Due to the subtype of triple-negative breast cancer, preoperative dose-dense epirubicin-cyclophosphamide chemotherapy was started. PEG-G was administered on day 3 after the first cycle of epirubicin-cyclophosphamide chemotherapy. On day 11, she had a fever (39.4 °C) and an elevated C-reactive protein level (27.1 mg/dL). Emergency computed tomography revealed diffused wall thickening of the aortic arch without any other signs of infection. Despite administering antibiotics, her general condition and laboratory findings deteriorated until day 18. Based on these observations, she was diagnosed with PEG-G-induced aortitis. Antibiotics were discontinued, and she was treated with prednisolone thereafter. Subsequently, her clinical symptoms and laboratory findings improved around day 39. A second computed tomography scan revealed a decrease in the aortic arch wall thickening, and she was discharged on day 43. CONCLUSIONS: We successfully treated PEG-G-induced aortitis using prednisolone. Although this side effect is rare, cancer patients receiving PEG-G for chemotherapy should be monitored for aortic inflammation.

Retarded differentiation of Leydig cells and increased apoptosis of germ cells in the initial round of spermatogenesis of rats with lethal dwarf and epilepsy (lde/lde) phenotypes.

The lde/lde rats show a severe dwarf phenotype with early postnatal lethality and a high incidence of epileptic seizure. Seizures are first detected in this model between 16 and 63 days of age, and mostly begin as wild running and progress to generalized tonic-clonic convulsions. Because our histological examination detected many extracellular vacuoles in the hippocampus and amygdaloid bodies of these animals at 28 days of age, these pathological alterations may be related to the epileptogenesis in lde/lde rats. In addition to these defects, male lde/lde rats have apparently smaller testes with reduced number of germ cells and poorly matured adult-type Leydig cells in comparison with wild-type controls. In the present study, we performed anatomical, histological, and endocrinologic examinations to characterize the testicular phenotype of lde/lde rats at 21, 28, 35, and 56 days of age. Male lde/lde rats showed severely retarded growth of the testes and accessory sex organs. Their seminiferous tubules were significantly smaller and contained markedly fewer germ cells at all time points examined as compared with controls. Significantly fewer Sertoli cells at 21 and 28 days of age, markedly decreased spermatocyte number at 28 days of age, and delayed appearance of spermatids at 56 days of age were observed in the testes of lde/lde rats. More TUNEL (T&T-mediated duTP-biotin nick-end labeling)-positive cells were detected in lde/lde seminiferous tubules, and the largest number of apoptotic cells was recorded at 28 days of age. The increases in 3beta-hydroxysteroid dehydrogenase-positive adult-type Leydig cells and 11beta-hydroxysteroid dehydrogenase-positive mature adult-type Leydig cells were also severely retarded in the testes of lde/lde rats. Consistent with these defects, significantly lower plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were detected in lde/lde males at 28 days of age, and weak immunostaining for FSH and smaller cytoplasm of LH-positive cells were detected in the anterior pituitary lobes of lde/lde males. Despite a normal level of plasma LH after 35 days of age, a significantly lower level of plasma testosterone was detected at 56 days of age. These results indicate that the normal lde allele is related to prepubertal elevations of gonadotropins and normal development of adult-type Leydig cells. Because lde/lde rats experience epileptic seizures during the period when the hypothalamus-pituitary-testicular axis is established, lde/lde rats would be useful as a model for reproductive disorder with pediatric epilepsy.

Embryonic pathogenesis of hypogonadism and renal hypoplasia in hgn/hgn rats characterized by male sterility, reduced female fertility and progressive renal insufficiency.

Congenital hypoplasia and dysplasia affect the postnatal development of organs, their physiological functioning in adulthood and the incidence of related diseases at an advanced age. Hypogonadic (hgn/hgn) rats are characterized by male sterility, reduced female fertility, progressive renal insufficiency and growth retardation, all controlled by a single recessive allele (hgn) located on chromosome 10. Since our previous studies indicated that the hypoplasia (dysplasia) of the affected organs was present at birth, we examined the embryonic pathogenesis. We mated hgn/hgn females to Brown Norway males and backcrossed F(1) males to hgn/hgn females. The resulting N(1) fetuses were genotyped using a hgn-linked microsatellite. Both sexes of hgn/hgn fetuses showed low body weight after embryonic day (ED) 15.5 and renal hypoplasia after ED 17.5. Their kidneys contained a reduced number of nephrons in a poorly formed nephrogenic zone and renal cortex. The hgn/hgn ovaries contained a small number of oogonia at ED 15.5 and oocytes after ED 17.5. Testicular growth defects were obvious after ED 17.5, and reduced numbers of Sertoli cells were detected at ED 19.5 and 21.5. The seminiferous cords in hgn/hgn testes contained more apoptotic and mitotic cells than those in +/hgn testes. These findings suggest that the phenotypes described in adult hgn/hgn rats result from embryonic hypogenesis, which continues to early postnatal stage and causes a reduction in functional tissues and cells. Since hgn/hgn rats have an insertion mutation in the microtubule-associated protein Spag5 gene, the embryonic hypogenesis described in hgn/hgn rats might result from defective cell proliferation.

