— Since 1972 the protective (quenching) effect of N3- and the enhancing effect of D2O have been used in combination to demonstrate 1O2 (1Δg) as the major reactive intermediate in the dye-sensitized photooxygenation of biomolecules in in vitro systems. Extended application to in vivo systems has recently begun, producing some results which generally support the involvement of 1O2 pathways in the photosensitization processes. The use of other 1O2 quenchers and acceptors has also been increasing. In the application of these techniques as diagnostic tests for 1O2 participation in in vivo systems, careful examination of the experimental conditions should be made with respect to the quenching capability and accessibility of D2O in the specified cellular environment. Furthermore, the diffusive nature of 1O2, generation sites, generation efficiencies, the location and type of sensitizers, and the reactivity of 1O2 with presumed target structures in the cell should also be taken into account in the interpretation. Recent studies illustrating the importance of these factors are discussed. Finally, a tentative picture of the mode of in vivo photodynamic activity of common dye sensitizers is outlined.

Cellular and subcellular mechanisms of photodynamic action: the 1o2 hypothesis as a driving force in recent research

A light quantum, upon absorption, can elevate the absorbing substance to an excited state and it, in turn, may affect a critical component of the cell, under certain conditions, by using this excitation energy Such a substance can either be exogeneous, or a normal component of the cell, and is called a photosensitizer The extent of the cellular effects produced by a photosensitizer is dependent upon a number of factors that include: (i) how easy the sensitizer is excited to a particular state by light of a given wavelength, (ii) how accessible the sensitizer is to the particular site for the relevant photobiological interaction in the cell, and (iii) the importance of the biological role played by the affected substrate in the cell Contributions of all these factors must be properly worked out before any systematic view on biological photosensitization can be constructed

Photodynamic Agents as Tools for Cell Biology

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) contain four major bases—adenine, guanine, cytosine, and thymine (uracil instead of thymine in RNA)—each of which strongly absorbs ultraviolet (UV) radiation with a maximum at about 260 nm. Therefore, UV irradiation of nucleic acids and related model compounds produces electronically excited bases that are able to undergo various photochemical reactions involving neighboring bases, sugar residues, surrounding water molecules, proteins, and bound organic ligands. The photochemical properties of minor bases and base analogues usually differ from those of the major normal bases. In view of this, the study of photochemical processes may well lead to important advances in the recognition of their biochemical functions.

Photochemistry of the Nucleic Acids

Photodynamic treatment of Kluyveromyces marxianus with chloroaluminum-phthalocyanine resulted in loss of clonogenicity. Several parameters were studied to identify targets that could be related to loss of colony-forming capacity. Inhibition of various plasma membrane-bound processes was observed, such as substrate transport and plasma membrane ATPase activity. Moreover, K+ loss from the cells was observed. Photodynamic treatment also reduced the activity of various enzymes involved in energy metabolism, thereby decreasing the cellular ATP level. It will be discussed however that none of these processes is likely to be related directly to loss of clonogenicity. Treatment with phthalocyanine and light resulted in a strong inhibition of the incorporation of 14C-phenylalanine in trichloracetic acid-precipitable material. The induction of the beta-galactoside utilization system was also strongly inhibited. The latter two processes did not recover during incubation, subsequent to photodynamic treatment. It is concluded that photodynamically induced inhibition of protein synthesis is a critical factor contributing to the loss of clonogenicity.

The effect of photodynamic treatment of yeast with the sensitizer chloroaluminum phthalocyanine on various cellular parameters.

