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

ABSTRACT

Photodynamic therapy (PDT) kills cells via the production of singlet oxygen and other reactive oxygen species. PDT causes chromosomal damage and mutation to cultured cells. However, DNA damage does not contribute to the phototoxic effect. To study the effect of Photofrin-PDT-induced DNA damage, we used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase and a human keratinocyte cell line to investigate photogenotoxicity and its prevention by tocopherol (TOC). This study shows that PDT induced DNA damage in HaCaT cells at doses allowing cells to survive 7 days after irradiation. [alpha]-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but did prevent Photofrin-PDT-induced DNA damage. However, the concentration of TOC that conferred protection (100 �M) was higher than is detected in human serum. Base oxidation was also measured using the comet assay. Although TOC could prevent frank DNA strand breaks caused by PDT, it was unable to decrease the level of base oxidation as revealed by enzyme-sensitive sites. It is suggested that the potential genotoxic risk from laser-PDT could be low, and that topical [alpha]-TOC at a high concentration may be useful in preventing some types of DNA damage without preventing acute photolysis after Photofrin-PDT.

Abbreviations: DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EtOH, ethanol; FPG, fonmamidopyrimidine DNA glycosylase; H^sub 2^O^sub 2^, hydrogen peroxide; ID^sub 50^, dose of radiation required to reduce the number of cells by 50%; LMP, low melting point; MTT, methylthiazoletetrazolium bromide; NRUA, neutral red uptake assay; PBS, phosphate-buffered saline; PDT, photodynamic therapy; PS, photosensitizer; ROS, reactive oxygen species; [alpha]-TOC, [alpha]-tocopherol; [alpha]-TOCA, [alpha]-tocopherol acetate.

INTRODUCTION

Photodynamic therapy (PDT) uses the combination of visible light, a pholosensitizer (PS) and oxygen to kill tumor cells. Without light, the PS has essentially no effect. PDT is believed to kill cells via the production of singlet oxygen and other reactive oxygen species (ROS), which overwhelm the numerous antioxidant defense mechanisms within the cell and cause oxidative damage to cellular macromolecules. Although the molecular events underlying PDT-induced cell death have still not been clearly defined, the principle cause of lethality is thought to be cell lysis due to oxidation and damage to membranes. Kelley et al. (1) showed that Photofrin-PDT resulted in consumption of membrane [alpha]-tocopherol ([alpha]-TOC) levels, followed by oxidation of lipid membranes and cell death. Metabolites of lipid oxidation can be both cytotoxic and genotoxic (2,3), and recently, ceramide produced as a result of PDT has been associated with apoptosis and phototoxicity (4). In vivo PDT results in tumor cell death either directly or indirectly via destruction of the vasculature due to the presence of PS in the blood (5-8).

In addition to membrane oxidation, damage to mitochondrial and cytoskeletal proteins, and also to DNA, has been reported. Unlike membrane damage, DNA damage caused by PDT has not been directly linked to lethal effects. PDT has been shown to cause DNA base damage, sister chromatid exchanges and cross-links (9-12). A number of reports have examined the mutagenicity of PDT in various human and animal cell lines. The mutagenic potential varies between cell types, possibly reflecting differences in repair capacity or damage surveillance mechanisms (10,13,14). With the increasing use of PDT in the treatment of cancer and also in benign conditions, the nature and relevance of DNA damage produced need to be determined. This is particularly important in areas where the photodynamic effect is such that DNA-damaged cells can survive.

Photofrin is currently the only Food and Drug Administration-approved PS available for the treatment of cancer. In addition to treatment of cancer in various organs, such as esophagus, lung and brain, it is also applied to patients with Barrett's esophagus, a precancerous condition, to prevent progression to carcinoma (15). The main side effect of Photofrin-PDT is skin photosensitivity, which can last for several weeks after treatment. To study the effects of Photofrin-PDT-induced DNA damage to skin, we have used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase (FPG protein) and a human keratinocyte cell line (HaCaT; [16]) as a model system to investigate photogenotoxicity and its prevention by TOC. The comet assay is a rapid, microelectrophoretic method for the quantitation of DNA damage in individual cells (17). This flexible technique can be used to detect strand breaks, apurinic sites, cross-links and also oxidized DNA bases if used in combination with bacterial base excision repair enzymes (18). We have also explored the role of [alpha]-TOC and its esterified derivative [alpha]-TOC acetate ([alpha]-TOCA) on PDT-induced cell death and related damage.