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
In recent years, increasing attention has been focused on the photosensitizing properties of porphyrins Fig.
1B and their analogues, such as chlorins and phthalocy- anines Jori and Spikes, 1983. Porphyrins are natural
compounds or close derivatives of such compounds, hence they are usually devoid of any appreciable intrin-
sic cytotoxicity in the absence of irradiation. Further- more, porphyrins are characterized by specific features
which make them especially useful photosensitizers of biological systems:
1. The ability to absorb essentially all the wavelengths of the solar spectrum in the UV and visible range.
In particular, porphyrins exhibit an intense absorption band the Soret band in the blue spectral region,
which represents the most intense component of the sun’s emission around midday Svaasand et al.,
1990. On the other hand, the red absorption bands of porphyrins are useful at dawn and sunset, when
wavelengths greater than 600 nm represent an important component of sunlight.
2. Several porphyrins yield long-lived triplet states with a high quantum yield .0.7 and therefore are quite
efficient photosensitizers. As a rule, the triplet state of porphyrins is efficiently quenched by oxygen. Hence
porphyrins typically cause cell inactivation through the generation of singlet oxygen type II pathway,
even though radical transfer processes may also be involved Ricchelli et al., 1993. This circumstance
enhances the scope and potential of porphyrins as photosensitizers, since they also express a high pho-
920 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925
toactivity in biological systems which are charac- terized by a low oxygen pressure.
3. The chemical structure of porphyrins can be modified at different levels, including i the substituents pro-
truding from the peripheral positions of the pyrrole rings or the meso-carbon atoms, ii the metal ions
possibly coordinated at the center of the tetrapyrrolic macrocycle, and iii the ligands axial to the metal
ion. In this way, it is possible to modulate the phys- ico-chemical properties of the porphyrin molecules
and control their partitioning among subcellular com- partments.
4. Hydrophobic porphyrins are localized at the level of the cell membranes including the plasma, mitochon-
drial and lysosomal membranes Ricchelli and Jori, 1986. As a consequence, the genetic material is not
involved in the photoprocesses leading to cell death. All the available evidence indicates that porphyrin
photosensitization of cells does not promote the onset of mutagenic effects, thereby minimizing the risk of
selecting photoresistant cell clones Bonnett and Berenbaum, 1989.
5. The extraction and isolation of porphyrins from natu- ral products, and their synthetic preparation often by
modification of natural porphyrins, are relatively simple procedures Ricchelli et al., 1995. Hence, the
overall commercial cost of porphyrins can be fairly low, of the order of 1–2 USg, which is a critical
factor for large-scale field utilization of porphyric insecticides. It is to be underlined that the uptake of
nanomoles of porphyrin is sufficient to cause a rapid mortality of several types of flies even under moder-
ate intensities of sunlight Ben Amor et al., 2000.
6. Porphyrins undergo fast photobleaching in sunlight as well as when exposed to artificial visible light sources
Rotomskis et al., 1997. The photodegradation pro- ducts do not appear to induce any appreciable toxic
or phototoxic effects in a variety of biological sys- tems. Thus, the rapid disappearance of porphyrins
from the environment strongly reduces the risk of widespread or persistent contamination.
7. Several porphyrins are presently used as photothera- peutic agents; toxicological studies have shown that
these dyes induce important damage to humans only upon uptake of at least 100 mgkg body weight, that
is far greater than the amount which is required for generating an extensive toxicity to insects Jori and
Reddi, 1991.
Two approaches have been devised for defining the scope and potential of porphyric insecticides. One
approach involves the administration of a large excess some hundred milligrams of 5- amino-levulinic acid
ALA, which is a metabolic precursor of heme, the prosthetic groups of hemoproteins Rebeiz et al., 1988;
Rebeiz et al., 1990b. The excess ALA through a feed- back mechanism inhibits the final step of the biosyn-
thetic pathway, namely the ferrochelatase-catalysed insertion of the Fe
2 +
ion into the tetrapyrrolic macrocy- cle. This leads to the accumulation of significant
amounts of protoporphyrin IX, a well-known photodyn- amic agent. As a consequence, the protoporphyrin-
loaded cells become photosensitive, especially at the level of mitochondria which represent the normal site of
heme biosynthesis. In general, the photosensitivity develops after a time interval of 3–4 h from the exposure
of the insect to ALA. The extent of the protoporphyrin accumulation can be enhanced by the presence of iron
chelating agents such as desferroxamine or phenanthro- line Rebeiz et al., 1990a.
A second strategy is based on the direct administration of a photodynamically active porphyrin in combination
with a bait. Typically, 1 h exposure of the insects to haematoporphyrin concentrations of the order of a
microgram per ml of bait are sufficient to obtain a 90– 100 decrease in the survival of widely diffused and
extremely noxious flies, such as Ceratitis capitata fruit fly, Bactrocera Dacus oleae olive fly and Stomoxys
calcitrans
stable fly Ben Amor et al. 1998a, 2000. The kinetics of post-irradiation fly death after 1 h
irradiation with light intensities corresponding to an early Autumn day at Mediterranean latitudes is shown
in Fig. 3. In general, the photosensitivity of porphyrin- loaded insects persists for about 48 h after the adminis-
tration of the photosensitizer has been interrupted.
The chemical structure Fig. 4 also has a profound influence on the photoinsecticidal activity of porphyrins.
As shown in Fig. 5, highly water-soluble porphyrins, such as tetracationic meso-substituted-N-methylpyridyl
porphine or tetraanionic meso-sulphonatophenyl por- phine, are very inefficient photoinsecticidal agents Ben
Fig. 3. Percent survival of three diptera, Ceratitis capitata I, Bac-
trocera Dacus oleae ×
and Stomoxys calcitrans j, after 1 h irradiation with white light at a fluence rate of 1220
µ E s
21
m
22
. The flies had been exposed to a bait containing 8
µ M haematoporphyrin.
Some mortality of the flies was observed already during the exposure to light Ben Amor et al. 1998a, 2000.
921 T. Ben Amor, G. Jori Insect Biochemistry and Molecular Biology 30 2000 915–925
Fig. 4. Chemical structures of meso-substituted porphyrins: TMP,
meso-tetra4N-methylpyridylporphine tetratosylate; DDP, meso-[di- cis
4N-methylpyridyl] cis-diphenylporphine ditosylate; TRP, meso- tri4N-methylpyridyl, monophenylporphine tritosylate; TCPP, meso-
tetra4-carboxyphenylporphine tetrasodium salt. The counterions are not indicated in the structures.
Fig. 5. Percent survival of Ceratitis capitata after 1 h exposure to
white light at a fluence rate of 1220 µ
E s
21
m
22
in the presence of 3.0
µ M TCPP H, TRP I, DDP G and haematoporphyrin HP
j Ben Amor et al., 1998b.
Amor et al., 1998b. Such inertness must be related to the anatomical distribution of these porphyrins, since
both of them are accumulated in markedly large amounts by the flies; moreover, the two porphyrins are efficient
generators of singlet oxygen and exhibit phototoxicity towards a variety of biological systems both in vitro and
in vivo Spikes, 1994. As has been observed for other classes of photosensitizers, the photoactivity of porphy-
rins increases with hydrophobicity and is particularly large in the case of amphiphilic derivatives, such as the
meso-cis-diphenyl,
meso-cis-di-N-methylpyridyl- porphine Fig. 5; the latter is more phototoxic than the
tricationic analogue or the dianionic hematoporphyrin even in the presence of a smaller uptake of the dicationic
porphyrin by the insects Ben Amor et al., 1998b.
6. Factors affecting the photoinsecticidal activity
