ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 294, No. 1, April, pp. 17-21, 1992 Effect of Lipophilic Chelators on Oxyradical-Induced DNA Strand Breaks in Human Granulocytes: Paradoxical Effect of 1,I 0-Phenanthroline’ H. Chaim Birnboim Department of Experimental Oncology, Ottawa Regional Cancer Centre, 501 Smyth Road, and Departments of Biochemistry and Microbiology/Immunology, University of Ottawa, Ottawa, Ontario KlH 8L6, Canada Received August 5, 1991, and in revised form October 30, 1991 Strand breaks can be produced in the DNA of intact granulocytes by a flux of oxyradicals (02 and HsOs) gen- erated by tetradecanoylphorbol acetate (TPA) or by a flux of HaOz generated by glucose oxidase. The mecha- nism by which such breaks are induced is still uncertain. Lipophilic chelators such as dipyridyl and l,lO-phen- anthroline (OP) strongly inhibit strand breaks induced by HsOs, presumably because of their ability to chelate intracellular iron. We now report that dipyridyl also partially inhibits strand breaks in TPA-stimulated granulocytes while a “copper-specific” lipophilic che- lator, neocuproine, has no effect. As opposed to these ef- fects, OP increases the number of strand breaks in TPA- stimulated granulocytes. Superoxide dismutase (SOD) (but not catalase) partially blocks this increase. Both the cell-impermeable chelator, EDTA, and neocuproine strongly block the increase also. In fact, in the presence of EDTA, OP behaves like dipyridyl and inhibits strand breaks. Preformed OPs-copper(D) complex causes DNA breaks in TPA-stimulated granulocytes. The paradoxical effect of OP may be explained by assuming that OP may form two different metal complexes, a DNA-damaging complex with copper or an inhibitory complex with iron. If copper(I1) and 0; are present, the first complex may form and the net effect may be an increase in strand breaks. If the formation of this complex is prevented by SOD, EDTA, or neocuproine, then OP may complex iron and the net effect may be (like dipyridyl) an inhibition of strand breaks. The source of the copper responsible for the formation of OP2-copper complex is unknown. 0 1992 Academic Press, Inc. The mechanism by which strand breaks are induced in cellular DNA by oxyradicals has been under investigation 1 The work was supported by a grant from the Medical Research Council of Canada to HCB. 0003-9S61/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. in many laboratories. H202 (either as a bolus or as a flux generated by glucose oxidase) or H202 plus 0; (generated by xanthine oxidase) has been most often used in these studies. Our laboratory was among the first to show that oxyradicals produced by phorbol ester-activated granu- locytes could cause DNA strand breaks in the producing cells as well as in target cells (1, 2). These results have been confirmed and extended to monocytes and macro- phages, which also generate 0, and HsOsafter appropriate stimulation (3-9). In general, it has been found that there is inhibition of DNA breaks on addition of catalase but not superoxide dismutase (SOD),2 implying a role for H202 but not 0,) other than as a precursor to H202. Meneghini and co-workers have reported that lipophilic “iron” che- lators such as dipyridyl (2,2’-bipyridine) and l,lO-phen- anthroline (OP) can effectively block the cytotoxic and genotoxic effects of H20z (10-14). Because they have ob- served that in vitro dipyridyl-iron and OP-iron complexes do not support the production of OH * by a Fenton-type reaction, they have argued that the protective effect of these chelators in intact cells is due to binding of intra- nuclear iron and have concluded that OH l is likely the ultimate genotoxic species. Similar protective effects of OP have been reported by others (7,9). We have also seen a protective effect of OP when glucose oxidase-generated H202 was the source of oxyradicals (15). However, the opposite effect, viz., enhancement of DNA strand breaks, was seen in phorbol ester-treated leukocytes (15) and in a murine mammary tumor line treated with resident peri- toneal macrophages (8). Some slight enhancement was also seen in HPETE-mediated DNA breaks in mouse fi- broblasts (16). The same author reported protection against tert-butyl hydroperoxide-induced chromosomal ’ Abbreviations used: OP, l,lO-phenanthroline; SOD, Cu,Zn-super- oxide dismutase; TPA, 12-0-tetradecanoylphorbol-13-acetate; DMSO, dimethyl sulfoxide. 17