Fig. 11.

Imprinting the signature of primary singlet oxygen from a photosensitizer is independent of peroxynitrite and H₂O₂. MKN-45  cells were treated with 5 μg/ml photofrin plus light for 20 min in the absence of inhibitors or in the presence of 2 mM histidine, 25 μM EUK-134, 25 μM FeTPPS, 100 μM AEBSF or a combination of AEBSF + histidine, AEBSF + EUK-134, AEBSF + FeTPPS. The cells were subjected to three cycles of washing and added to untreated tumor cells at increasing percentages. The percentages of apoptotic cells were determined 4 h after beginning of the coculture. In addition, cells that had been pretreated with photofrin in the absence of inhibitors were added to untreated cells and the coculture was performed in the presence of histidine (dashed lind with diamond). This result confirms the differentiation between the action of primary and secondary singlet oxygen after treatment with the singlet oxygen generator photofrin. It also shows that imprinting the signature of primary singlet oxygen can only be inhibited by histidine, but not by EUK-134 or FeTPPS. This finding is expected and confirms the significance of the inhibitors used in our studies. This finding is contrasted by imprinting the signature of primary singlet oxygen generated through the interaction between H₂O₂ and nitrite, as shown in preceding figures. These reactions are dependent on H₂O₂ and peroxynitrite. Statistical analysis: A: The inhibitory effects of EUK-134, FeTPPS and AEBSF are highly significant (p < 0.001) and without significant variation among themselves. Inhibition by this group of inhibitors is highly significantly (p < 0.001) different from inhibition by histidine, which is highly significant by itself (p < 0.001). B: Only AEBSF causes highly significant inhibition (p < 0.001).