Masanobu Kaido

Dose dependent effects of inhaled ethylene oxide on spermatogenesis in rats.

Male Wistar rats were exposed to ethylene oxide (EO) at concentrations of 50, 100, or 250 ppm for six hours a day, on five days a week for 13 weeks. Dose effect relations of inhaled EO on spermatogenesis were evaluated from testicular and epididymal weights, histopathological changes and lactate dehydrogenase X (LDH X) activity in the testis, and sperm counts and sperm head abnormalities in the epididymis. At 250 ppm, a decrease in epididymal weights, slight degenerations in the seminiferous tubules, decreased sperm counts, and increased numbers of abnormal sperm heads in the tail of the epididymis were found; these were not seen at lower doses. When the abnormal sperm heads were classified into immature types and teratic types, the number of immature heads increased only at 250 ppm. On the other hand, the teratic type had increased at doses of 50 and 100 ppm EO when compared with the control group. Hence, subchronic inhalation of EO at low concentrations affects spermatogenesis in rats.

Testicular toxicity and alterations of glutathione metabolism resulting from chronic inhalation of ethylene oxide in rats

Wistar male rats were exposed to ethylene oxide (EtO) at a concentration of 500 ppm, 6 hr a day, 3 days a week, for 2, 4, 6, or 13 weeks. Testicular toxicity and changes in glutathione metabolism in the testis were investigated. The relative weights of the testes and the epididymes of the EtO-exposed group decreased in a time-dependent manner. Light microscopic examination revealed degeneration and exfoliation of germ cells. Although the severity of damage became apparent over the course of exposure, some seminiferous tubules showed germ cell recovery at 13 weeks compared with 6 weeks. There was no alteration in plasma testosterone concentration. Glutathione reductase (GR) activity decreased during the entire examination period, and recovery from the decrease was not achieved by addition of flavin adenine dinucleotide (FAD). On the other hand, glutathione peroxidase (GPx) activity decreased at 2 weeks, and then increased at 6 and 13 weeks. In spite of alterations in the glutathione redox cycle, the level of reduced glutathione (GSH) in the testes was not affected. Glutathione S-transferase activity, measured with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, increased at 6 and 13 weeks and, measured with 1,2-epoxy-3-(p-nitrophenoxy)propane, increased at 4, 6, and 13 weeks. These data indicate that chronic inhalation of EtO induces testicular atrophy. Alterations in the glutathione redox cycle and glutathione S-transferase activity might play important roles in the toxicity and the detoxifying mechanism of the testis.

Preventive effects of methylcobalamin on the testicular damage induced by ethylene oxide

In this study, the effects of methylcobalamin on testicular damage induced by ethylene oxide (EtO) were studied. When Wistar male rats inhaled EtO at 500 ppm, 6 h a day, 3 days a week, for 6 weeks, testicular damage was observed histopathologically and by some other parameters. Subcutaneous injection of methylcobalamin at 500 μg/kg, 5 times/week was found to ameliorate the damage. However, the degree of the methylcobalamin effect differed among the parameters examined in this study. Decrease in testicular weight due to EtO exposure was completely prevented by methylcobalamin, and decrease in testicular mature spermatid count and LDH-X activity was fairly well prevented. The degree of prevention of alteration in the epididymis, such as epididymal weight, epididymal sperm count and sperm abnormality rate, was significant but not complete. EtO caused apparent alterations in glutathione metabolism in the testes, but methylcobalamin did not affect such alterations induced by EtO. From these results, it has been determined that methylcobalamin has definite preventive effects on testicular toxicity of EtO.

