UV-A damage to DNA causes genetic instability of non-irradiated normal
chromosomes
A. Urushibara1), S. Kodama2), A. Yokoya1)
1): Research Gr. for Radiation and Biomolecular Science, JAEA 2): Osaka Prefecture Univ.

Ionizing radiation induces a variety of DNA lesions such as
chemical modifications or bonding scission. These lesions arethought to be strongly relevant to radiobiological effects, suchas mutation induction or cell death. Even when the irradiatedcell survive, the progeny of them after a number of cell division
is found to frequently undergo chromosome aberrations, socalled genetic instability. The instability is likely caused by themalfunction of the cellular activities that maintain genome innormal status. Thus it might play a crucial role in radiationinduced carcinogenesis. Although many studies have been
focused the genetic instability with regard to preventing cancer,
the underlying mechanism still remains unclear. Recently,
localized lesions, so called clustered DNA damage site,
consisting of two of more oxidative nucleobase lesions,apurinic/apyrimidinic (AP) sites, or breaks of one of the twostrands of a DNA molecule (SSBs), have been known to bestrongly related to raidobiological effects. Particularly
nucleobase lesions in the clustered damage site play animportant role in induction of mutations [1]. In this study we
examined how the nucleobase lesions are involved in the geneticinstability.
In order to discriminate the effect of nuclobase lesions fromthat of AP sites or SSBs, we exposed human choromosome 21
in “microcells” to Ultra Violet-A radiation (UV-A; 365nm),
which has been known to induce oxidative base lesions. We
used a microcell-fusion technique to transfect a specificchromosome with UV-A damage into non-irradiated cells. After
UV-A irradiation with a dose of 400 or 4000 kJ/m2, themicrocells were fused to non-irradiated mouse m5S cells(recipient cells). The fused microcell-hybrid cells were cultured
in selective medium for about one month, and then, several
clones were established. Each clone is originated from anindividual cell that has an irradiated human chromosome. The
stability of the irradiated and unirradiated human chromosomes
and unirradiated mouse chromosomes in the microcell hybrids
over 20 population doublings post irradiation were examined bywhole-chromosome painting and fluorescence in situ
hybridization (WCP-FISH). We used a probe specific for humanchromosome 21. Microscopic images were analyzed for bothhuman and mouse chromosomes as shown in Fig. 1.
The ploidy (the number of sets of chromosomes in a cell) of
the mouse recipient cells increased, and chromosomal
aberrations occurred not only in the UV-A-irradiated humanchromosome but also in the non-irradiated mouse chromosomes.
The frequencies of these abnormalities increased with the UV-A
dose received by the transferred human chromosome. In contrast,in the control experiment, in which an non-irradiated humanchromosome was transferred, the micro-cell hybrids remained
diploid, and the frequency of chromosomal aberrations in boththe transferred human chromosome and recipient mouse
chromosomes remained nearly normal. These results indicatethat a chromosome harboring oxidative nucleobase damage
induced by UV-A irradiation is unstable and transmits
instability to chromosomes of non-irradiated recipient mousecells.

Although the mechanism of the genetic instability of nonirradiated normal chromosomes has still been less understood,
certain types of oxidative nucleobase damage might stronglyinvolved in this phenomenon. One of the major nucleobase
damage induced by UV-A irradiation is oxidative guanine, 8oxo guanine. We suggest that candidate target of UV-A
radiation in chromosome is likely to be telomere, because it
consists of a guanine (G)-rich repeated sequence, TTAGGG, at
the terminus of chromosomes. This specific DNA terminal
structure does not code any genes, but is thought to play animportant role in partition of chromosome pairs during mitosis(cell division). Thus the telomere instability increases theprobability of chromosomal instability over many cell divisions
rather than prompt cell death. The fused microcell-hybrid cellswith destabilized telomere of the human chromosome 21 mightundergo incorrect cell division, and finally cause failure of thedivision resulting polyploidy, or fusion of chromosomes even if
they are normal, as observed in this study.
The obtained results that the UV-A damage to DNA
significantly affects unirradiated normal chromosomes have asignificant impact in the research field of radiation
carcinogenesis, as well as in cancer therapy. Details of theincomplete cell division originated from oxidative nucleobase
lesions, and mechanism of the chromosome aberrations should
be addressed in future studies. Particularly failure of function ofcell division machineries, such as centromere formation, will be
an important point to be focused.
We thank Dr. T. Tsutsui for providing us with the cells. This
work was supported in part by a Grant-in-Aid for Young
Scientists (B) (No. 20710045) from the Ministry of Education,Culture, Sports, Science, and Technology (MEXT).
ABCD

Fig.1 Examples of chromosome aberrations.
Microscopic images obtained as representative delayed
chromosome aberrations and their schematic figures (insets) areshown. UV-A irradiated human and unirradiated mouse
chromosomes were stained with red and blue dyes, respectively.
Arrowheads indicate centromeres. A UV-A irradiated humanchromosome fused to an unirradiated mouse chromosome (A). A
dicentric chromosome derived from two human chromosomes (B),and two mouse chromosomes (C). A multicentric chromosome (D).

References[1] M. Noguchi et al., Mutat. Res. 732, 34 (2012).
[2] A. Urushibara et al., Mutat. Res. 766, 29 (2014).