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Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light

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Abstract

DNA was isolated from the cotyledons of cucumber seedlings irradiated with ultraviolet (UV)-C (254 nm) or UV-B+UV-A (280–360 nm; maximum energy at 312 nm) at various fluence rates and durations. Following enzymatic hydrolysis of DNA, the content of 8-hydroxy-2′-deoxyguanosine [(8-OHdG), 8-oxo-7,8-dihydro-2′-deoxyguanosine], a well-established biomarker closely identified with carcinogenesis and aging in animal cells, was determined using a high-performance liquid chromatograph equipped with an electrochemical detector. The levels of 8-OHdG increased with UV-C and UV-B irradiation in a fluence-dependent manner. This increase was also observed in etiolated cotyledons that had been excised from dark-grown cucumber seedlings and then cultured in vitro under UV light: monochromatic UV light at 270 nm or 290 nm increased the 8-OHdG level considerably, while UV at wavelengths above 310 nm had only small effects. In situ detection of H2O2 and quantification of H2O2 in plant extracts revealed that H2O2 accumulated in cotyledons irradiated with UV light. These results suggest that UV irradiation induces oxidative DNA damage in plant cells.

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Abbreviations

CPD:

Cyclobutane pyrimidine dimer

CTAB:

Hexadecyltrimethylammonium bromide

DAB:

3,3′-Diaminobenzidine

dG:

Deoxyguanosine

ECD:

Electrochemical detector

HPLC:

High-performance liquid chromatography

8-OHdG:

8-Hydroxy-2′-deoxyguanosine

(6–4)photoproduct:

Pyrimidine-(6–4)-pyrimidone photoproduct

ROS:

Reactive oxygen species

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Acknowledgements

The irradiation by monochromatic UV light experiments were performed in the framework of the Cooperative Research Program of the National Institute for Basic Biology for the Okazaki Large Spectrograph. We thank Dr. M. Watanabe and Mr. S. Higashi of the Large Spectrograph Laboratory at the National Institute for Basic Biology for assistance with UV irradiation. This work was supported in part by a Grant-in-Aid from the Japan Society for the Promotion of Science (Research for the Future Program, JSPS-RFTF96L00602) and from the Global Environment Research Fund of the Environment Agency of Japan.

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Authors and Affiliations

  1. Course of Environmental and Biological Sciences, Graduated School of Science and Engineering, Hokkaido Tokai University, Sapporo, 005-8601, Japan

    Kaori Watanabe & Naohiro Yamada

  2. Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, 5-1-1 Minami-sawa, Minami-ku, Sapporo, 005-8601, Japan

    Yuichi Takeuchi

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  1. Kaori Watanabe
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  2. Naohiro Yamada
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  3. Yuichi Takeuchi
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Correspondence to Yuichi Takeuchi.

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Watanabe, K., Yamada, N. & Takeuchi, Y. Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light. J Plant Res 119, 239–246 (2006). https://doi.org/10.1007/s10265-006-0266-2

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