Mechanism of photodynamic activity of pheophorbides.

Plasmid DNA is efficiently photocleaved by sodium pheophorbides (Na-Phdes) a and b in the absence of oxygen as well as in the presence of oxygen. Fluorescence microscopic observation shows a rapid incorporation of Na-Phde a into nuclei, mitochondria, and lysosome of human oral mucosa cells. In contrast Na-Phde b is incorporated only into the plasma membrane. The photodynamic activity of these pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures and involves two types of reactions: (i) direct electron transfer between DNA bases (especially guanine) and pheophorbide singlet excited state, and (ii) indirect reactions mediated by reactive oxygen species, including singlet oxygen whose production from molecular oxygen is sensitized by the Na-Phdes triplet state. A preliminary report has appeared in "Photodynamic Therapy of Cancer II," Proc. SPIE 2325, 416-424 (1994).

Quantum yield of singlet oxygen production by monomeric and aggregated forms of hematoporphyrin derivative.

Hematoporphyrin derivative (HpD) is a complex mixture of monoporphyrinic compounds, including hematoporphyrin, and oligomers containing up to eight porphyrin units. In methanol a sensitizer concentration-independent quantum yield of 0.64 is measured for the HpD-induced production of singlet molecular oxygen O2 (1Delta(g)). This finding is consistent with the dye components remaining unassociated in this solvent. In water pH 7.4 HpD consists of a complex mixture of non-aggregated and self- and cross-aggregated monoporphyrinic and oligomeric species, and the quantum yield of O2 (1Delta(g)) formation decreases significantly with increasing HpD concentration due to the lower quantum yield of aggregates. These variations can be quantitatively described in terms of a monomer-dimer equilibrium, with quantum yields of 0.64 and 0.11, respectively, for monomers and dimers. The yields of unassociated species are identical in methanol and water.

Mechanistic and kinetic aspects of photosensitization in the presence of oxygen.

Determining whether the first step of photooxygenation is Type I or Type II is a necessary prerequisite in order to establish the mechanism of photodynamic action. But this distinction is not sufficient, because other processes, both consecutive and competitive, commonly participate in the overall mechanism. Thus, in both Type I and Type II reactions, the initial products are often peroxides that can break down and induce free radical reactions. These aspects of photosensitization are discussed and illustrated by sensitizer/substrate systems involving (1) only radical reactions (decatungstate/alkane) and (2) reactions of singlet oxygen occurring in competitive and consecutive processes and possibly followed by radical reactions (methylene blue/2'-deoxyguanosine). Two other previously investigated systems involving, respectively, a Type II interaction followed by radical processes (methylene blue/alkene) and Type II reactions, some of which being competitive or consecutive (rose bengal/alkene), are briefly reconsidered.

Kinetics of the competitive degradation of deoxyribose and other biomolecules by hydroxyl radicals produced by the Fenton reaction.

The objective of this work is to reexamine the competitive degradation of deoxyribose by hydroxyl radicals (.OH) produced by the reaction between H2O2 and Fe(2+)-EDTA. The .OH radicals produced attack deoxyribose (D, rate constant kD) and eventually an .OH scavenger (S, rate constant kS). First, we examined the effect of [D], [H2O2], [Fe(2+)-EDTA], [EDTA]/[Fe2+] ratio and reaction time on the rate of D degradation, measured as the absorbance of the chromogen formed between the product of the reaction D + .OH (malondialdehyde) and thiobarbituric acid. In particular, it was showed that under our experimental conditions ([D] = 3 mM, [H2O2] = 0.85 mM, [Fe2+] = 0.13 mM), the rate of overall process is first order in Fe2+, zero order in H2O2 and is maximal for a ratio [EDTA]/[Fe2+] = 1.1. Second, the kinetics of .OH radical reaction in competition experiments between D and S (mannitol) was investigated. The results show that the ratio of the rates of D degradation in the absence (VD) and in the presence (VDS) of S should be represented by VD/VDS = 1 + ks[S]/(kD[D] + kx) where kx accounts for the rate of .OH reactions with other reagents such as Fe(2+)-EDTA, H2O2 etc . . . After having determined kx for each set of experimental conditions, we obtained the values of kS/kD by determining the variations of VD/VDS as a function of [S] and [D]. By taking kD = 1.9 x 10(9) M-1s-1 a value of kS = 1.9 x 10(9) M-1s-1 was obtained, very close to that obtained by pulse radiolysis. Finally, the validity of the established relation was confirmed for other biomolecules (methionine, k = 5.6 x 10(9)M-1s-1 and alanine, k = 3.3 x 10(8) M-1s-1). By contrast, it was not applicable to cysteine, thiourea and mercaptoethanol which was attributed to an interaction of the latter scavengers with Fe2+ and/or H2O2.

