A STUDY OF THE UPTAKE OF TOLUIDINE BLUE O BY PORPHYROMONAS-GINGIVALISAND THE MECHANISM OF LETHAL PHOTOSENSITIZATION

The purpose of the study was to determine the distribution of the phot osensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization o f this organism. The distribution of TBO was determined by incubating P, gingivalis with tritiated TBO (H-3-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, 8.7, 5.4, 1.9, 5.7 and 0.3%, respec tively. The involvement of cytotoxic species in the lethal photosensit ization induced by light from a helium-neon (HeNe) laser and TBO was i nvestigated by using deuterium oxide (D2O), which prolongs the lifetim e 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 D2O and H2O (saline solutions), respectively, at a light d ose of 0.44 J (energy density = 0.22 J/cm(2)), suggesting the involvem ent of singlet oxygen, Decreased kills were attained in the presence o f increasing concentrations of L-tryptophan, The effect of lethal phot osensitization on whole cell proteins was determined by measuring tryp tophan fluorescence, which decreased by 30% using 4.3 J (energy densit y = 4.3 J/cm2) of light. Effects on the OM and PM proteins were determ ined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, The re was evidence of change in the molecular masses of several PM protei ns and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscop ic studies showed that there was coaggregation of P. gingivalis cells when sensitized and then exposed to laser light, These results suggest that lethal photosensitization of P, gingivalis may involve changes i n OM and/or PM proteins and DNA damage mediated by singlet oxygen.