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Avarol-induced DNA Strand Breakage in Vitro and in Friend Erythroleukemia Cells¹

Werner E. G. Müller², Dusan Sladi, Rudolf K. Zahn, Karl-Heinz Bässler, Nikola Dogovi, Horst Gerner, Miroslav J. Gasi and Heinz C. Schröder

Institut für Physiologische Chemie [W. E. G. M., R. K. Z., K-H. B., H. G., H. C. S.], Universität, Duesbergweg 6, 6500 Mainz, West Germany, and Faculty of Science [D. S., N. D., M. J. G.], Department of Chemistry, Belgrade, Yugoslavia

The hydroquinone-containing cytostatic compound avarol inhibits predominantly growth of those cell lines which have a low level of superoxide dismutase. The substrate of this enzyme, the superoxide anion, was found to be formed during the in vitro oxidation reaction of avarol to its semiquinone radical in the presence of oxygen. Under the same incubation conditions plasmid DNA (pBR322) was converted from the fully super-coiled circular form mainly to the nicked circular form, indicating that the compound causes primarily single-strand breaks. Using Friend erythroleukemia cells (FLC) it was found that avarol induces a dose-dependent DNA damage; the maximum number of DNA strand breaks was observed at 5 h after addition of the compound to the cells. Removal of avarol resulted in a rapid DNA rejoining with biphasic repair kinetics [first half-time, 8 min (90% of the breaks) and a second half-time, 40 min (10% of the breaks)]. When the degree of avarol-induced DNA damage in FLC was compared with the drug-caused inhibition of cell growth a close correlation was established. Avarol displayed no effect on dimethyl sulfoxide-induced erythrodifferentiation of FLC as determined by the benzidine reaction and by dot blot hydridization experiments. From incubation studies of FLC with [³H]avarol no hint was obtained for the formation of an adduct between DNA and the compound. The subcellular distribution of [³H]avarol was studied in liver cells after i.v. application of the compound. The predominant amount of the compound was present in the cytosolic fraction; little avarol was associated with plasma membranes, nuclei, and mitochondria. Using (a) oxidative phosphorylation and (b) oxygen uptake as parameters for mitochondrial function, no effect of the compound on the activity of this organelle was determined.

These results suggest that avarol forms superoxide anions (and in consequence possibly also hydroxyl radicals) especially in those cells which have low levels of superoxide dismutase. Moreover, evidence is provided that the active oxygen species cause DNA damage resulting in the observed cytotoxic effect.

¹ Supported in part by a grant from the Deutsche Krebshilfe e.V. (W.E.G.M.) and by a grant from the Bundesministerium für Forschung und Technologie (German-Yugoslavic cooperation program) under the coordination of the Internationales Büro Gesellschaft für Kernenergieverwertung in Schiffbau und Schiffahrt Geesthacht. This paper is dedicated to Prof. Dr. Hamao Umezawa.

² To whom requests for reprints should be addressed.

Received 4/13/87. Revised 7/31/87. Revised 9/10/87. Accepted 9/18/87.