Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin.

Inherent deficiencies of Leishmania in heme biosynthesis were genetically complemented for delta-aminolevulinate-inducible biosynthesis and accumulation of light-excitable uroporphyrin. The phototoxic flagellar immobilization and cytolysis phenotypes and porphyrin mobilization noted previously were further analyzed biochemically and cytologically to delineate the mechanism of phototoxicity and detoxification in this monoporphyric model. Under optimal conditions of induction for approximately 3 days, cells remained viable but became increasingly uroporphyric, peaking at > or =90% of the population by approximately day 2; thereafter, a small population of less porphyric or aporphyric cells emerged. On exposure to light, the flagella of porphyric cells were immobilized in milliseconds, and singlet oxygen became detectable in their lysates. Both photosensitive phenotypes increased proportionally with the cellular uroporphyric levels and were susceptible to inhibition by azide, but not by D-mannitol. Brief irradiation of the uroporphyric cells produced no appreciable protein degradation but inactivated cytosolic neomycin phosphotransferase and significantly bleached cytosolic green fluorescent protein, which was azide reversible. These cells were irreparably photodamaged, as indicated by their subsequent loss of membrane permeability and viability. This is the first in situ demonstration that early inactivation of functional proteins by singlet oxygen initiates the cytolytic phototoxicity in uroporphyria. Detoxification appears to involve endocytic/exocytic mobilization of uroporphyrin from cytosol to "porphyrinosomes" for its eventual extracellular expulsion. This is proposed as the sole mechanism of detoxification, since it is attributable to the reversion of porphyric to aporphyric cells during uroporphyrinogenesis and repeated cycles of this event plus photolysis selected no resistant mutants, only aporphyric clones of the parental phenotypes. Further characterization of the transport system for uroporphyrin in this model is expected to benefit not only our understanding of the cellular mechanism for disposal of toxic soluble wastes but also potentially the effective management of human uroporphyria and the use of uroporphyric Leishmania for vaccine/drug delivery.

Selective in vivo tumor localization of heptacarboxylic porphyrin isomer I in a bladder tumor model: a novel technique to modulate porphyrin localization.

We were the first to report that uroporphyrin isomer I is a superior tumor localizer when compared with hematoporphyrin derivative. In the present study, we have examined the tumor localization of heptacarboxylic porphyrin isomer I (hepta-P) using a bladder tumor model. We have also compared it to that found with uroporphyrin isomer I (Uro-P). We now show, for the first time, that (hepta-P) isomer I can be selectively retained in bladder malignant cells, a novel observation which has not yet been described by other investigators. Furthermore, we have provided a novel technique to modulate and manipulate blood protein binding to porphyrin in a controlled manner, such that the tumor localization properties can be effectively utilized without prolonged retention in the skin and to produce high uptake in the tumor, i.e., a higher therapeutic ratio. The biodistribution of hepta-P in different organs is presented.