Rousset N, Keminon E, Eléouet S, Le Néel T, Auget J L, Vonarx V, Carré J, Lajat Y, Patrice T
Département Laser, Laboratoire de Photobiologie des Cancers, Hôpital Laënnec, Nantes, France.
J Photochem Photobiol B. 2000 Jul;56(2-3):118-31. doi: 10.1016/s1011-1344(00)00053-1.
Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.
在日本、美国和法国，卟吩姆钠光动力疗法（PDT）已被批准用于某些特定应用，目前正在考虑批准使用如间-(四羟基苯基)氯卟啉（m-THPC）等第二代强效光敏剂。尽管光敏剂可能定位于细胞质或质膜内，但在光动力反应早期核膜可能会受损，从而导致DNA损伤。因此，评估主要用于治疗早期高分化癌症的m-THPC-PDT的安全性至关重要。在此背景下，通过比色MTT法在C6细胞上研究了m-THPC的毒性和光毒性，以确定半数致死剂量（10 J/cm2照射时为2.5 μg/ml m-THPC，25 J/cm2照射时为1 μg/ml）以及诱导约25%细胞死亡的PDT剂量。单细胞电泳（一种计算尾矩的彗星试验）用于评估在进行PDT试验时给予LD25或更高剂量后小鼠胶质母细胞瘤C6细胞中的DNA损伤和修复情况。这些结果与通过共聚焦显微荧光光谱法测定的m-THPC核分布相关。在无光照情况下，m-THPC未能诱导C6细胞的尾矩发生显著变化，而m-THPC-PDT在照射后立即诱导DNA损伤。尾矩的增加并非呈线性（0 - 1 μg/ml m-THPC时曲线斜率为43，1 - 3 μg/ml时为117），但对于1 μg/ml m-THPC孵育，其平均值随光照剂量（0、10或25 J/cm2）和孵育时间（1至4小时每小时）而增加。然而，培养的小鼠胶质母细胞瘤细胞在4小时后能够进行显著的DNA修复，在37℃处理后孵育24小时，未发现明显的残留DNA损伤。对于相似毒性，光照剂量增加似乎比m-THPC剂量增加的遗传毒性更小。我们的结果表明，m-THPC PDT似乎是一种安全的治疗方法，因为DNA修复似乎未受损，且仅在致死性PDT剂量下才会发生DNA损伤。然而，彗星试验无法让我们确定是否未发生突变、光加合物或氧化损伤，因此这一点将通过另一种致突变性试验进行验证。
