STUDENT ABSTRACTS - Environmental UV Radiation: Impact on Ecosystems and Human Health and Predictive Models - page 276

Repair processes in plant following UV-irradiation photoreactivation of DNA
damages
NADIA GONCHAROVA
International Sakharov Environmental University, Minsk, Belarus

Photoreactivation is a substantial increase of survival of UV-irradiated cells under the
effect of visible light. Object of this study are the shoots of barley ( Hordeum vulgare L.). The
seeds shot in darkness in thermostat at 24-26 °C. Westinghouse FS-40 and Philips TL 40/12
lamps were used for UV irradiation. Doses were measured with an UVX-radiometer dosimeter
(UV Ltd., USA). To assess the duration of cellular cycle and proliferative activity after UV
irradiation and in normal conditions, the shoots with 0.5-1 cm long radicles were incubated with
3 H-methionine (0.2 MBq/ml) for 30 min. They were washed, put into the solution of unmarked
thymidine and incubated in darkness. One and half hour after UV irradiation and every 2 hours
during the following 25 hours the radicles were fixed with an ethanol/acetic acid solution (3:1)
and stained with Falgen. The fixed preparations from crushed radicles were covered with a special
photoemulsion for 7 days. The number of mitoses was calculated in developed preparations. The
duration of separate phases and of whole cellular cycle was calculated by the curves of marked
mitoses. The principal radicles were fixed and stained with acetocarmine, and, after maceration,
the mitotic index (MI; %) was determined from the mitosis number in temporary preparations.
The shoots were irradiated with short-wave UV radiation at 3.5 and 5.4 10 3 J/m 2 doses. The

principal radicles were fixed every 2-3 hours in a 20 hours period. The chromosome aberrations
were analyzed by means of the anaphase method in temporary preparations stained with
acetocarmine and crushed. Immediately after UV irradiation the shoots were irradiated with
photoreactivating light of different spectral composition (300-390 nm, 330-460 nm, 380-1500 nm)
and fixed 7-20 hours after irradiation. DNA was isolated from marked plants. UV-induced
damages of DNA sites were determined by their sensitivity to the action of UV endonuclease. It
was found that the photoreactivating light is able to induce chromosome aberrations by itself and
to inhibit cell proliferation. Mainly the aberrations of chromatid were observed in UV-irradiated
cells. Single fragments constituted 85% of chromosome aberrations. The maximum
photoreactivating effect was observed in cells irradiated in the S phase; the minimum in cells
irradiated in the G 1 phase. In cells irradiated in phases G 1 and S, the photoreactivation increased
the yield of chromosome aberrations by 21% and 55%, respectively. The photoreactivating light
and UV irradiation did not change the mitotic activity of the cells. This suggests that the decrease
of the number of chromosome aberrations after photoreactivation is connected to DNA
photoreactivation. The observed photoreactivation process might indicate that a small part of UV-
induced thymine dimers constitutes the primary damage of chromosome aberration. We tested this
hypothesis determining the number of sites vulnerable to UV-endonuclease action. There was a
significant degradation of DNA irradiated at doses of 2.2-17.0

x

10 2 J/m 2 , which allowed to
determine the number of sites vulnerable to UV-endonuclease. A linear dependence was found
between the number of sites and the UV dose.
The comparison between the curves of the UV-dose dependence of site number and of
chromosome aberrations indicates a correlation between induction of DNA damage and induction
of chromosome aberrations.

x