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A Study of the Uptake of Toluidine Blue O by Porphyromonas gingivalis and the Mechanism of Lethal Photosensitization
Author(s) -
Bhatti Manpreet,
MacRobert Alexander,
Meghji Sajeda,
Henderson Brian,
Wilson Michael
Publication year - 1998
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1998.tb09694.x
Subject(s) - chemistry , singlet oxygen , sodium dodecyl sulfate , tryptophan , photosensitizer , oxygen , rose bengal , nuclear chemistry , photochemistry , biophysics , chromatography , biochemistry , organic chemistry , biology , amino acid
The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO ( 3 H‐TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium‐neon (HeNe) laser and TBO was investigated by using deuterium oxide (D 2 O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L‐tryptophan. There were 9.0 log 10 and 2 log 10 reductions in the presence of D 2 O and H 2 O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm 2 ), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L‐tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/ cm 2 ) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggre‐gation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.