Figure 16.

Induction of bystander effects by CAP and PAM. (A) MKN-45 cells were treated with CAP for 1 min, followed by an incubation step of 25 min in the same medium. After a washing step of three cycles, increasing percentages of pretreated cells were added to untreated tumor cells (open squares). The percentages of apoptotic cells in these cocultures were determined at the indicated times. This basis approach was modified by either adding the NOX1 inhibitor AEBSF (100 µM) during CAP treatment and initial incubation (open crosses), and in this way blocking the generation of secondary ¹O₂, or adding AEBSF and the ONOO⁻ decomposition catalyst FeTPPS (25 µM) (open diamonds), and in this way allowing to focus on ¹O₂ derived directly from the gaseous phase of CAP, as the generation of ¹O₂ from long-lived species in the medium and secondary ¹O₂ generation by the cells was prevented under these specific conditions. All modifications were studied in parallel in the presence of histidine during pretreatment (closed symbols) to allow the evaluation of the role of ¹O₂. (B) The experiment was performed in an analogous mode to A, with the exception that the cells were not treated with CAP in medium, but were mixed (50%) with PAM that had been prepared by treating medium with CAP for 1 min. The results show that maximal bystander inducing potential is achieved when CAP or PAM treatment is applied without experimental interference with primary or secondary ¹O₂ generation (open squares). Prevention of secondary ¹O₂ generation causes a dramatic loss of bystander effect-inducing cells both after CAP and PAM treatment (open crosses). This effect is essentially due to primary ¹O₂ generated by long-lived species (i. e. H₂O₂ and NO₂⁻). Direct treatment of cells in medium in the presence of AEBSF and FeTPPS (Fig. 11A) allows to detect the effect of ¹O₂ derived from the gaseous phase of CAP (open diamonds) which is lower than the effect by ¹O₂ generated by long-lived species (open crosses). In assays treated with the PAM regime (Fig. 11B), no effects due to ¹O₂ derived from the gaseous phase of CAP were detected, consistent with the short life time of ¹O₂.