Reaction and quenching of singlet molecular oxygen with esters of polyunsaturated fatty acids.

The rate constants for the reactive (kR) and unreactive (kQ) interaction of singlet molecular oxygen with three esters of polyunsaturated fatty acids (PUFA: cis-methyl oleate, MO; cis-methyl linoleate, MLA and cis-ethyl linolenate, ELN) are determined. The values of the ratio kQ/kR are 0.51, 0.26 and 0.20 for MO, MLA and ELN, respectively. This variation results principally from that of kR because the values of kQ are only slightly different (1.24 x 10(4) M-1 s-1 for MO and approximately 1.0 x 10(4) M-1 s-1 for MLA and ELN). It is shown that the rate constant kQ characterizes mainly an interaction with the unreactive part of the molecule rather than with the double bonds (solventlike quenching). Contrary to the already reported case of 1,5-polyenes for which kQ << kR, the present results and those obtained from a number of literature data show that for PUFA and their esters, neither kR+kQ nor kR are proportional to the total number of double bonds or of methylene groups adjacent to the double bonds. Instead, a linear correlation is observed by plotting kR vs the number of methylene groups adjacent to two double bonds. It is deduced that contrary to a common assumption, biallylic hydrogens have a reactivity higher than that of singly allylic hydrogens (reactivity ratio 1.19). The consequence of this result on the estimation of relative contributions of singlet oxygen and radical mechanisms to oxidation processes is discussed. Moreover, the whole of these results allows prediction of the values of kR and kQ for all unsaturated fatty acids (and their esters) of similar structure.

Photosensitization of L1210 leukaemic cells by argon laser irradiation after incubation with haematoporphyrin derivative and rhodamine 123.

The selectivity and efficacy of photodynamic therapy (PDT) may be improved by the combined use of photosensitizers in a similar manner to the combined use of drugs in cancer chemotherapy. Two photosensitizers (haematoporphyrin derivative (HPD) and rhodamine 123 (Rh-123) were analysed which can be irradiated at the same wavelength (514 nm), are preferentially taken up by tumour tissue and are not specific for the same target (membrane for HPD, mitochondria for Rh-123). The analysis of the phototoxic effects in surviving fractions showed a dependence on dose for both products and a dependence on incubation time for HPD but not Rh-123. The lethal dose for 50% cell death (LD50) for HPD increased from 25 to 56 J cm-2 when the HPD dose was reduced from 2.5 to 1 micrograms ml-1 for the same incubation time. When the incubation time was increased from 15 to 45 min, the surviving fraction decreased by 37% and 17% for doses of 1 and 2.5 micrograms ml-1 respectively. For low doses (0.5 and 1 microgram ml-1), the toxicity of the two photosensitizers added simultaneously was weaker than for Rh-123 alone, whereas for high doses (2.5 micrograms ml-1) the surviving fraction was less than that obtained with Rh-123 alone. These results were compared with the light energy absorbed, the quantum yield of singlet oxygen and Rh-123 uptake as determined by flow cytometry analysis.

[Photo-oxygenation sensitized by dyes]. / Photooxygénations sensibilisées par les colorants.

This paper discusses the mechanism of photooxygenation reactions sensitized by dyes, such as: rose bengal, methylene blue, tetraphenylporphin and chlorophyll a. First it is shown more particularly that: the quantum yields of singlet oxygen O2(1 delta g) production gamma delta, and of intersystem crossing singlet S1-triplet T1, gamma is, are not always equal and that the possibilities gamma delta greater than or less than gamma is may occur or are observed; the processes S1 + O2(3 sigma)----T1 + O2(3 sigma) and T1 + O2(3 sigma)----S0 + O2(1 delta g) are mainly if not fully responsible for the quenching of the singlet and triplet excited states of the sensitizer by oxygen. Thereafter, clear indication is given of the considerable complication of the photooxygenation which may arise from particular properties of the investigated substrate A (or of other compounds present in the reaction medium) and which may result in a decrease of the oxygenation quantum yield phi O2. It is shown that this lowering of phi O2 is due to that of gamma delta and/or of phi A, (the probability that O2(1 delta g) yields an oxygenation product AO2) since phi O2 = gamma delta phi A. The latter effect can be induced by the quenching of singlet oxygen by the dye, a process which is quite general and which must be always taken into account in kinetic studies.