Nobuo Egami

Life-span Studies on Spontaneous Tumor Development in the Medaka (Oryzias latipes)

A total of 961 medaka, separated chronologically from the first to the fifth year of life, were examined for spontaneous tumor development. While no liver tumors were found in either male or female medaka under the age of 1 year and the incidence in 2‐year‐old fish was relatively low (males 1.9% and females 1.7%), they became more common with advancing age. The incidence was higher in females than in males from 3 to 5 years of age, reaching 7.1% in 5‐year‐old female stock. These liver tumors included a total of 12 adenomas and 9 hepatocellular carcinomas. The hepatocellular carcinomas were histologically well differentiated and were all observed in female medaka. Spontaneous tumors occurring in organs other than the liver were rare and sporadic. Four squamous cell carcinomas, 5 melanomas and 4 lymphosarcomas were observed with no sexual or pronounced age bias being evident. The squamous cell carcinomas developed in the surface epithelium with local invasion into the dermis. Melanomas occurred in the abdominal cavity and demonstrated systemic invasion into various parts of the body. Three out of the 4 lymphosarcomas arose from the inner part of the operculum suggesting that these tumors were of thymic origin. They also showed extensive invasion. The data indicate a particular susceptibility of older female medaka to liver but not other tumor development.

Effects of Irradiation on Germ Cells and Embryonic Development in Teleosts

Publisher Summary This chapter discusses the effects of irradiation on germ cells and embryonic development in teleosts. Sterility and weight reduction of testes after irradiation are mainly because of actual cell loss, and the order of decreasing sensitivity of spermatogenic cells is from the most sensitive spermatogonia, through primary and secondary spermatocytes and spermatids, to sperm, which are quite radioresistant. Even in morphologically homogeneous populations of spermatogonia, heterogeneity of sensitivity exists. The results of local irradiation of the testes are different from those of irradiation of the ovaries. The radiosensitivity of gametogenesis in the ovary and testis cannot be directly compared, except probably from the viewpoint of genetic effects of radiation. Most studies concerning radiosensitivity of fish embryos have so far been performed for the end points of hatchability and lethality during development. This is mainly because of the simplicity of the technique. Many irradiated larvae that appear normal at hatching carry radiation damage that becomes evident at some future stage.

Photoreactivation of ultraviolet light-induced damage in cultured fish cells as revealed by increased colony forming ability and decreased content of pyrimidine dimers.

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV‐irradiated (5 J/m2) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV‐irradiated (15 J/m2) cells to radiation from white fluorescent lamps reduced the amounts of thymine‐containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV‐irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark‐repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.

Survival and Photoreactivability of Ultraviolet-Irradiated Cultured Fish Cells (CAF-MM1)

The sensitivity to ultraviolet light (uv) and photoreactivating ability of cultured fish clone cells (CAF-MM1) were investigated. Dose-survival relationship curves were obtained using the colony-forming technique at various postirradiation temperatures (33, 26, and 20/sup 0/C). At 26/sup 0/C the values of D/sub 0/, D/sub q/, and the extrapolation number (n) were 1.74 J/m/sup 2/, 2.62 J/m/sup 2/, and 4.5, respectively; no marked differences in these values were found among different temperatures. Visible light illumination after uv irradiation produced a marked increase in survival. No photoreactivation effects were observed beyond about 30 h. Caffeine increased uv sensitivity of the CAF-MM1 cells, and from the results it is suggested that the cells have some caffeine-sensitive dark repair mechanisms.

Effect of corticosteroids on the thymus of the fish Oryzias latipes.

Morphology and development of the thymus in fish Oryzias latipes was studied in response to corticosteroids. Adult fish were subjected to different concentrations of two corticosteroids, deoxycorticosterone acetate (DOCA) and cortisone acetate (CA). Histology of the thymus and thymocyte counts in treated fish were compared to normal ones. There was no change in histology of thymus due to CA treatment even those subjected to 10 mg/l water which is a lethal dose of DOCA. In contrast, the effect of DOCA at low doses (0.01 and 0.1 mg/l water) was examined and resulted in reduction of thymus volume due to depopulation of thymocytes from the entire gland. Thymus subjected to DOCA at a high dose (1 mg/l water) caused destruction of all thymocytes and reticular cells resulting in complete disappearance of the thymus. There was dose dependent mortality associated with DOCA. The maximum lethal dose was 10 mg/l causing death of all treated fish within a few hours. This information is relevant to the use of CS in fish therapy in response to stress.

