Cytotoxicity But No Mutagenicity In Bacteria With Externally Generated Singlet Oxygen

W. Robert Midden; Thomas A. Dahl; Philip E. Hartman

doi:10.1117/12.942699

19 February 1988 Cytotoxicity But No Mutagenicity In Bacteria With Externally Generated Singlet Oxygen

W. Robert Midden, Thomas A. Dahl, Philip E. Hartman

Proceedings Volume 0847, New Directions in Photodynamic Therapy; (1988) https://doi.org/10.1117/12.942699
Event: Cambridge Symposium on Optics in Medicine and Visual Image Processing, 1987, San Diego, CA, United States

Abstract

Singlet oxygen is believed to be an important intermediate responsible for the cytotoxicity of HpD phototherapy. It has been recognized as a possible intermediate in photosensitization for more than 20 years. However, it has been difficult to obtain conclusive evidence of its biological characteristics in the past because most of the methods available for its generation that are compatible with biological systems also generate other reactive intermediates whose effects are difficult to distinguish from singlet oxygen. We have used a recently devised separated-surface-sensi-tizer (S-S-S) system for singlet oxygen generation' to measure the cytotoxicity and mutagenicity of singlet oxygen in bacteria. The S-S-S system employs rose bengal as a sensitizer immobilized on one surface of a glass plate. The glass plate is placed sensitizer-side down a small distance (< 1.5 mm) above a microscopically flat membrane (MilliporeTM or NucleoporeTM) that carries a monocellular layer of bacteria. The sensi-tizer-coated plate is illuminated from above to generate singlet oxygen at the surface of the sensitizer. The singlet oxygen thus generated can diffuse the short dis-tance to the surface of the membrane to react with the bacteria. Because of the short lifetime of singlet oxygen in air, increasing the distance between the sensitizer and the membrane causes a decline in the amount of singlet oxygen reaching the membrane according to a function derived from the Einstein-Smoluchowski equation for net displacement by diffusion. Plotting the log of the effect measured (e.g., cytotoxicity) vs. the square of the distance gives a straight line. The slope of this line can be used to calculate the gas phase half life of the intermediate responsible for the observed effects. We have found that bacteria are rapidly killed in the illuminated S-S-S system and that the gas phase half life of the agent responsible for cell killing is the same as that of singlet oxygen. This observation and other simple chemical tests have conclusively estab-lished that singlet oxygen is responsible for the cytotoxicity observed with bacteria. Dosimetry measurements allow us to estimate that singlet oxygen is at least 104 times more potent as a cytotoxin for Salmonella bacteria than hydrogen peroxide, on a molar basis. We have not observed mutagenicity in these bacteria exposed to sufficient singlet oxygen to kill 60-90% using a variety of bacterial strains and assays.

Citation Download Citation

W. Robert Midden, Thomas A. Dahl, and Philip E. Hartman "Cytotoxicity But No Mutagenicity In Bacteria With Externally Generated Singlet Oxygen", Proc. SPIE 0847, New Directions in Photodynamic Therapy, (19 February 1988); https://doi.org/10.1117/12.942699

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