Shea C R, Hefetz Y, Gillies R, Wimberly J, Dalickas G, Hasan T
Wellman Laboratories of Photomedicine, Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston 02114.
J Biol Chem. 1990 Apr 15;265(11):5977-82.
In order to elucidate the photophysical mechanisms of cellular phototoxicity sensitized by doxycycline, MGH-U1 human bladder carcinoma cells in vitro were treated with 20.7 microM doxycycline and irradiated with either a pulsed (lambda = 355 nm, pulse duration = 24 ps) or a continuous wave (lambda = 351 nm) laser. Cumulative radiant exposure and irradiance were systematically varied in experiments with both lasers. Phototoxicity was assessed by epifluorescence microscopy of unfixed cells using rhodamine 123 labeling of mitochondria. With the continuous wave source, the cumulative radiant exposure required for induction of phototoxic injury was independent of irradiance. With the 24-ps-pulsed source, a significantly lower cumulative radiant exposure was required to induce the phototoxicity when the peak irradiance was 5.8 x 10(7) or 1.3 x 10(8) watts cm-2 compared with when peak irradiance was either lower (6.0 x 10(6) watts cm-2) or higher (7.6 x 10(8) watts cm-2). The measured fluorescence lifetimes of doxycycline in buffered saline solution were longer than the laser pulse duration of 24 ps. The increased efficiency of photosensitization at the optimal peak irradiance in the ps domain appears to result from sequential multiphoton absorption involving higher excited states of the singlet manifold. At the highest irradiance studied, on the other hand, reduced efficiency of photosensitization is attributed to increased photodegradation of doxycycline from higher excited states by processes such as photoionization. A model consistent with these observations is presented along with calculations, based on simple rate equations, that fit the essentials of the proposed model.
为了阐明强力霉素致敏的细胞光毒性的光物理机制，用20.7微摩尔的强力霉素处理体外培养的MGH-U1人膀胱癌细胞，并用脉冲激光（波长=355纳米，脉冲持续时间=24皮秒）或连续波激光（波长=351纳米）照射。在两种激光实验中，系统地改变了累积辐射暴露量和辐照度。使用罗丹明123对线粒体进行标记，通过对未固定细胞进行落射荧光显微镜检查来评估光毒性。对于连续波光源，诱导光毒性损伤所需的累积辐射暴露量与辐照度无关。对于24皮秒脉冲光源，当峰值辐照度为5.8×10⁷或1.3×10⁸瓦/平方厘米时，与峰值辐照度较低（6.0×10⁶瓦/平方厘米）或较高（7.6×10⁸瓦/平方厘米）时相比，诱导光毒性所需的累积辐射暴露量显著更低。在缓冲盐溶液中测得的强力霉素荧光寿命长于24皮秒的激光脉冲持续时间。在皮秒范围内最佳峰值辐照度下光致敏效率的提高似乎是由于涉及单重态高激发态的顺序多光子吸收。另一方面，在研究的最高辐照度下，光致敏效率降低归因于强力霉素从高激发态通过诸如光电离等过程的光降解增加。提出了一个与这些观察结果一致的模型，并基于简单的速率方程进行了计算，这些计算符合所提出模型的要点。
