Frequency and size of micronuclei induced in gill cells of medaka fish (Oryzias latipes) after whole-body exposure to clastogenic chemicals.

Medaka fish (Oryzias latipes) were whole-bodily treated with various doses of mitomycin C (MMC), ethylmethanesulfonate (EMS), cyclophosphamide (CP), diethylnitrosamine (DEN), or colchicine (COL) for 24 h, and the frequency of micronucleated cells (MNCs) was measured in the gills at 24 and 48 h after treatment. In the present experiments, MMC, CP, and DEN were recorded as efficient inducers of micronuclei at both sampling times, and none of the MNC frequencies recorded with these agents at 24 h significantly exceeded the corresponding frequency at 48 h. For EMS and COL, positive responses were recorded only 48 h after treatment. By comparison with the time-course data reported for radiation-induced MNCs in the same MN assay system, the clear responses observed at the 48-h time point for all the chemicals used were regarded as evidence of their delayed effects on micronucleus (MN) formation. The mean sizes of micronuclei induced after exposure to COL was significantly larger by a factor 2 as compared with that induced by X-irradiation, whereas those determined for the other four chemicals were almost equal to that induced by X-irradiation. These results demonstrate that the medaka gill-cell MN assay can detect chemically-induced chromosome damage, either directly or after metabolic activation, and spindle malfunction, and provide a basis for further development of the present assay system for testing cytogenetic activities of chemical agents.

Attempt at cloning high-quality goldfish breed 'Ranchu' by fin-cultured cell nuclear transplantation.

The viability of ornamental fish culture relies on the maintenance of high-quality breeds. To improve the profitability of culture operations we attempted to produce cloned fish from the somatic nucleus of the high-quality Japanese goldfish (Carassius auratus auratus) breed 'Ranchu'. We transplanted the nucleus of a cultured fin-cell from an adult Ranchu into the non-enucleated egg of the original goldfish breed 'Wakin'. Of the 2323 eggs we treated, 802 underwent cleavage, 321 reached the blastula stage, and 51 reached the gastrula stage. Two of the gastrulas developed until the hatching stage. A considerable number of nuclear transplants retained only the donor nucleus. Some of these had only a 2n nucleus derived from the same donor fish. Our results provide insights into the process of somatic cell nuclear transplantation in teleosts, and the cloning of Ranchu.

Multiple sex chromosome system of X1X1X2X2/X1X2)Y type in lutjanid fish, Lutjanus quinquelineatus (Perciformes).

The karyotype and other chromosomal markers as revealed by C-banding and Ag-staining were studied in Lutjanus quinquelineatus and L. kasmira (Lutjanidae, Perciformes). While in latter species, the karyotype was invariably composed of 48 acrocentric chromosomes in both sexes, in L. quinquelineatus the female karyotype had exclusively 48 acrocentric chromosomes (2n = 48) but that of the male consisted of one large metacentric and 46 acrocentric chromosomes (2n = 47). The chromosomes in the first meiotic division in males showed 22 bivalents and one trivalent, which was formed by an end-to-end association and a chiasmatic association. Multiple sex chromosome system of X(1)X(1)X(2)X(2)/X(1)X(2)Y type resulting from single Robertsonian fusion between the original Y chromosome and an autosome was hypothesized to produce neo-Y sex chromosome. The multiple sex chromosome system of L. quinquelineatus appears to be at the early stage of the differentiation. The positive C-banded heterochromatin was situated exclusively in centromeric regions of all chromosomes in both species. Similarly, nucleolus organizer region sites were identified in the pericentromeric region of one middle-sized pair of chromosomes in both species. The cellular DNA contents were the same (3.3 pg) between the sexes and among this species and related species.

Relative biological effectiveness of fission neutrons for induction of micronucleus formation in mouse reticulocytes in vivo.

Following whole-body irradiation of ICR mice with various doses of fission neutrons or X-rays, the frequency of micronuclei (MNs) in peripheral blood reticulocytes was measured at 12 h intervals beginning immediately after irradiation and ending at 72 h after irradiation. The resulting time-course curve of MN frequency had a clear peak 36 h after irradiation, irrespective of the type of radiation applied and the dose used. The MN frequency, averaged as the unweighted mean over the experimental time course, showed a linear increase with increasing dose of either fission neutrons or X-rays. The linear response to X-rays supports reported conclusion that induction of MN formation in reticulocytes is a dose-rate independent phenomenon. The relative biological effectiveness (RBE) of fission neutrons to X-rays for MN induction was estimated to be 1.9 +/- 0.3. This value is considerably lower than the RBE value of 4.6 +/- 0.5 reported for the same fission neutrons for induction of lymphocyte apoptosis in the thymus of ICR mice that represents dose-rate independent, one-track event. Based on these results, we propose that MNs increased in reticulocytes after irradiation mostly represent acentric fragments caused by single chromosome breaks, and that some confounding factor is operating in erythroblasts for the formation of aberrations from non-rejoining DNA double-strand breaks more severely after high-LET radiation than after low-LET radiation.

Dose- and time-dependent responses for micronucleus induction by X-rays and fast neutrons in gill cells of medaka (Oryzias latipes).

Medaka fish (Oryzias latipes) were exposed to various doses of X-rays or fast neutrons, and the frequency of micronucleated cells (MNCs) was measured in gills sampled at 12- or 24-hr intervals from 12 to 96 hr after exposure. The resulting time course of MNC frequency was biphasic, with a clear peak 24 hr after exposure, irrespective of the kind of radiation applied and the dose used. The half-life of MNCs induced in the gill tissues by the two exposures fluctuated around 28 hr, with no significant dose-dependent trend for either X-ray- or neutron-exposed fish. As assayed 24 hr after exposure, the MNC frequency increased linearly over the control level with increasing doses of both X-rays and fast neutrons. The relative biological effectiveness (RBE) of fast neutrons to X-rays for MNC induction was estimated to be 4.3 +/- 0.6. This value is close to the RBE value of 5.1 +/- 0.3 reported for fast neutron induction of somatic crossing-over mutations in Drosophila melanogaster that arise from recombination repair of DNA double-strand breaks. These results and other data support our conclusion that the medaka gill cell micronucleus assay is a reliable short-term test for detecting potential inducers of DNA double-strand breaks.

Susceptibility of male and female medaka (Oryzias latipes) fish to spontaneous and X-ray induced micronucleus formation in gill cells.

The frequency of micronucleated cells (MNCs) was measured in acridine-orange (AO) stained RNA-rich gill cells from male and female medaka (Oryzias latipes) fish of known body weight. Spontaneous MNC frequencies were not significantly correlated with body weight, despite the fact that the heaviest of the 30 fish used outweighed the lightest by a factor of 3. Average MNC frequencies were identical in males and females at 0.8 per thousand. An X-ray dose of 4 Gy increased the frequency of MNCs over the spontaneous level in all 30 of the fish used, reaching a level of 7.2 per thousand on average when assayed 24 h after exposure. In X-ray treated fish, MNC frequency and body weight were not significantly correlated, nor was there any difference between the sexes. These and other results support our primary conclusion that AO-staining is suitable for the medaka micronucleus assay in gill cells, and indicate that male and female medaka fish are similarly and size-independently susceptible to both spontaneous and X-ray induced micronucleus formation in gill cells.