Camptothecin (CPT) is an anticancer drug that inhibits topoisomerase I (Topo 1), an enzyme closely linked to cell division, by forming a ternary DNA-CPT-Topo I complex. However, it has been shown that UVA-irradiated CPT in the absence of Topo I produces DNA damage. It has been proposed that free radicals are the key species responsible for the DNA cleavage. It has also been shown that the presence of metal ions enhances the activity of several anticancer drugs. Therefore, we attempt here to explore and identify free radicals generated in these processes. We describe a detailed spectroscopic study of UVA-irradiated CPT and the Cu(II)-CPT complex. From the low-temperature EPR spectrum of the Cu(II)-CPT complex, a proximity between the Cu(II) ion and the 20-hydroxy group of the lactone E ring of CPT is proposed. Upon irradiation (lambda = 365 nm) of the Cu(II)-CPT complex in deoxygenated dimethyl sulfoxide (DMSO), the EPR signal of Cu(II) measured in situ at room temperature shows formal first-order exponential decay with a formal half-life of 11 min. By the use of a specific Cu(I) chelating agent, neocuproine, it was shown that, during this process, Cu(II) is reduced to Cu(I). When the photochemical experiments are repeated in oxygen-saturated DMSO solutions, analogous phenomena are observed, characterized by a formal half-life of 16 min for Cu(II), except that there is an induction period of similar to3 min. Application of the spin-trap agent 5,5-dimethyl-l-pyrroline N-oxide (DMPO) shows that during this induction period the only radical formed is the superoxide radical, trapped as the (DMPO)-D-.-O-2(-) adduct. The loss in EPR signal intensity of the Cu(II)-CPT complex upon irradiation is accompanied by the appearance of a new EPR signal at g approximate to 2.0022. Application of the spin traps nitrosodurene (ND) and DMPO revealed that the main radical product formed upon continuous irradiation of CPT in DMSO solutions is the hydroxyl radical (trapped in DMSO as the (CH3)-C-. adduct). Application of 2,2,6,6-tetramethyl-4-piperidinol has revealed that irradiation of CPT in aerated DMSO solution also leads to the formation of singlet oxygen (O-1(2)). In the Cu(II)-CPT system, the formation of methyl radicals is suppressed, and the generation of two new radical adducts originating from camptothecin ring cleavage is identified. A mechanism of photochemically generated radicals that include the superoxide radical and the radical cation of the {Cu(II)...CPT.+) complex followed by the reduction of Cu(II) to Cu(I) is proposed. The EPR experiments on irradiated CPT in the absence of copper (II) support the importance of the 20-hydroxy group of the lactone E ring in the antitumor activity of the drug mediated through the initially generated hydroxyl radical. In the presence of Cu(H), there is blockage of the 20-hydroxy group of CPT, and the generation of hydroxyl radicals is strongly suppressed. In this case, there is a mixture of radicals of various origin generated as a result of irradiation, which are capable of causing DNA damage. We propose that the superoxide radical, hydroxyl radical, singlet oxygen, and carbon-centered radicals generated from CPT in close proximity to DNA can cause considerable damage to DNA. These findings would help to explain the experiments indicating that the photoactivated camptothecin interacts specifically with guanines, consistent with preferential stimulation of topoisomerase I cleavage at sites that bear a guanine at their 5'-DNA terminus. Taken together, our spectroscopic experiments indicate that CPT is a promising photosensitizer and that radicals and singlet oxygen generated upon illumination play a central role in DNA cleavage and in the induction of apoptosis in cancer cells.