Protective mechanism of metallothionein against copper-1,10-phenanthrolineinduced DNA cleavage

Metallothionein (MT) has been shown to protect DNA against cleavage induced by a variety of mutagenic agents. The mechanism has been attributed to its ability to either chelate transitional metals that participate in the Fent on reaction, or scavenge free radicals by means of the abundant cystenyl re sidues of the proteins. In the present study, the protective action of MT a gainst DNA cleavage by the copper- 1,10-phenanthroline [(OP)(2)Cu+] complex was studied in situ. At 0.1 mu M, MT inhibited the (OP)(2)Cu+ induced DNA cleavage by about 50% (IC50 approximate to 0.1 mu M). At 2.5 mu M, the clea vage activity was completely inhibited. Similar to MT, cysteine can protect against DNA cleavage by (OP)(2)Cu+ (IC50 of approximately 3 mM), however, its action was 1500-fold less efficient than MT. The combined action of MT and cysteine was additive. Reduced glutathione (1 and 10 mM) did not protec t the (OP)(2)Cu+ induced DNA cleavage. Sodium azide could inhibit the cleav age only at high concentrations (IC40 approximate to 25 mM). Spectrophotome tric analysis showed that MT can inhibit the formation of the DNA[(OP)(2)Cu +] complex possibly by chelating Cu. It can also cause a dissociation of th e complex after it was formed. In the later case, the mechanism through whi ch MT protects against the DNA cleavage might occur when MT fitted in close ly with the complex, competing with the hydroxyl groups of the nucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reactio n. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.