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Journal of photochemistry and photobiology. B, Biology

DNA damage after phototherapy in wounded fibroblast cells irradiated with 16 J/cm(2).


PMID 19101162

Abstract

Phototherapy or biomodulation is a remarkable therapy that has become more popular and widely used in the treatment of a variety of medical conditions, such as slow to heal wounds, pain, soft tissue injuries and skin trauma. It has been shown to induce DNA damage; however this damage appears to be repairable. This study aimed to determine the effects of phototherapy induced DNA damage and activation of the DNA repair gene methylpurine DNA glycosylase (MPG). DNA integrity was assessed using the comet assay, with and without formamidopyrimidine glycosylase (Fpg). For the comet assay, wounded human skin fibroblast cells (WS1) were irradiated twice, once at 30 min and again at 72 h with 5 or 16 J/cm(2) using a diode laser at 636 nm and cellular responses were assessed 1 or 24h post-irradiation. Real time reverse transcriptase polymerase chain reaction (RT-PCR) assessed MPG expression and three reference genes namely; beta Actin (ACTB), Glyceraldehyde three phosphate dehydrogenase (GAPDH) and Ubiquitin c (UBC). Wounded cells were irradiated once (30 min) with 16 J/cm(2), and MPG expression was assessed at 0, 3 and 8h post-laser irradiation. At both 1 and 24h, wounded cells irradiated with 5 J/cm(2) showed insignificant DNA damage compared to control cells, while irradiation with 16 J/cm(2) showed significant damage. However, 24h post-irradiation these cells showed a significant decrease in damage compared to cells left to incubate for 1h. This observation was attributed to activation of DNA repair mechanisms. Real time RT-PCR showed that ACTB was not influenced by cell culture conditions or laser irradiation, and MPG expression was not detected. In conclusion, irradiation with 5 J/cm(2) did not produce additional DNA damage, while damage to cells irradiated with 16 J/cm(2) was repairable by mechanisms other than MPG. This study also showed that ACTB can be used as a reference gene in laser experiments, using parameters set out in this study.