Izuo Tobari

Chromosomal analysis in mouse eggs fertilized in vitro with sperma exposed to ultraviolet light (UV) and methyl and ethyl methanesulfonate (MMS and EMS)

Chromosome aberrations were analyzed at the first-cleavage metaphase of mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) as well as to methyl and ethyl methanesulfonate (MMS and EMS). The frequencies of chromosome aberrations markedly increased with dose of UV as well as with concentration of MMS and EMS. In the UV-irradiation group, the frequency of chromosome-type aberrations was much higher than that of chromatid-type aberrations. About 90% of chromosome aberrations observed in the eggs following MMS and EMS treatment to sperm were chromosome type in which the frequency of chromosome fragments was the highest. The effects of UV on the induction of chromosome aberrations were clearly potentiated by post-treatment incubation of fertilized eggs in the presence of Ara-C or caffeine, but the effects of MMS and EMS were not pronounced by post-treatment of Ara-C or caffeine. The results indicate a possibility that UV damage induced in mouse sperm DNA is reparable in the eggs during the period between the entry of sperm into the egg cytoplasm and the first-cleavage metaphase.

Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes.

To study the repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes, the potentiating effects of 3 well-known repair inhibitors, arabinofuranosyl cytosine (ara-C), 3-aminobenzamide (3AB) and caffeine, on the frequency of induced chromosome aberrations were examined in eggs fertilized with X-irradiated sperm or in eggs irradiated with X-rays at the mature oocyte stage immediately before fertilization. Gametic treatment, fertilization and embryo culture were carried out in vitro. Ara-C treatment was done only in the pre-DNA replication period, while treatment with 3AB and caffeine was continuous from fertilization to the first-cleavage metaphase. The induction of chromosome aberrations by exposing sperm or oocytes to X-rays was remarkably potentiated by post-treatment incubation in the presence of each of the 3 inhibitors. This result indicates the possibility that X-ray damage induced in sperm or oocytes is reparable in the fertilized eggs and that various types of repair processes are involved.

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation.

The induction of chromosome aberrations in eggs of mice fertilized with X-irradiated sperm was performed by using an in vitro fertilization technique. Capacitated mature sperm was irradiated with various doses of X-rays and cytological analysis of the first cleavage metaphase of in vitro fertilized eggs was made. The frequencies of chromosome aberrations increased exponentially with dose and the dose-response relationship for overall breaks fitted well to a quadratic equation. The chromosome aberrations were mainly chromosome-type (82.1%), and the majority of aberrations were fragments.

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.

A mouse-cell mutant sensitive to ionizing radiation is hypermutable by low doses of γ-radiation

Abstract The mutant mouse lymphoma cell M10, which is sensitive to methyl methanesulfonate and ionizing radiation, was compared with the parental L5178Y cells for mutation induction after γ-irradiation. The rate of induced mutations to 6-thioganine resistance in L5178Y cells was 2−3 x 10 −7 per R, as determined after exposure ranging from 25 to 500 R. The induced mutation frequency per unit dose per locus in M10 cells was about 4 times higher than that in L5178Y cels at the lower doses of exposure (25–75 R), but it declined sharply at the higher doses of γ-rays (100–150 R). The rate of induced mutation per unit cell killing in M10 cells was nearly the same as that in L5178Y cells when they were compared at the levels of lower cell killing.

Radiation-induced chromosome aberrations in lymphocytes from man and crab-eating monkey: The dose-response relationships at low doses

To obtain information on the relation between yield of chromosome aberrations and dose at low-dose levels, experiments were conducted with 5, 10, 20, 30 and 50 rad of 137Cs gamma-rays, on lymphocytes from man and crab-eating monkey (Macaca fascicularis). The dose-response relationship for dicentrics was obtained from the combined data of these low-dose experiments with those of our previous ones at high doses (100-400 rad). When the difference between observed yields and those expected from the linear-quadratic model were computed, the dose-response curve had a good fit for man, but not for the monkey. The linear regression lines between 0 and 30 rad were calculated, because the expected values of alpha/beta for man and monkey would be about 100 and 60 rad. The human data gave a satisfactory fit to a linear model, i.e., a linear increase in aberration frequency with dose, whereas this was not so for those of the monkey. Furthermore, there was some suggestive evidence for the existence of a plateau in dicentric yields between 10 and 30 rad for the monkey and between 20 and 30 rad for human lymphocytes, but more data would be needed to verify this suggestion, particularly for human lymphocytes.

