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Much attention in recent years has been given to the antigenotoxicity of chlorophyll. Chlorophyll, however, is known to be converted into pheophytin, pyropheophytin, and pheophorbide in processed vegetable food and following ingestion by humans. Studies were conducted on the antimutagenic and tumoricidal potencies of these compounds. All the chlorophyll derivatives tested exhibit identical antimutagenic effect towards 3-methylcholanthrene (3-MC), suggesting that the porphyrin nucleus may complex directly with the mutagen. It does not exclude, however, another mechanism of activity involving inactivation the enzymatic transformation of 3-MC. In contrast, the action of N'-nitro-N'-nitrosoguanidine (MNNG) depends upon structural differences between the chlorophyll derivatives. It is significantly lower when the phytol-containing pheophytin and pyropheophytin are tested as to that of the phytol-lacking pheophorbide. The higher concentrations of the chlorophyll derivatives were required to reduce the mutagenicity of MNNG than needed for 3-MC. The cytotoxicity of chlorophyll derivatives against tumor cells also was evaluated. The cellular uptake and inhibition of myeloma cell multiplicity were found to be greater for pheophorbide than for pheophytin. Calculated on the amount of cell associated chlorophyll derivative, however, pheophytin was more cytostatic/cytotoxic than pheophorbide. The results presented in this report indicate that food sources that yield chlorophyll derivatives may play a significant role in cancer prevention. Teratogenesis Carcinog. Mutagen. 19:313-322, 1999.

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The preparation of chlorophyllin copper complex (CCC), shown to be a tumor promoter in an animal model (Nelson, R.L. (1992) Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model. Anticancer Res., 12, 737-740), also inhibits the activities of direct- and indirect-acting mutagens in the Salmonella assay and exhibits cytostatic and cytocidal effects toward myeloma cells. Data from elemental analyses, spectrophotometry and reversed-phase high-performance liquid chromatography indicate that CCC preparations generally used in antimutagenic/anticarcinogenic experiments are variable, complex mixtures of structurally distinct porphyrins lacking copper in some instances. This variability of the composition may be a cause for the differences reported for the tumor promotion activity of CCC.

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Pheophorbide a is a photocytotoxic agent. To develop a tissue-specific, intracellularly targeted photoactive system, pheophorbide a was incorporated into immunoliposomes coated with a monoclonal antibody (T-43) directed against the T-24 bladder tumor cell line. The efficacy of this system was studied in vitro using the human bladder tumor cell line MGH-U1. Uptake and localization were determined by the fluorescence of the immunoliposome markers within biochemically resolved subcellular components. The results demonstrate localization of the immunoliposome markers within the lysosomes of the tumor cells. Specific monoclonal antibody enhancement of the immunoliposomes uptake by MGH-U1 cells was demonstrated by the use of soluble T-43 monoclonal antibody as a competitive inhibitor. Pheophorbide-a-loaded immunoliposomes were shown to be photocytotoxic towards MGH-U1 cells at concentrations equivalent to photosensitizer at 500 ng ml-1. Treated cells, when protected from light, showed no cytotoxicity. These results demonstrate that uptake of pheophorbide-a-containing immunoliposomes by target cells and subsequent delivery to the lysosomes cause photoactivated killing of tumor cells. The utilization of immunoliposomes for intracellular lysosomal targeting of photoactive drugs to tumor cells constitutes a potentially valuable approach to photodynamic therapeutics.

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We propose the use of acetoxymethyl esters of pH-sensitive amphipathic photosensitizers (PS) for photodynamic therapy (PDT). These compounds may be applicable for PDT involving endocytosis of lipophilic carriers leading to lysosomal uptake of the esterified PS by target cells. Partial and/or total enzymatic de-esterification may result in the extralysosomal distribution of the photoactive agents, possibly culminating in a multisite photochemical response. We report here the synthesis and properties of chlorin e6 triacetoxymethyl ester (CAME) and pheophorbide a acetoxymethyl ester (PAME). Chlorin e6 and pheophorbide a are photocytotoxic chlorins that possess free carboxylate groups and exhibit optimum wavelengths of excitation substantially red shifted relative to hematoporphyrin derivative. Acetoxymethyl esterification of chlorin e6 and pheophorbide a was accomplished with bromomethyl acetate. High-performance liquid chromatography allowed for the purification of PAME, in 87% purity, and CAME, in 63% yield and 94% purity, as well as the detection of the presumed mono- and diesters of chlorin e6 as transient intermediates in the synthesis of CAME. The ultraviolet-visible absorption, fluorescence excitation and emission, NMR and mass spectra of the chlorin e6 triester are consistent with those expected for CAME. The pH-sensitive amphipathicity of pheophorbide a and chlorin e6 but not CAME was demonstrated using a water/1-octanol partition assay. The production of pheophorbide a from PAME and the sequential formation of the di- and monoesters and free chlorin e6 from CAME, by the action of lysosomal esterases obtained from cancer cells, demonstrate the potential of cellular enzymes to convert the lipophilic esters to pH-sensitive amphipathic PS.(ABSTRACT TRUNCATED AT 250 WORDS)

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Pheophorbide a-induced photo-oxidation, in vitro, of cytochrome c oxidase and cytochrome c results in irreversible modifications to both protein components. Photo-oxidation of cytochrome c, as exhibited by change in its heme oxidation state, displays exponential kinetics and is detected with a lag period. Both the photo-induced inactivation of the enzyme, and destruction of the substrate ability of cytochrome c occur as complex multi-process events. Under similar experimental conditions, the loss of the substrate capability of cytochrome c develops approximately three times faster than inactivation of the enzyme. The slight lag in the photo-oxidation of cytochrome c is due to pheophorbide a-induced superoxide production. However, the relative amount of photo-oxidant produced is considerably more effective than the cytochrome c reducing capacity of the superoxide. Neither hydroxyl radical nor hydrogen peroxide are involved in the photo-oxidation of the heme function. The possibilities of heme oxidation by a singlet oxygen mediated pathway or direct electron abstraction involving the heme or apoprotein are not excluded. It is proposed that a multi-site oxidation of numerous reduced energy cofactors within cells may augment collateral enzyme inactivation in maximizing photosensitizer-induced cytotoxicity. Accordingly, amphipathic photosensitizers, capable of accessing both lipid and aqueous compartments containing reduced cofactors, may be more effective agents for photodynamic therapy than those which exhibit a high specificity of subcellular localization.

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