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

We also found that [alpha]-TOCA prevented PDT-induced DNA damage. Esterification of the hydroxyl group at position 6 prevents reaction with lipid -OO^sup *^ because the ester is unable to donate hydrogen to the lipid radical. However, [alpha]-TOCA can protect membranes when present at high doses, and this has been attributed either to a structural effect or to a result of influence of the composition of membrane fatty acids (30). It is also known that human skin can metabolize [alpha]-TOCA to [alpha]-TOC (31); however, it cannot be determined from the present study whether metabolism occurred in the HaCaT cells, thus accounting for the apparent protective effect of [alpha]-TOCA or whether there are other mechanisms responsible. It has previously been shown in hamster V79 cells that [alpha]-TOCA can attenuate DNA damage caused by H^sub 2^O^sub 2^, although not as effectively as [alpha]-TOC (32). In addition, Ouedraogo and Redmond (33) have shown under different experimental conditions to the present study that [alpha]-TOCA can inhibit strand breaks caused by deuteroporphyrin-PDT. Interestingly, this group also reported a decrease in malondialdehyde as a marker of lipid peroxidation in the presence of [alpha]-TOCA.

That [alpha]-TOC was not able to substantially prevent cell lysis in the present study could be due to the fact that it was consumed rapidly as a result of overwhelming lipid peroxidation during PDT, leading to membrane breakdown. In the process, intermediates of DNA damage were attenuated but not completely prevented. This is borne out by the fact that protection against DNA strand breaks disappeared at higher doses of PS or light (or both), implying that damage was delayed. The present results also suggest that [alpha]-TOC and [alpha]-TOCA only exerted their protective effects toward DNA strand breaks at subphototoxic doses of PDT.

Finally, the modified version of the comet assay was used to detect DNA base oxidation. Oxidative damage to DNA has been proposed as a possible risk factor for cancer development (34,35), ageing and various degenerative diseases (30). Oxidized bases are not revealed by the standard comet assay protocol unless bacterial DNA glycosylases are used to excise the modified base, thus creating a strand break that is then detectable by the technique (18). Endonuclease III detects mainly modified pyrimidine bases, whereas FPG protein detects mainly modified purine bases, including the potentially mutagenic 8-oxo-7,8-dihydroguanine, which is considered to be a good indicator of oxidative stress. Under our experimental conditions, we found the modified assay to have a higher variation between independent experiments as previously reported (36), and that the background level of base oxidation was lower when detected with endonuclease III than with FPG protein. Endonuclease III-sensitive sites increased with Photofrin-PDT. This was not as evident for FPG protein. It is possible that the number of enzyme-sensitive sites was underestimated by measuring DNA damage immediately after PDT. We might have been better able to resolve base damage if the cells had been allowed to repair frank strand breaks before addition of the enzyme (18). Kipp and Young (36) have also suggested that enzyme-sensitive sites could be masked by high levels of strand breaks.