Effect of Photofrin on DNA Strand Breaks and Base Oxidation in HaCaT Keratinocytes: A Comet Assay Study6, The

Photochemistry and Photobiology, Jan 2004 by Woods, J A, Traynor, N J, Brancaleon, L, Moseley, H

As well as measuring acute phototoxicity, survival after PDT was determined by a colony-forming assay. Figure 3 shows that 1 �g/mL Photofrin irradiated with 1 J/cm^sup 2^ red light resulted in approximately 50% cell survival 7 days after irradiation. There was a high variation in cell survival at this dose. However, at the higher doses of PDT, consistently no colonies grew out over the 7 day assay period, indicating that although populations of cells were apparently viable 24 h after treatment, they were unable to propagate and therefore presumably any cells with DNA damaged under these conditions would not pose a potential hazard.

Photogenotoxicity of Photofrin

The standard alkaline comet assay protocol was used to establish whether Photofrin (1 �g/mL) generated DNA strand breaks when irradiated with 630 nm laser light. There was a dose-related increase in DNA migration with increasing irradiation dose (Fig. 4); however, the breaks produced at the higher irradiation doses (10 and 25 J/cm^sup 2^) would have been caused partly by cell death. A significant (P

Effect of [alpha]-TOC on photogenotoxicity

In contrast to the effect on acute phototoxicity, addition of 100 �M [alpha]-TOC attenuated DNA strand breaks caused by Photofrin-PDT by 65% (P

Effect of endonuclease III and FPG protein

Using the modified comet assay, we examined the effects of repair enzymes on DNA base damage elicited by the subphototoxic dose of Photofrin-PDT. Considering DNA strand breaks (buffer-only samples), neither the irradiated control nor nonirradiated, Photofrin-treated samples were different from the nonirradiated control. In contrast, Photofrin-PDT generated a significant increase (P

Effect of [alpha]-TOC on FPG protein-sensitive sites

The effect of [alpha]-TOC or [alpha]-TOCA (100 �M) on FPG protein-sensitive sites was investigated (Fig. 7). In the absence of enzyme, both TOC reduced PDT-induced strand breaks as previously shown (Fig. 5). In the presence of repair enzyme the combined level of strand breaks and enzyme-sensitive sites revealed did not differ from the PDT-only treated samples. This suggests that enzyme-sensitive sites formed during PDT were not prevented by [alpha]-TOC or its ester, and the formation may actually have been enhanced by [alpha]-TOC.

DISCUSSION

PDT is a form of photochemotherapy that uses a PS, oxygen and visible light to kill cells. The exact mechanism by which cell death occurs depends on many different factors including cell type, oxygen concentration and subcellular localization of the PS. Photofrin is administered intravenously at a dose of 2 mg/kg body weight approximately 48 h before laser treatment. The peak plasma concentration after injection has been found to reach 15 � 3 �g/ mL, falling to around 2.6 �g/mL after 48 h (product insert). Thus, the concentration of Photofrin used in the present study (1 �g/mL) is similar to that found in the plasma of a patient undergoing PDT. The minimum phototoxic concentration of protoporphyrin has been calculated to be 2-5 �g/g tissue (28), which is less than, but in line with, the intracellular content calculated under the present experimental conditions. It has previously been demonstrated that prolonged (>18 h) incubation with Photofrin results in subcellular localization to the cytoplasmic organelles, including the mitochondria (6). Our own observations showed that fluorescence was visible throughout the cytoplasm.

This study shows that Photofrin-PDT induced DNA damage at very low doses that allowed a proportion of cells to survive 7 days after irradiation. [alpha]-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but was able to prevent Photofrin-PDT-induced DNA damage. However, the concentration of TOC that conferred protection (100 �M; [asymptotically =]50 �g/mL) was much higher than is detected in normal human serum (the concentration in serum is in the region of 10 �g/mL [27]). At these high concentrations, [alpha]-TOC has been shown to affect activation of membrane signaling proteins such as protein kinase C via nonantioxidant behavior (29). Moreover, the protective effect was lost at higher doses of light (5 J/cm^sup 2^) or PS (10 �g/mL Photofrin; data not shown) (or both). [alpha]-TOC is lipid soluble and concentrates in the interior of cell membranes where it is the major antioxidant. It interacts with singlet oxygen, the hydroxyl radical and importantly, lipid peroxy- and alkoxy radicals. TOC may be particularly important in organs such as the brain, which is relatively poor in the antioxidant defense enzymes catalase, superoxide dismutase and glutathione peroxidase.