The effect of photodynamic treatment on the yeast Kluyveromyces marxianus with aluminum-phthalocyanines has been studied. It was found that the nonsulfonated sensitizer caused light-dependent loss of colony-forming capacity, whereas the mono- and tetrasulfonated forms did not induce loss of clonogenicity. The effect of the nonsulfonated sensitizer increased with longer preincubation periods of cells with the dye. Formation of cellattached, mostly intracellularly localized monomelic sensitizer also increased with time. The amount of cell-bound multimeric nonsulfonated phthalocyanine did not vary with time. Experiments designed to specifically increase the amount of cell-attached monomers led also to an increased photoinactivation of the cells. It is therefore concluded that the photodynamic effect of the nonsulfonated Al-phthalocyanine is mediated by the monomeric form of the dye.

Photodynamic treatment of yeast with chloroaluminum-phthalocyanine : role of the monomeric form of the dye

Die RNS-Synthese inSaccharomyces- Zellen wird bis zu 10 Std nach photodynamischer Behandlung mit Thiopyronin und vergleichsweise auch nach Rontgenbestrahlung untersucht. Wahrend die RNS-Synthese selbst nach Einwirkung hoher Rontgendosen bis zu 2 Std nach Bestrahlung in allen Zellen nur geringfugig gehemmt ist, dann aber in nicht mehr teilungsfahigen Zellen stark inhibiert wird, erfolgt die Blockierung der RNS-Synthese nach photodynamischer Behandlung in nicht mehr teilungsfahigen Zellen sofort, wahrend die uberlebenden, teilungsfahigen Zellen offensichtlich in ihrem Stoffwechsel nicht beeintrachtigt sind. Die Ergebnisse werden als weitere Bestatigung der Tatsache gewertet, das die photodynamische Wirkung in Hefezellen nicht wie nach Rontgenbestrahlung hauptsachlich durch eine DNS-Schadigung verursacht wird, sondern wahrscheinlich durch sofortige Inaktivierung plasmatischer Strukturen.

[Synthesis of RNA in Saccharomyces after photodynamic treatment or irradiation with x-rays (author's transl)].

The content and the synthesis of membrane bound and free ribosomes in growing yeast cells was investigated after photodynamic treatment of the cells with thiopyronine and visible light. It was shown that the synthesis of ribosomes is inhibited after photodynamic treatment and that as a consequence, the content of ribosomes in the cell is diminished.

Thiopyronine sensitized photodynamic effect on ribosome synthesis and ribosome content in Saccharomyces cells.

Abstract After incubation of Saccharomyces cells in [3H] thiopyro nine solution the photodynamically high effective dye was detected only to about 1% in the nucleus of the cell. 99% of the dye is distributed in the cytoplasm.

Verteilung von [3H]Thiopyronin in Hefezellen (Saccharomyces cerevisiae) / Distribution of [3H]Thiopyronine in Yeast Cells (Saccharomyces cerevisiae)

Abstract After the inhibition of protein synthesis in Saccharomyces cells with cycloheximid, the content of membrane-bound ribosomes decreases significantly, whereas the content of free ribosomes remains the same as the untreated control for a longer period of time. This is further evidence that in yeast cells protein synthesis takes place only on membrane-bound ribosomes.

Über den Gehalt an membrangebundenen und freien Ribosomen in Saccharomyceszellen nach Hemmung der Proteinsynthese mit Cycloheximid / The Content of Membrane-Bound and Free Ribosomes in Saccharomyces after the Inhibition of Protein Synthesis with Cycloheximid

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[Synthesis of RNA in Saccharomyces after photodynamic treatment or irradiation with x-rays (author's transl)].

Thiopyronine sensitized photodynamic effect on ribosome synthesis and ribosome content in Saccharomyces cells.

Verteilung von [3H]Thiopyronin in Hefezellen (Saccharomyces cerevisiae) / Distribution of [3H]Thiopyronine in Yeast Cells (Saccharomyces cerevisiae)

Über den Gehalt an membrangebundenen und freien Ribosomen in Saccharomyceszellen nach Hemmung der Proteinsynthese mit Cycloheximid / The Content of Membrane-Bound and Free Ribosomes in Saccharomyces after the Inhibition of Protein Synthesis with Cycloheximid