Detection and cytotoxicity of cisplatin-induced superoxide anion in monolayer cultures of a human ovarian cancer cell line

Superoxide anions (O-2(-)) generated by cisplatin [cis-diamminedichloroplat inum (II), DDP] were determined by measuring the chemiluminescence from the luminescence probe, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a] pyrazin-3-one (methyl Cyprdina luciferin analog, MCLA), in monolayer cultur es of a human ovarian cancer cell line (A2780) in physiological saline at p H 7.0. In a time-course study, chemiluminescence of MCLA (C-MCLA) showed a peak level at 10 min and a background level at 60 min after the addition of DDP. The intensity of C-MCLA increased with increasing concentrations of D DP or MCLA in a limited concentration range, and was significantly correlat ed (r=0.960) with the number of A2780 cells. DDP-induced C-MCLA was complet ely inhibited by the addition of the O-2(-) scavenger, superoxide dismutase (SOD). However, SOD did not decrease DDP cytotoxicity in terms of clonogen ic cell survival. These findings suggest that DDP generates extracellular O -2(-), probably by interaction with the cellular membrane in A2780 cells, a nd O-2(-) does not lead to cellular damage. reactive oxygen species (ROS) p lay a crucial role in DDP-induced cytotoxicity [7, 9, 21], although the maj or mechanism of DDP cytotoxicity is the inhibition of DNA synthesis through inter- and intrastrand crosslinking [23]. Several studies have demonstrate d that DDP induces ROS in murine macrophage monolayers [13, 15, 16] and hum an polymorphonuclear leukocytes [1]. However, in these studies the primary function of these phagocytes, e.g. the release of O-2(-) and H2O2, was inve stigated. The present study was performed to evaluate the involvement of RO S in DDP cytotoxicity in cancer cells. We have previously reported that the interaction of DDP with purified cellular DNA generates O-2(-) in cell-fre e systems [5, 6]. In the course of our efforts to detect ROS released from living cells as a result of DDP treatment, we found a simple method of dete cting DDP-induced O-2(-) in a human ovarian cancer cell line, cultured as a monolayer, by measuring the chemiluminescence from the luminescence probe, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[ 1,2-a]pyrazin-3-one (meth yl Cypridina luciferin analog, MCLA), and found that O-2(-) induced in this way did not lead to cellular damage.