γ-Ray-induced reciprocal translocations in spermatogonia of the crab-eating monkey (Macaca fascicularis)

Abstract The yield of translocations induced by γ-rays in the crab-eating monkey (Macaca fascicularis) spermatogonia were studied by cytological analysis in spermatocytes derived from them. The frequencies of translocations were 0.09% at 0 Gy, 1.9% at 1 Gy, 2.5% at 2 Gy and 1.3% at 3 Gy, showing a humped dose-response curve with a peak yield around 2 Gy. No remarkable inter-seasonal or inter-animal variations in the induction of translocation were observed. The frequencies in the crab-eating monkey were significantly higher than those in the same Macaca genus, the rhesus monkey (Macaca mulatta) (van Buul, 1976, 1980). This inter-species difference in radiosensitivity might be affected by the condition of spermatogonial stem cells at the time of exposure to radiation, depending on the seasonal change in spermatogenetic activity.

Characteristics of γ-ray-induced chromosomal aberrations in mutagen-sensitive mutants of L5178Y cells

We have examined the chromosomal radiosensitivities of an ionizing-radiation-and MMS-sensitive mutant (M10), and a UV- and 4NQO-sensitive mutant (Q31), isolated from mouse lymphoma L5178Y cells, with regard to killing effects. In the first mitoses after 100 R gamma-irradiations, it was found that M10 cells were highly radiosensitive in terms of chromosomal aberrations accompanying longer mitotic delay (3 h); the frequencies of both chromatid-type and chromosome-type aberrations were, respectively, about 7 and 4 times higher than that of wild-type L5178Y cells. Furthermore, chromatid exchanges, particularly triradials, isochromatid breaks with sister union, and chromatid gaps and breaks were markedly enhanced at G1 phase of M10 cells. In contrast, the chromosomal radiosensitivity of Q31 cells after 100 R irradiation was similar to that of L5178Y cells. On the other hand, spontaneous aberration frequencies (overall breaks per cell) of M10 and Q31 cells were, respectively, 5.1 and 2.2 times higher than that of wild-type L5178Y cells. The chromosomal hypersensitivity to gamma-rays in M10 cells is discussed in the light of knowledge obtained from ataxia telangiectasia cells.

Chromosome aberrations induced by tritiated water or 60Co γ-rays at early pronuclear stage in mouse eggs

The induction of chromosome aberrations in mouse eggs by exposure to HTO beta-particles and 60Co gamma-rays at the early pronuclear stage was examined at the first-cleavage metaphase by using an in vitro fertilization technique. Eggs at the pronuclear stage were exposed to beta-particles in a chemically defined medium containing tritiated water (HTO) for 2 h at 3-5 h after insemination. Other eggs at the same stage were exposed to gamma-rays from 60Co during the same period. The dose-response relationships for frequencies of chromosome aberrations per egg were fitted to a linear-quadratic model for HTO beta-particles, and to a linear model for 60Co gamma-rays. The chromosome aberrations were mainly chromosome-type, and the majority of all aberrations were fragments. RBE values of HTO beta-particles relative to 60Co gamma-rays and acute X-rays, which were estimated from the ratio of the linear regression coefficients over 0.05-Gy range, were 2.0 and 1.6, respectively.

Dose-response relations for dicentric yields in G0 lymphocytes on man and crab-eating monkey following acute and chronic γ-irradiations

A comparison has been made of dicentric yields in GO lymphocytes between man and crab-eating monkey, Macaca fascicularis, after acute and chronic gamma-irradiations. With acute irradiation (49.6 rad/min) there was no significant difference between them, but for the chronic irradiation (17.1 rad/h) a significant difference was observed between the species. When the dose-response relations were fitted to the linear-quadratic model (Y = alpha D + beta D2), the species-difference observed for chronic irradiation was almost entirely due to change in the value of beta. In addition, after chronic irradiation the beta-value for monkey was almost negligible, but that for man was significant. Post-irradiation incubation experiment showed that cells with dicentrics were partly eliminated during the course of chronic irradiation, because there were appreciable reductions of dicentric yields (ca. 25% for both man and monkey at 400 rad) together with mitotic indices (ca. 30% and 60% for man and monkey, respectively, at 400 rad). Accordingly, it would be reasonable to postulate that GO repair for dicentrics other than selection mechanism must play a major role in the effects of low dose rate. It can be further suggested that GO-repair capacity for chromosal damages leading to dicentrics may be different among different primate species.