Ceruloplasmin (Cp) was found to promote the oxidative damage to DNA in vitro, as evidenced by the formation of 8-hydroxy-2'-deoxyguanosine and strand breaks, when incubated with a cysteine metal-catalyzed oxidation system (Cys-MCO) comprised of Fe(3+), O(2), and cysteine as an electron donor. The capacity of Cp to enhance oxidative damage to DNA was inhibited by hydroxyl radical scavengers such as sodium azide and mannitol, a metal chelator, diethylenetriaminepentaacetic acid, a spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and catalase. Ceruloplasmin also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation. Incubation of Cp with Cys-MCO resulted in an increase in the content of carbonyl groups and the significant alteration of the ferroxidase activity, as well as the proteolytic susceptibility. The deoxyribose assay and the salicylate hydroxylation assay showed that hydroxyl free radicals were generated in the reaction of Cp with Cys-MCO. The release of a portion of Cu from Cp was observed, and conformational alterations were indicated by the changes in fluorescence spectra. Based on these results, we interpret the enhancing effect of Cp on DNA damage and mutagenicity induced by Cys-MCO as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged Cp, and H(2)O(2) produced by Cys-MCO. The release of Cu from Cp during oxidative stress could enhance the formation of reactive oxygen species and could also potentiate cellular damage.