Effect of amifostine (Ethyol®) on the development of extraembryonic blood vessels in chick embryos

Aim

Systemic and local side effects can limit radioas well as chemotherapy in patients suffering from neoplastic discaccs One possibility to reduce the therapy-dependent side effects is to attenuate radical induced alterations of normal healthy tissue by application of antioxidants. Preclinical and clinical studies demonstrated the ability of amifostine to protect normal, but not neoplastic, tissues from cytotoxic effects of chemotherapy or irradiation. The purpose of the present study was to establish whether amifostine (Ethyol®) can affect microvessel density in vivo.

Material and Methods

For this study fertilized crossbred ldWhite-Plymouth-Rocks x Sussexrd eggs were used. After 48 hours of incubation 0.05 ml solution containing 25.7 μg (approximately 120 μM) amifostine were injected next to the germ disc. Taking into account the mean surface area of the area vasculosa and the embryo, this corresponds to a dose of 26 μg/cm². As controls, the area vasculosa of eggs treated with 0.05 ml NaCl 0.9% were used. Twenty-four and 48 hours after injection of amifostine or NaCl photographs and video microscopic pictures from treated areas and controls were taken and evaluated for vascular density. Results of vascular density are given as vascular intersections per mm² (VIS/mm²).

Results

There was a significant (p < 0.001) difference in vascular density with a mean microvessel count of 30.40 (± 12.84 SD) VIS/mm² in the NaCl control and 53.69 (±24.56 SD) VIS/mm² in the amifostine-treated area vasculosa.

Conclusion

The results show that amifostine induced an increase in vascular density in the rapidly proliferating area vasculosa of the early chick embryo.     

Structural chromosomal aberrations,aneuploidy, and mosaicism in early cleavage mouse embryos derived from spermatozoa exposed to γ-rays

Purpose:?To quantitatively and qualitatively investigate the changes in chromosomal aberrations during early cleavage in mouse embryos derived from γ-irradiated spermatozoa.

Materials and methods:?Mature males were exposed to 2?Gy or 4?Gy of ¹³⁷Cs γ-rays, and their spermatozoa were used to produce embryos via in vitro fertilisation (IVF). The metaphase chromosomes were prepared from one-cell, two-cell, and four-cell embryos. In the chromosome preparations from two-cell and four-cell embryos, the separation of the sister blastomeres was precluded by treatment of the embryos with concanavalin A. The incidence of embryos with structural chromosomal aberrations, aneuploidy, or mosaicism was estimated. The fates of the different types of γ-ray-induced structural chromosomal aberrations were also investigated in those embryos.

Results:?The exposure of spermatozoa to 2?Gy or 4?Gy γ-rays caused structural chromosomal aberrations in 25.9% and 35.7% of the resultant one-cell embryos, respectively. At two-cell embryonic stage, the incidence of structural chromosomal aberrations was 17.4% in the 2?Gy group and 27.1% in the 4?Gy group. At the four-cell embryonic stage, although the incidence of control embryos with structural chromosomal aberrations was considerably high, the net incidence of embryos with radiation-induced structural chromosomal aberrations was similar to that at the one-cell stage. The incidence of aneuploidy was high in two-cell and four-cell embryos after both doses of γ-rays. The incidence of mosaicism increased significantly in dose- and embryonic-stage-dependent manners. Anaphase lag, and the degeneration and non-disjunction of the aberrant chromosomes were frequently observed in aneuploid and mosaic embryos.

Conclusions:?Mouse sperm DNA is highly vulnerable to γ-rays. The structural chromosomal aberrations of sperm origin are unstable in their behaviour and structure during cleavage, and therefore cause secondary aneuploidy and mosaicism in the early cleavage embryos.     

Numerical chromosome abnormalities in 8-cell embryos generated from γ-irradiated male mice in the absence and presence of vitamin E

Purpose:?To investigate the effects of γ-rays on male NMRI mice, in the absence or presence of vitamin E, on abnormalities in chromosome number in 8-cell embryos generated after mating with non-irradiated female mice.

Materials and methods:?The 8 – 11 week old male NMRI mice were irradiated whole body with 4 Gy of γ-rays alone or in combination with 200 international units (IU)/kg vitamin E administered 1 h prior to irradiation. After 4 days, they were mated at weekly intervals with superovulated, non-irradiated female mice in successive 6 weekly periods. About 68 h post coitous (p.c.), 8-cell embryos were fixed on slides using standard methods in order to screen for abnormalities in chromosome number.

Results:?In control embryos, 8% of metaphases were aneuploid whereas in embryos generated from irradiated mice, the frequency of aneuploidy increased dramatically at all post irradiation sampling times (p < 0.001). Administration of vitamin E one hour before irradiation, significantly decreased chromosomal aberrations in all 6 groups (p < 0.05).

Conclusion:?Data indicate that γ-irradiation affects spermatogenesis and causes DNA alterations in sperm that may lead to chromosome abnormalities in subsequent embryos. Administration of vitamin E before irradiation effectively reduced the frequency of chromosomal abnormalities. The mechanism(s) by which vitamin E reduces genotoxic effects of radiation could be via radical scavenging or antioxidative effects.