Resistance of a cultured fish cell line (CAF-MM1) to. gamma. irradiation

Fish are generally more resistant to whole-body ionizing radiation than mammals. To study the radiosensitivity of fish in vitro, CAF-MM1 cells derived from the fin of the goldfish, Carassius auratus, were used. The survival parameters of CAF-MM1 obtained after ..gamma.. irradiation at 26/sup 0/C were 325 rad for D/sub o/, 975 rad for Dq, and 15 for n. No mammalian cell line with such a low sensitivity in the presence of O/sub 2/ has been reported. It was found that the large initial shoulder of the survival curve was paralleled by substantial repair of sublethal damage as evidenced by split-dose experiments. This low sensitivity to ..gamma.. irradiation did not change upon the administration of caffeine or postirradiation illumination, although these treatments were effective after uv irradiation. The decrease in the mitotic index in CAF-MM1 occurred immediately after irradiation, and it recovered within a very short time. This indicated that the duration of G2 arrest was shorter than that observed in mammalian cells. The data also suggest that the resistance of fish to whole-body irradiation is attributable to resistance at the cellular level.

Age related changes in morphology of the thymus of the fish, Oryzias latipes.

The thymus of the teleost fish Oryzias latipes is a paired structure found at the dorsoposterior part of the gill chamber. In 3-month-old fish, the thymus shows a great development. The thymus displays atrophy during aging, and the thymus involution continues until 5 years of age. Male thymus shows heavier involution than female thymus of the same age. Emigration of thymus cells takes place at all ages but increases with age.

Absence of systematic polyploidization of hepatocyte nuclei during the ageing process of the male medaka, Oryzias latipes.

Abstract The DNA contents of hepatocyte nuclei of young, medium and old male fish, Oryzias latipes, were measured by the Feulgen-DNA cytofluorometric method to examine the age-changes in DNA content-based ploidy classes. Since the erythrocytes, which coexist with the hepatocytes in smear preparations of the liver, are nucleated in fish, these were excluded from the measurement by their cytoplasmic yellow hemoglobin fluorescence on excitation with 405 nm light. In all age-groups studied the distribution of nuclear DNA contents was rather broad, but no systematic age-associated polyploidization could be found in the fish hepatocyte nuclei. The present results, which are in contrast with those of mouse hepatocyte nuclei, are discussed from a comparative point of view.

The Unique Responses of the Primordial Germ Cells in the Fish Oryzias Latipes to Gamma-rays

The effects of gamma-radiation on the development of the primordial germ cells (PGCs) of medaka embryos (Oryzias latipes) during the early stages of development were quantitatively examined and compared to the effects on the intestinal cells. The PGCs develop in three stages: an extra-gonadal proliferative stage (1-2.5 days after fertilization), a mitotically inactive stage after the termination of the migration into the gonad (2.5-4.5 days), and an extensive proliferative stage (between 4.5 days and hatching). A dose-rate effect was absent in the PGCs, regardless of their mitotic activity, when dose rates were 2.5 and 0.14 Gy/min. The radiation effect on the PGCs was not reduced by hypoxia and was not enhanced by heat treatment during the proliferating stages. Conversely, radiation resistance was induced in the PCGs during the mitotocally inactive stage by hypoxia and, unexpectedly, by heat treatment. From the present data, we conclude that the PGCs have a small repair ability, and we discuss the radiation resistance induced in the PGCs by hypoxia and heat treatment.

Effects of γ-irradiation on the rejection of transplanted scale melanophores in the teleost, Oryziaslatipes

The effects of γ-irradiation on allograft rejection in the teleost, Oryziaslatipes, were examined at 25°C. The survival of melanophores in the transplanted scale was observed as an index of rejection. Allografts were rejected in non-irradiated fish within 7 days. In the γ-irradiated recipients (2kR), the grafts were rejected more slowly, but still within 20 days. The γ-ray effects, however, disappeared almost completely within 25 days after the irradiation. If the same recipient again received transplants, the secondary response occurred clearly and the melanophores were rejected very rapidly. The secondary response was suppressed by γ-rays if the fish was irradiated just before the second transplantation. Immunologic memory against the first transplants disappeared within 30 days, a period shorter than that of mammals.