Tsvetan G. Gantchev

CATALASE INACTIVATION FOLLOWING PHOTOSENSITIZATION WITH TETRASULFONATEDMETALLOPHTHALOCYANINES

Abstract— Catalase (CAT) in solution or incorporated in erythrocytes and K562 leukemic cells is inactivated during photosensitization with tetrasulfonated metallophthalocyantnes (MePcS4). The effect of added scavengers and D20 showed that both singlet oxygen and free radical species are involved in this process. Evidence was found that direct interactions of ground or excited‐stated photosensitizers with CAT are not responsible for CAT inactivation. Specific techniques to probe early damage to the CAT structure involved optical and EPR spectroscopy, HPLC and polyacrylamide gel electrophoresis analyses. Different primary events of photosensitized protein damage included oxidation of cysteine residues as well as other amino acids, as demonstrated by the formation of carbon‐centered free radicals and the loss of absorbance at λ= 275 nm. In parallel, we detected degradation of the CAT heme groups, accompanied by release of Fe(II) ions in solution. These combined phenomena initiate cross‐linkages between CAT subunits and subsequent degradation of the protein with formation of irreversible aggregates in solution. Phthalocyanine‐mediated photoinactivation of cell‐bound CAT results in loss of protection against accumulating H202, providing an additional pathway of phototoxicity.

DNA interstrand cross-links induced by ionizing radiation: an unsung lesion.

The induction of DNA interstrand cross-links by ionizing radiation has been largely ignored in favour of studies on double-strand break formation and repair. At least part of the problem is technical; it is difficult to detect and quantify interstrand cross-links when the same agent forms both cross-links and single strand breaks because the detection of interstrand cross-links generally involves a denaturation step. Our group has studied the induction of interstrand cross-links following irradiation of DNA containing bromouracil at specific sites. We found that the formation of interstrand cross-links requires the presence of a few (3-5) mismatched bases, comprising the bromouracil. In the absence of mismatched bases, no radiation-induced cross-linking was observed; however, even in the absence of bromouracil, cross-linking still occurred, albeit at a lower efficiency. Our molecular modelling studies demonstrate that the mobility of the bases in the mismatched region is essential for the cross-linking process. Thus, our hypothesis is that ionizing radiation induces DNA interstrand cross-links in non-hybridized regions of DNA. Some obvious examples of such DNA regions are replication forks, transcription bubbles and the D-loop of telomeres. However, an abundance of studies have made it clear that there must be many single-stranded regions in the genome, such as hairpins and cruciforms. For example, alpha satellite DNA, in centromere regions of human chromosomes, forms hairpins. Thus, a variety of non-B DNA structures (hairpins, slipped DNA and tetrahelical structures) exist in the genome and should be susceptible to the formation of radiation-induced interstrand cross-links. Although interstrand cross-links have thus far been virtually ignored in radiation biology, it will be worthwhile to develop methods to detect their presence following exposure of cells to biologically relevant levels of ionizing radiation, since, on a per lesions basis, they are probably more toxic than double-strand breaks.

Enhancement of etoposide (VP-16) cytotoxicity by enzymatic and photodynamically induced oxidative stress.

The effects of glutathione (GSH) depletion by buthionine sulfoximane (BSO) or by photosensitization-induced oxidative stress using metallo-phthalocyanines (MePcS4) on etoposide (VP-16) cytotoxicity against K562 human leukemic cells were investigated. Both treatments enhanced VP-16 toxicity in a markedly synergistic way, as revealed by combination index analysis procedure. Synergistic drug interactions were accompanied by a supra-additive induction of DNA strand breaks. The proposed role of intracellular GSH in preventing metabolic transformations of VP-16 and thus decreasing its toxicity was confirmed by electron spin resonance (ESR) monitoring of the accumulation of the VP-16 phenoxyl radical in cell cytoplasm subjected to GSH depletion. Taken together the results emphasize the beneficial effect of GSH-related oxidative stress in enhancement of etoposide toxicity and possibly in its anticancer applications.

Photocytotoxicity and intracellular generation of free radicals by tetrasulphonated Al- and Zn-phthalocyanines

The photosensitizing properties of tetrasulphonated Al- and Zn-phthalocyanines (AlPcS4 and ZnPcS4) in lymphoma cells were studied as a function of the pre/post-illumination incubation time. Photocytotoxicity increased with incubation time, ranging from a transient cell-cycle arrest to cell killing. Under all experimental conditions, the phototoxicity of ZnPcS4 was markedly higher than that of AlPcS4. The primary photoprocesses initiated by metallo-phthalocyanines (MePcS4) in the cells were probed with DMPO/esr spin-trapping techniques. Under all incubation conditions the intracellularly bound MePcS4 sensitized formation of three different types of DMPO spin-adducts: DMPO/OH (hydroxyl radical), DMPO/R (organic carbon-centred radical(s)) and an unidentified simple nitroxyl, referred to as DMPO/ox. The yields of trapped radicals depended on the length of the incubation with the dyes prior to illumination and the formation of spin-adducts was shown to be intracellular. The ability of DMPO to protect cells from the photocytotoxic effects of Al- and ZnPcS4, combined with the generation of carbon-centred spin-adducts is direct evidence for the involvement of free-radical-mediated damage of cellular constituents.

