Synthetic dyes, including thiazines, acridines, xanthenes, and a phenazine, were used as models for studying the photodynamic effect. In the presence of physiological reductants, the illuminated dyes produced superoxide (O2-), hydrogen peroxide (H2O2), and hydroxyl free radical (OH.)[Please note that the dot after 'OH' should be midway up the space, not like a period, as html is displaying here and throughout the thesis.]. Suitable reductants included NADH, glutathione, GMP, cysteine, tryptophan, and tyrosine. The production of OH. was dependent on chelated iron or on copper. Effective iron chelators included EDTA, DTPA, dipyridyl, and phenanthroline, and biologically significant compounds such as ATP, ADP, succinate, citrate, and DNA. DNA intercalation of the dyes did not prevent oxidization of NADH or the production of O2- or OH.. Hydroxyl radical scavengers competed effectively in the assays for the OH.. While in the Haber-Weiss reaction superoxide reduces iron, which is oxidized by H2O2 to produce OH., excited state reduced dyes appeared capable of reducing the iron, so that SOD was only partially inhibitory of OH. production. Catalase prevented production of OH..
Similar results were found for substrates and metals in an assay of single strand scission of DNA mediated by the dyes. Amounts of strand scission seen were dependent on concentrations of iron or dye. Cysteine, NADH, GTP, dGMP, tryptophan, and tyrosine were all able to provide electrons for the strand scission reaction. Furthermore, the single strand scission of DNA by the dyes was prevented by scavengers of O2-, H2O2, or OH. which are poor scavengers for singlet oxygen (1O2), and substitution of deuterium oxide for water, which exacerbates any damage produced by 1O2, did not increase damage.
The physiological reductant glutathione in E. coli was depleted by exposure to illuminated dye. Lethality of the dyes was reduced by enhanced levels of catalase or endonuclease IV provided by plasmid-coded genes, indicating that H2O2 is an important mediator of toxicity and that DNA is an important target, while OH. scavengers prevented kill, indicating that the OH. is also an important mediator of phototoxicity. Finally, DNA damage in vivo was reduced by an OH. scavenger. In conclusion, toxicity in the photodynamic effect is mediated by reduced oxygen species, particularly OH., and DNA damage probably underlies this toxicity.
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