Reduced mitotic activity and increased apoptosis of fetal sertoli cells in rat hypogonadic (hgn/hgn) testes.

Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes.

Duplicated insertion mutation in the microtubule-associated protein Spag5 (astrin/MAP126) and defective proliferation of immature Sertoli cells in rat hypogonadic (hgn/hgn) testes.

Male rats with hypogonadism (hgn/hgn) experience sterility from testicular dysplasia, which is controlled by a single recessive gene, hgn. The postnatal growth of the seminiferous tubules was severely affected. In this study, we localized the hgn locus to a 320 kb region on rat chromosome 10 and detected the insertion of a 25 bp duplication into the sixth exon of the sperm-associated antigen 5 (Spag5/astrin/MAP126) gene, which codes for a microtubule-associated protein. This mutation results in a truncated Spag5 protein lacking the primary spindle-targeting domain at the C terminus. Immunological staining with antibodies to markers for Sertoli and germ cells during the early postnatal period indicated that the abnormal mitosis with dispersed chromosomes in hgn/hgn testes occurs in proliferating Sertoli cells. Therefore, apoptotic Sertoli cell death would result from the disorganization of the spindle apparatus caused by defective Spag5. These findings suggested that the Spag5 is essential for testis development in rats and that the hgn/hgn rat is a unique animal model for studying the function of Spag5.

Apoptotic Sertoli cell death in hypogonadic (hgn/hgn) rat testes during early postnatal development.

AIM: To determine the involvement of apoptotic cell death in postnatal pathogenesis in mutant strain of hypogonadic (hgn/hgn) rats testes. We evaluated the numbers and types of cells undergoing apoptotic cell death. METHODS: Tissue sections were stained by the TUNEL method for in situ detection of apoptotic cells, with specific antibodies used as markers of testicular somatic and germ cells. RESULTS: We found that apoptosis in the hgn/hgn testes during the early postnatal period occurred primarily in Sertoli cells, which should actively proliferate during this stage of differentiation. These findings strongly suggest that the normal allele of hgn is involved in the direct or indirect control of differentiation and proliferation of Sertoli cells. CONCLUSION: To our knowledge, this is the first report demonstrating early postnatal apoptosis of Sertoli cells, suggesting that the hgn/hgn rat is a unique model for the study of Sertoli cell deficiency.

Influence of the hypogonadism (hgn) locus on female reproduction and ovarian development in an altered genetic background.

Background and Aims: The hypogonadic rat (hgn/hgn) shows male sterility controlled by a single recessive gene hgn. The hgn/hgn females detected by the presence of renal hypoplasia in the HGN inbred strain show a reduced fertility. Recently, the gene responsible for male hypogonadism was mapped on chromosome 10 by a linkage analysis using only male backcross progeny. Because backcross females could not be categorized as affected or normal because of variations in their renal sizes, we could not examine female fertility in the backcross progeny. In the present experiment, we examined whether the gene mapped on rat chromosome 10 has any influences on female reproduction and ovarian development. Methods: The assumptive hgn/hgn females were identified in the backcross progeny by microsatellite markers closely linked to the hgn locus. Postnatal body growth, the weights of reproductive organs, estrus cycle and ovarian histology were examined. In addition, backcross embryos were genotyped, and their body growth and ovarian development was examined. Results: The hgn/hgn females showed growth retardation, a short reproductive life and an abnormal ovarian histology as adults. Regarding embryonic development, hgn/hgn females showed body growth retardation and ovarian hypoplasia. Conclusion: The mutation of the hgn mapped on chromosome 10 causes not only male sterility but also female reduced fertility related to ovarian hypoplasia beyond the altered genetic background. (Reprod Med Biol 2006; 5: 227-234).

Dysplastic development of seminiferous tubules and interstitial tissue in rat hypogonadic (hgn/hgn) testes.

The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.