Spesia Mariana B, Caminos Daniel A, Pons Patricia, Durantini Edgardo N
Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro. 3, X5804BYA Río Cuarto, Córdoba, Argentina.
Photodiagnosis Photodyn Ther. 2009 Mar;6(1):52-61. doi: 10.1016/j.pdpdt.2009.01.003. Epub 2009 Feb 7.
Photodynamic inactivation (PDI) of Escherichia coli has been studied in cultures treated with zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc(+4)) to obtain insight about the mechanism of damage. This phthalocyanine is rapidly bound to cells, reaching a value of approximately 0.8 nmol/10(6) cells when the cultures were incubated with 2 microM sensitizer. After 30 min of irradiation, a 4 log decrease of E. coli survival was observed. The photocytotoxic action was investigated in plasmid and genomic DNA by electrophoretic analysis. Absorption spectroscopic studies showed that this cationic phthalocyanine interacts strongly with DNA (K(DNA)=4.7 x 10(6)M(-1)). Photocleavage of calf thymus DNA sensitized by ZnPPc(+)4 was not found even after long irradiation periods. Similar results were also observed in genomic DNA extracted from E. coli cells after PDI treatment. Modifications of plasmid DNA isolated from bacteria were only observed after long irradiation periods. However, under these conditions transmission electron microscopy of the PDI bacteria revealed an aggregation of cytoplasmic macromolecules and irregularities in cell barriers. Also, scanning electron microscopy showed a shrunken appearance in cells after PDI. Even so, release of intracellular biopolymers was not detected by absorption. On the other hand, outer and inner membranes permeabilization assays showed an increase in the permeability. Consequently, alterations in the cell membrane functionality induced by ZnPPc(+4) appear to be the major cause of E. coli inactivation upon PDI.
已在用锌（II）2,9,16,23 - 四[4 - （N - 甲基吡啶氧基）]酞菁（ZnPPc(+4)）处理的培养物中研究了大肠杆菌的光动力灭活（PDI），以深入了解损伤机制。这种酞菁能迅速与细胞结合，当培养物与2μM敏化剂孵育时，其结合量达到约0.8 nmol/10(6)个细胞。照射30分钟后，观察到大肠杆菌存活率下降了4个对数级。通过电泳分析研究了质粒和基因组DNA中的光细胞毒性作用。吸收光谱研究表明，这种阳离子酞菁与DNA强烈相互作用（K(DNA)=4.7×10(6)M(-1)）。即使经过长时间照射，也未发现由ZnPPc(+)4敏化的小牛胸腺DNA的光切割现象。在PDI处理后的大肠杆菌细胞中提取的基因组DNA中也观察到了类似结果。仅在长时间照射后才观察到从细菌中分离出的质粒DNA的修饰。然而，在这些条件下，对PDI处理后的细菌进行透射电子显微镜观察发现细胞质大分子聚集且细胞屏障出现不规则现象。此外，扫描电子显微镜显示PDI处理后的细胞外观收缩。即便如此，通过吸收未检测到细胞内生物聚合物的释放。另一方面，内膜和外膜通透性测定表明通透性增加。因此，ZnPPc(+4)诱导的细胞膜功能改变似乎是PDI作用下大肠杆菌失活的主要原因。
