Main classes of photoactivatable insecticides

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

Several photodynamic sensitizers have been shown to act as efficient photoinsecticidal agents upon activation 918 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925 Table 1 Main biological targets of photosensitized processes Cell constituent Target Modified chromophore Main photoproducts Protein Tryptophan Indole Hydroxyindoles, kynurenine Tyrosine Phenol Quinones, melanin-type pigments Histidine Imidazole Endoperoxide Methionine Thioether Methionine sulphoxide Cysteine Thiol Cystine, cysteic acid Unsaturated lipids Oleic, linoleic, linolenic, Carbon–carbon double bond Allylic hydroperoxides, arachidonic acids endoperoxides Steroids Cholesterol Carbon–carbon double bond 5 α - or 7 α -hydroperoxide Nucleic acids Guanosine Purine Ketone-type degradation products with either natural or artificial UV or visible light wave- lengths. A list of the main classes of photoinsecticides is given in Table 2. Undoubtedly, xanthene dyes have been most frequently and extensively studied largely due to the systematic investigations carried out by Heitz and co-workers Heitz, 1987; Heitz, 1997a. Eventually, phloxin B, a polyhalogenated fluorescein spiro- benzofuran-13H,9H-xanthen-3-one-29,49,5,79-tetra- bromo-4,5,6,7-tetrachloro-39,69-dihydroxy disodium salt has been developed for commercial use as a pestic- ide Dowell et al., 1997; Heitz, 1997a. While fluor- escein can be considered as the parent compound of this class of photosensitizers, several derivatives can be syn- thetically prepared by the insertion of up to eight halogen atoms into selected positions of the aromatic macrocycle Fig. 1A. All the xanthenes exhibit intense absorption bands in the green region of the visible spectrum; the maximal absorbance falls in the 480–550 nm interval, while the exact position of the peak shifts to the red as the number of the halogen substituents and their atomic weight increase. These parameters also affect the photo- dynamic efficiency of xanthenes, since the presence of heavy bromine or iodine atoms enhances the yield of Table 2 Selected examples of photodynamic sensitizers which have been used as photoinsecticidal agents Class Typical examples Targets References Xanthenes Rose bengal House fly, Aedes larvae Carpenter et al. 1984 Erythrosin B House fly, face fly Fairbrother et al. 1981 Phloxin B Culex mosquito larvae Fondren et al. 1979 Rhodamin 6G Fire ant, House fly Fondren and Heitz 1978b Heitz 1987 Phenothiazines Methylene blue Yellow mealworms Lavialle and Dumortier 1978 Cabbage butterfly Furanocoumarins Xanthotoxin Black swallowtail larvae Cunat et al. 1999 Angelicin Swallowtail butterfly Robinson 1983 Thiophenes α -Terthienyl Aedes mosquito, Blackfly larvae Guillet et al. 2000 Acridines Acridine red Acridine looper Robinson 1983 Various Hypericin Fruit fly Armitage 1998 Cercosporin Aedes mosquito larvae Bosca and Miranda 1998 Benzopyrene Black fly larvae Cunat et al. 1999 Polyacetylenes Fig. 1. Schematic chemical structure of A xanthene dyes and B porphyrin dyes. 919 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925 intersystem crossing to the reactive triplet state of the dye Jori and Reddi, 1991. Thus, other conditions being the same, the greatest photosensitizing activity is dis- played by tetraiodo xanthene derivatives, such as rose bengal and erythrosin B. As shown in Fig. 2, both these dyes are significantly more efficient than their tetra- bromo analogue eosin yellow in photosensitizing the killing of Musca domestica. In general, xanthenes exert their phototoxic effects through the generation of reactive oxygen species largely, singlet oxygen. However, one cannot rule out the parallel occurrence of radical-involving processes owing to the well-known photolability of covalent bonds between halogens and aromatic rings Spikes and Mack- night, 1970. Xanthenes are typically localized in cell membranes, so that at a molecular level xanthenes mostly photosensitize the cross-linking of membrane proteins and the formation of hydroperoxides from unsaturated lipids, thereby markedly increasing the osmotic fragility of cells Pooler and Valenzeno, 1979. Very similar considerations are found for acridines, which efficiently absorb light wavelengths around 550 nm and largely act via the generation of activated oxygen species Rossi et al., 1981. One important difference is represented by the tendency of acridines to yield a heterogeneous subcellular distribution pattern, involving both the partitioning in specific organelles such as lyso- somes and the interaction with the phosphate groups in double-stranded DNA Briviba et al., 1997, which enhances the possibility of photosensitized damage to the genetic material. Both furocoumarines and thiophenes preferentially absorb near-UV light, which has a low penetration power into most biological tissues Anderson and Parr- ish, 1992. Thus, these photosensitizing agents promote the damage mainly at the level of superficial tissue lay- Fig. 2. Effect of different xanthene dyes on the percent survival of Musca domestica , upon exposure to sunlight after 12 h free access to a bait with 0.125 photosensitizer concentration. Eosin yellow × ; rose bengal j; erythrosin B I. Adapted from Fondren et al. 1978, 1979. ers. However, the photodamaged area can become quite extensive since these compounds, once electronically excited, can promote radical processes which signifi- cantly amplify the initial damage through the induction of chain reactions. Furocoumarines may also intercalate among DNA bases with the formation of covalent pho- toadducts and the consequent inhibition of cell repli- cation Armitage, 1998. Lastly, phenothiazines Boyle and Dolphin, 1996 and hypericin Bosca and Miranda, 1998 are characterized by relatively intense absorption bands in the orange–red spectral region. Both these classes of photosensitizers therefore allow the direct damage of tissue compart- ments located at depths of several millimetres below the surface where light initially impinges. Moreover, several constituents of these classes produce the highly reactive singlet oxygen with a quantum yield greater than 0.6; hence, the overall photodamage is most often confined within the microenvironment of the photosensitizer. This makes it important to control the biodistribution of such photosensitizers as closely as possible.

5. Porphyric photoinsecticides