Effects of megadoses of pyridoxine on spermatogenesis and male reproductive organs in rats

Although it has been indicated that many neurotoxicants also cause reproductive toxicity, the reproductive toxicity of megadoses of pyridoxine, which is a neurotoxicant, has not been studied. In this paper, we studied the effects of megadoses of pyridoxine on male reproductive organs. Pyridoxine hydrochloride, 125 mg/kg, 250 mg/kg, 500 mg/kg or 1000 mg/kg, daily, was intraperitoneally injected into Wistar male rats 5 days a week for 2 or 6 weeks, and its effects on the male reproductive organs were investigated. After 2 weeks of administration, absolute weights of the testis in the 500 and 1000 mg/kg epididymis in all the exposed groups and prostate gland in the 1000 mg/kg group decreased, and mature spermatid counts in the testis decreased in the 1000 mg/kg group. After 6 weeks administration, the absolute and relative weights of the testis, epididymis, prostate gland and seminal vesicle decreased in the 500 mg/kg and 1000 mg/kg groups, and mature spermatid counts in the testis and sperm counts in the epididymis decreased in these groups. Among the marker enzymes of the testicular cells, LDH-X activity decreased, and β-glucuronidase activity, cytochrome P-450 content and cytochrome b5 content increased in the 1000 mg/kg group. Plasma testosterone concentration did not significantly alter in all the exposed groups. From these results, it was concluded that megadoses of pyridoxine affected the spermatogenesis and decreased reproductive organ weights in the rat.

Diffuse Pleural Rhabdomyosarcoma with Persistent Pleural Effusion

A unique case of embryonal rhabdomyosarcoma arising at the left pleura of a 7‐year‐old Japanese girl is reported. The present case was characterized by persistent pleural effusion, and the malignant cells incidentally found in it were the first diagnostic clue. The tumor showed a rare growth pattern involving diffuse thickening of the parietal pleura. Biopsy of the thickened parietal pleura upon thoracotomy revealed embryonal rhabdomyosarcoma largely composed of immature mesenchymal cells. Immunohistochemical demonstration of creatinine phos‐phokinase MM was most helpful among several types of immunostain for the histopathological diagnosis. Ultra‐structurally, thin filaments with primitive Z bands could be seen in some tumor cells. Intensive clinical examination revealed only diffuse thickening of the parietal pleura, which was reduced by chemotherapy. This is the first documented case of rhabdomyosarcoma arising at the pleura. Previous reports of rhabdomyosarcoma arising at unusual sites are reviewed and the histogenesis of this tumor is briefly discussed. Acta Pathol Jpn 39: 803 809, 1989.

Testicular damage by high doses of vitamin B6 (pyridoxine) in rats: A light and electron microscopical study

Male Wistar rats were administered daily intraperitoneal injections of 125, 250, 500, and 1000 mg/kg/day of vitamin B6 for 2 and 6 weeks and the histogenesis of the testicular damage was investigated. A reduction of germ cells was not prominent in the 2-week groups, whereas a delay in spermiation, degeneration of elongated spermatids, and Sertoli cell alterations were observed in the 500- and 1000-mg groups, although generally, these were relatively mild. Ectoplasmic specializations (ES), tubulobulbar complexes, and endoplasmic reticulum (ER) in the apical processes of Sertoli cells were irregularly arranged and their disappearance was also retarded. The Sertoli cell cytoplasm was often retracted and condensed. In the 6-week groups, no histological change in the testis was noted with the 125-mg dose. The retardation in spermiation and Sertoli cell alterations similar to those in the 500-mg dose 2-week group were observed in the 250-mg group. In the 500- and 1000-mg groups, germ cells were generally degenerated and markedly reduced in number. Multinucleate germ cells were mingled with anisocytotic germ cells, and openings of intercellular bridges were occasionally found. Sertoli cells also showed more severe alterations, such as focal disappearance of ES in earlier than ordinary stages, marked dilation of the ER, and markedly condensed or electron-lucent cytoplasm. These results suggest that the Sertoli cell damage may induce diverse germ cell degeneration in which retardation of spermiation occurs first.

