0024908 - ÚEB 2006 RIV NL eng J - Článek v odborném periodiku
Gichner, Tomáš - Mukherjee, A. - Wagner, E. D. - Plewa, M. J.
Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis.
[Hodnocení jaderného DNA difúzního testu k detekci apoptózy a apoptózy.]
Mutation Research - Genetic Toxicology and Environmental Mutagenesis. Roč. 586, č. 1 (2005), s. 38-46. ISSN 1383-5718. E-ISSN 1879-3592
Grant CEP: GA ČR GA521/05/0500
Výzkumný záměr: CEZ:AV0Z50380511
Klíčová slova: DNase-I * Ethyl methanesulphonate * Hydrogen peroxide
Kód oboru RIV: EB - Genetika a molekulární biologie
Impakt faktor: 2.188, rok: 2005

We applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328-337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apo-ptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA da-mage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) fol-lowed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 degrees C for 1-15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bro-mide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treat-ments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants. (c) 2005 Elsevier B.V. All rights reserved.

Difuzní jaderní test je doporučen k rozlišení apoptotického a nekrotického poškození DNA podle struktury jader u savčích buněk. Tento test jsme aplikovali na jádra z listů a kořenů tabáku (Nicotiana tabacum var. xanthi). Po aplikaci peroxidu vodíku na jádra z kořenů se průkazně zvýšil průměr jader, způsobený poškozením DNA, po 24 h však bylo poškození DNA kompletně napraveno. To dokazuje, že poškození DNA není podmíněno apoptózou nebo apoptózou , která není reparovatelná, ale reparovatelnou genotoxicitou. Ani po aplikaci DNase-1 a hyperthermie nebylo možno rozlišit podle struktury mezi apoptotickou, nekrotickou a genotoxickou fragmentaci DNA.
Trvalý link: http://hdl.handle.net/11104/0115379