15. Studies on Heavy Ion Radiation-induced Chromosome Aberrations on an lonizing Radiation Sensitive Mutant Mouse Cell Line by Atomic Force Microscope

Masahiro Murakami, Masako Minamihisamatsu, Koki Sato*, and Tsamu Hayata (*Kinki Univ.)

Keywords: chromosome aberration, heavy ion radiation, atomic force mtcroscope

Chromosome damage occurs following exposure of cells to ionizing radiation. The various types of chromosome aberrations, i.e. chromosome breaks, chromosome exchange, chromatid gaps, chromatid breaks etc., have been classified by light microscopy. Heavy ion radiation [classified as high linear energy transfer (LET) radiation] , which densely deposits its energy along the path of particle radiation, causes clustered DNA breaks and various types of chromosome aberration.

Recently, atomic force microscope (AFM) imaging has been applied to radiation research for biological systems. AFM imaging revealed the specific induction of small DNA fragments by neutron irradiation. Shortening of plasmid DNA after -irradiation was observed by AFM.

We applied a new method employing AFM for nanometer-level structural analysis of chromosome damage induced by heavy ion irradiation. An X-ray sensitive mutant mouse cell line, SL3-147, was exposed to heavy ion [neon (an initial energy of 400 MeV/n) and carbon ion (an initial energy of 135 MeV/n)] radiation, then the fine structures of chromosome aberrations were visualized by AFM. After irradiation, the cells were kept under 5% CO₂ at 37 C in a culture medium containing 0.05 g/ml of colcemid for 1.5h or 20h, and then collected. The cells were treated with a hypotonic solution of 0.O75M KCI, fixed with methanol-acetic acid (3: 1) and then air-dried slides were prepared. The AFM can visualize the detailed structure of chromosomes on Giemsa stained or unstained samples. The surface of unstained chromosomes was rougher than that of those stained with Giemsa. A fibrous structure was observed on the unstained chromosomes. The structure of the break point induced by neon (LET=1OOkeV/m, l.5Gy) or carbon ion (LET 1OOkeV/m, 1.5Gy) radiation was imaged by AFM. The fibrous structure was also observed in these break points. The section analysis revealed that chromosome fragments were connected by the chromatin fiber inside the chromatid gap. On the other hand, chromosomes with chromatid breaks were disconnected at the break region. These observations indicated that AFM is a useful tool for analysis of heavy ion radiation-induced chromosome a berrations.