Combination toxicity of etoposide (VP-16) and photosensitisation with a water-soluble aluminium phthalocyanine in K562 human leukaemic cells

Etoposide (VP-16) is an anti-cancer drug commonly used against several types of tumours and leukaemia, either alone or in combination chemotherapy. Photodynamic therapy (PDT) is another, relatively new modality for treatment of various malignancies. The interactions between VP-16 and PDT, using aluminium tetrasulphophthalocyanine as photosensitiser, in K562 human leukaemic cells were investigated. Cell responses to individual and combined drug treatment under different experimental conditions revealed synergistic drug toxicity. The latter was evident from various events of cell response, including supra-additive accumulation of cells in G2/M cell cycle phase and endonucleolytic DNA fragmentation (apoptosis). The involvement of the cellular antioxidant system in the synergistic interactions of photosensitisation and VP-16 is proposed.

Metallophthalocyanines Photosensitize the Breakdown of (Hydro)peroxides in Solution to Yield Hydroxyl or Alkoxyl and Peroxyl Free Radicals via Different Interaction Pathways

Interactions of organic peroxides (R′OOR) and hydroperoxides (R′OOH), including H2O2, with excited triplet and singlet state metallophthalocyanines (MPc, M = Zn, Al) have been studied by T–T absorption decay and fluorescence quenching. The ensuing photochemical processes result in decomposition of (hydro)peroxides as assessed by photo‐EPR (electron paramagnetic resonance) and spin trapping. In argon‐saturated apolar solutions and low MPc concentrations, alkoxyl free radicals (·OR) were identified as the primary products of (hydro)peroxide breakdown. Similarly, photosensitized decomposition of symmetric disulfides results in the formation of sulfur‐centered radicals. In air‐free aqueous solutions, ROOH photosensitization always gave rise to a mixture of hydroxyl and peroxyl radical (·OOR) adducts in varying molar ratios. At high MPc concentrations, both in polar and in apolar solutions, the most abundant products of ROOH decomposition were identified as ·OOR. This indicates a change in the predominant interaction pathway, most likely mediated by MPc exciplexes and involving H‐atom abstraction from ROOH by MPc‐cation radicals. The prevalence of MPc singlet vs. triplet state interactions was confirmed by the much higher singlet quenching rate constants (log kq up to 9.5; vs. log kT≤ 4.5). In contrast to the triplet quenching, singlet quenching rates were found to depend on the (hydro)peroxide structure, following closely the trend of varying ·OR yields for different substrates. Thermodynamic calculations were performed to correlate experimental results with models for electronic energy and charge transfer processes in agreement with the Marcus theory (Rhem and Weller approximation) and Savéants model for a concerted dissociative electron transfer mechanism.

[63] Interactions of phenoxyl radical of antitumor drug, etoposide, with reductants in solution and in cell and nuclear homogenates: Electron spin resonance and high-performance liquid chromatography

Publisher Summary Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers, including testicular and small lung cancers and lymphoma. Mechanisms of cytotoxicity of VP-16 may involve DNA strand cleavage or topoisomerase II inhibition accompanied by formation of DNA-topoisomerase cross-links and DNA strand breaks. The VP-16 molecule contains a hindered phenolic group, which is crucial for its antitumor activity. Several enzymatic systems (peroxidase, tyrosinase, prostaglandin synthase, cytochrome P-450) and exogenous sources of peroxyl radicals (for example, azo initiators) activate VP-16 via one-electron oxidation of the phenolic group to yield reactive metabolites (quinones) capable of irreversible binding to macromolecular targets (DNA and proteins) and generating hydroxyl radicals in the presence of metal catalysts. An essential step in the process of VP-16 activation is the formation of its phenoxyl radical that can be further either converted to oxidation products or reduced by intracellular reductants to the initial phenolic form.

Interactions of photo-excited zinc phthalocyanine with (hydro)peroxides: triplet vs. singlet state pathways

Abstract Photo-excited Zn-phthalocyanine (ZnPc) interactions with four (hydro)peroxides (ROOR′) were studied by T–T absorption decay and fluorescence quenching. Triplet state interactions are weak (logkq∼4) and independent from the quencher used, while singlet quenching is variable (logkq 4.5–9.4). EPR and spin-trapping show that at low ZnPc concentrations the primary decomposition products are alkoxyl radicals. At high ZnPc concentrations, photosensitization of ROOH gives rise to peroxyl radicals. The (hydro)peroxide reducing potentials were estimated following AM1 MO-computations. Our experimental data and theoretical predictions (Marcus–Saveant theory) are consistent with a charge transfer mechanism to account for the alkoxyl radical formation.

Probing the interactions of the solvated electron with DNA by molecular dynamics simulations: II. bromodeoxyuridine-thymidine mismatched DNA

AbstractThe interaction of solvated electrons