Mechanisms of photodynamic sensitization

917 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925

3. Mechanisms of photodynamic sensitization

Upon absorption of UV or visible photons, a photo- sensitizer can be promoted to a variety of electronically excited states. However, the efficiency of the photosensi- tizing action is generally dependent on the photophysical properties of the lowest excited triplet state 3 Sens, which is reached via intersystem crossing from the initially formed excited singlet state 1 Sens. The 3 Sens species is most often characterized by a lifetime in the microsecond to millisecond range, hence it can play a major role in diffusion-controlled processes Carmichael and Huh, 1986. Several deactivation pathways are poss- ible for 3 Sens; those which are of utmost importance from a photobiological point of view can be schematized as follows. 1. Electron transfer to or from a substrate with suitable redox properties, e.g. 3 Sens1Sub→Sens ·+ 1Sub · − This pathway, defined as a type I mechanism Jori and Reddi, 1991, leads to the generation of radical inter- mediates, which in turn can undergo further reactions with other substrates, solvent molecules, or oxygen. The latter process results in the formation of oxidized pro- ducts. A particular case occurs when oxygen acts as an electron acceptor: 3 Sens1O 2 →Sens ·+ 1O 2 · − The superoxide anion has a relatively low level of reac- tivity. However, under certain experimental conditions, it can be converted to very reactive and cytotoxic spec- ies, such as the hydroxyl radical and hydrogen peroxide Bensasson et al., 1983. 2. Energy transfer to any substrate whose triplet state energy lies at a lower level compared with the photosen- sitizer triplet state; this pathway is defined as a type II mechanism Jori and Reddi, 1991: 3 Sens1Sub→Sens1 3 Sub Most components of cells and tissues are not suitable acceptors of electronic energy from 3 Sens, since their triplet states are too energetic. One notable exception is represented by oxygen; this ubiquitous component of biological systems can be readily promoted to its excited singlet state, whose energy level lies at only 22.5 kcal above the triplet ground state and which is endowed with a high cytotoxicity: 3 Sens1 3 O 2 →Sens1 1 O 2 The high reactivity of 1 O 2 is partly due to its long life- time 3–4 µ s in aqueous media, several tens of microse- conds in lipid environments which allows this species to diffuse over relatively long distances before being deactivated Wasserman and Murray, 1989. Both type I and type II photosensitization mechanisms generate electrophilic species, hence the most photosen- sitive targets are represented by electron-rich biomolec- ules Vilensky and Feitelson, 1999. Table 1 shows that of the naturally occurring amino acids only those which possess aromatic or sulphur-con- taining side chains are readily photooxidized. Other rap- idly attacked moieties include carbon–carbon double bonds of unsaturated lipids and steroids, as well as the heterocyclic ring of guanosine nucleotides. At a cellular level the photosensitizing action is characterized by an additional selectivity, since the overall photoprocess is generally confined to the microenvironment of the pho- tosensitizer owing to the tendency of the photogenerated intermediates to react with a large variety of targets Moan et al., 1995. Thus, it appears essential to control the subcellular distribution of the photosensitizing agent. In this connection, a critical role is performed by the chemical structure of the photosensitizer, and in parti- cular by its degree of hydrophobicity Jori and Reddi, 1993. This parameter is usually measured by the par- tition coefficient between n-octanol and water. Thus, moderately or highly lipophilic dyes partition coef- ficient .8–10 become preferentially associated with the cell membranes; at short incubation times the plasma membrane represents the main binding site, while at longer times significant photosensitizer concentrations are recovered from other subcellular membranes includ- ing the mitochondrial and lysosomal membranes, the Golgi apparatus and the rough endoplasmic reticulum. For in vivo administration, amphiphilic photosensitizers which are sufficiently water-soluble, yet are charac- terized by the presence of a hydrophobic matrix facilitat- ing the crossing of the lipid domains of cell membranes proved to be particularly useful. Hydrophilic photosensi- tizers show a more complex pattern; although such com- pounds, especially if electrically charged, undergo ionic interactions with charged groups at the cell surface, the possibility exists of their internalization by both active or passive diffusion processes. Thus, cationic photosen- sitizers are often localized in mitochondria, while anionic dyes e.g. carboxylated derivatives are accumulated at the lysosomal level Spikes, 1994; Jori, 1996. Lastly, some photosensitizers, such as furocoumarins, can reach the cell nuclei and bind to the DNA bases Armitage, 1998. The subcellular localization of a photosensitizer determines the efficiency, as well as the mechanism of photoinduced cell inactivation; in particular, it controls the relative weight of apoptotic and random necrotic pathways which are eventually responsible for cell death He et al., 1994

4. Main classes of photoactivatable insecticides