Involvement of both Type I and Type II mechanisms in Gram-positive and Gram-negative bacteria photosensitization by a meso-substituted cationic porphyrin

A meso-substituted cationic porphyrin (TMPyP) showed a photocytotoxicity against Gram-positive and Gram-negative bacteria. In order to determine the mechanism involved in the phototoxicity of this photosensitizer, electron paramagnetic resonance (EPR) experiments with 2,2,6,6-tetramethyl-4-piperidone (TEMP), a specific probe for singlet oxygen, and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were carried out with illuminated TMPyP. An EPR signal characteristic of TEMP-singlet oxygen (TEMPO) adduct formation was observed, which could be ascribed to singlet oxygen (O-1(2)) generated by TMPyP photosensitization. The signal for the DMPO spin adduct of superoxide anion (DMPO-OOH) was observed in DMSO solution but not in aqueous conditions. However, an EPR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-OH) was observed in aqueous conditions. The obtained results testify a primary hydroxyl radical ((OH)-O-center dot) generation probably from superoxide anion (O-2(center dot-)) via the Fenton reaction and/or via Haber-Weiss reaction. Gram-positive and Gram-negative bacteria inactivation by TMPyP photosensitization predominantly involved Type II reactions mediated by the formation of O-1(2), as demonstrated by the effect of quenchers for O-1(2) and scavengers for (OH)-O-center dot (sodium azide, thiourea, and dimethylsulphoxide). Participation of other active oxygen species cannot however be neglected since Type I reactions also had a significant effect, particularly for Gram-negative bacteria. For Gram-negative bacteria the photoinactivation rate was lower in the presence of superoxide dismutase, a specific O-2(center dot-) scavenger, and/or catalase, an enzyme which specifically eliminates H2O2, but was unchanged for Gram-positive bacteria. The generation of O-1(2), O-2(center dot-) and (OH)-O-center dot by TMPyP photosensitization indicated that TMPyP maintained a photodynamic activity in terms of Type I and Type II mechanisms. (C) 2008 Elsevier Ltd. All rights reserved.