Testicular Damage Caused by Inhalation of Ethylene Oxide in Rats: Light and Electron Microscopic Studies

Although testicular damage caused by ethylene oxide vapor (EtO) has been previously reported, the morphological changes occurring in seminiferous tubules remain unclear. We examined the time course of the testicular lesion induced by EtO in order to clarify its morphogenesis. Wistar rats were exposed to 500 ppm EtO for 6 hr per day, 3 times per week for 2, 4, 6, or 13 weeks through inhalation. In the 2-week exposure group, Sertoli cells often showed condensation and retraction of the cytoplasm, and dilatation of the endoplasmic reticulum (ER). In apical Sertoli cells, processes which encapsulated the heads of elongate spermatids, ectoplasmic specializations, and tubulobulbar complexes were often deformed and many elongate spermatids were degenerated. In the 4- and 6-week exposure groups, many degenerated Sertoli cells were present, and deformed germ cells, sometimes with multinucleation, appeared to make direct contact with each other without interlocation of Sertoli cell lateral processes. A few scattered immature Sertoli cells were evident in the 6-week exposure group. In the 13-week exposure group, seminiferous tubules containing almost all types of germ cells reappeared, mixed with atrophic tubules containing Sertoli cells only. In the former tubules, Sertoli cells often possessed regularly regenerated lateral processes, which were interposed between germ cells. These results indicate that the germ cell damage may be associated with damage to Sertoli cells. In spite of the intermittent exposure, focal regeneration of Sertoli cells appeared after 6 weeks of exposure to EtO and preceded patchy recovery of germ cells. Therefore, the data suggest that Sertoli cell regeneration may permit regeneration of germ cells.

Changes in Spermatozoa due to Large Doses of Pyridoxine (Vitamin B6)

To investigate the changes of spermatozoa by high doses of vitamin B6, (B6), the alterations in spermatozoa and testis of rats after the administration of high doses of B6 were evaluated quantitatively and morphometrically. Wistar rats of 11 weeks of age were intraperitoneally injected with 63,125,250 and 500 mg/kg of B6 daily 5 times per week for 6 weeks. Using the spermatozoa taken from the epididymis and ductus deferens, the number, motility and nuclear morphology of spermatozoa were examined. After preparing 7 parameters for the nuclear morphology, the morphometry was performed by an IBAS version 2 (Zeiss) image analysis system. The number of spermatocytes and spermatids in representative stages of spermatogenesis was counted per Sertoli cell histologically. Mild deformation of spermatozoa nuclei occurred in the 63 mg or more exposure groups. In the 125 and 250 mg groups, the decrease in number as well as motility of spermatozoa together with slight decrease of spermatids in late maturation phase (mature spermatids) and the delay in spermia‐tion appeared. Phagocytosis of mature spermatids by Sertoli cells was clearly increased in the 250 mg group. The alteration and the decreased number of spermatozoa are suggested to have mainly resulted from alteration of mature spermatids and the increased phagocytosis of mature spermatids by Sertoli cells. Computer‐assisted morphometry of spermatozoa nuclei was useful not only to evaluate morphological changes objectively but also to discern them early. Acta Pathol Jpn 42: 861–869, 1992.

[Evaluation of testicular damage by flow cytometry: testicular atrophy caused by ethylene oxide].

The new method using flow cytometry was applied to analyse the testicular toxicity of ethylene oxide, and the usefulness of this method is discussed. When Wistar male rats were exposed to ethylene oxide for six hours a day, three times a week for six weeks, the testicular weights of the exposed group significantly decreased. When the cells of these testes were stained by propidium iodide and analysed by flow cytometry, four peaks which corresponded to maturation phase spermatids (less than C), the other haploid cells (C), diploid cells (2C) and tetraploid cells (4C) were obtained. Calculating the ratio of the percentage of less than C, C and 4C to that of 2C, the ratio of less than C of the exposed group decreased by 72.9%, 2C by 53.5% and 4C by 5.1% when compared with the control group. As these changes were almost consistent with that of histopathological examinations, we are able to conclude that more mature germ cells were affected by ethylene oxide. This method by flow cytometry is thought to be objective, quantitative and convenient to evaluate testicular damage by chemicals.