Porphyrins and related compounds as photoactivable insecticides. 3. Laboratory and field studies

Photochemistry and Photobiology, Feb 2000 by Amor, T Ben, Bortolotto, L, Jori, G

ABSTRACT

The exposure of populations of Ceratitis & capitata (fruit fly), Bactrocera oleae (olive fly) and Stomoxis calcitrans (house fly) to a bait containing umolar concentrations of porphyrin-type photosensitizers resulted in a significant accumulation of the porphyrin by the insects and a consequent development of photosensitivity upon exposure to visible light. The photoinsecticidal activity appeared to increase with increasing hydrophobicity of the porphyrin molecule: thus, the amphiphilic dicationic meso-di(cis4N-methyl-pyridyl)-cis-diphenyl-porphine (n-octanol/water partition coefficient = 20) was markedly more efficient than its tricationic analogue or the dianionic hematoporphyrin (n-octanol/water partition coefficient = 12). The observed large decrease in the acetylcholinesterase activity of the photosensitized flies suggests that the damage of the nervous system gives an important contribution to the phototoxic action of porphyrins. Studies with C capitata indicate that the photoinsecticidal action of porphyrins can be utilized to control the population of noxious insects also in open field conditions.

Abbreviations: BCA, bicinchoninic acid; DDP, meso-di(cis-4Nmethyl-pyridyl)-cis-diphenyl-porphine ditosylate; HP, hematoporphyrin; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TRP, tri(4-N-methyl-pyridyl)monophenyl-porphine tritosylate.

INTRODUCTION

A considerable body of data has so far been accumulated regarding the potential of photodynamic sensitizers (ie. near-UV- or visible light-absorbing polycyclic dyes that require the presence of oxygen (1) in order to exert their photosensitizing effect in biological systems) to act as phototoxins against a variety of noxious insects (2,3). Notable successes have been obtained by employing several classes of compounds, such as thione derivatives of natural or synthetic coumarins, thiophenes, furanochromones and xanthenes (4). In particular, one photoactivatable compound, namely phloxine B, has been approved in the USA for field applications (5).

Recently, the possibility of using some members of the porphyrin family as photoactivatable insecticides has been examined. Porphyrins have the favorable feature of absorbing essentially all the wavelengths included in the near-UV and visible portion of the sunlight emission spectrum, hence (at least in principle) they should be endowed with a high photosensitizing efficiency in field applications: for most free base or diamagnetic metal ion-coordinated porphyrins the quantum yield for the generation of the long-lived reactive triplet state is higher than 0.6 (6). Two approaches have been used to test the efficacy of porphyrin photoinsecticides: (1) the modulation of the endogenous heme metabolism with an aim to induce the accumulation of large amounts of endogenous porphyrins, such as protoporphyrin IX, in the insect body and make insects photosensitive (7); (2) the direct administration of selected porphyrins to the insects in association with suitable baits (8,9).

Both approaches yielded very promising results for controlling the population of a large number of insects, ranging from the olive and fruit fly to the larvae of com earn eamworm and cabbage looper. On the basis of These findings, it certainly appears worthwhile to undertake more detailed investigations with porphyric photoinsecticides in order to assess the role of the different experimental parameters controlling the response of insects to their photodynamic action, to elucidate the mechanism of the photodamage induced by the visible light/photosensitizer combination and to optimize the efficacy of the overall photoprocess.

MATERIALS AND METHODS

Porphyrins. Hematoporphyrin IX (HP)t was obtained from Porphyrin Products (Logan, UT) and used as received. The HPLC analysis that was performed (10) on Waters C-18 columns using the eluent system acetonitrile-2.5 mM tetrabutylammonium phosphate (pH 2.5)-methanol (3:2: 1, vol/vol/vol) showed that the sample used in the present investigation had a purity of about 90%; the impurities were represented by protoporphyrin IX, hydroxyethyl-vinyl-deuteroporphyrin IX and oligomeric HP ethers/esters. The HP concentration was determined by diluting a known volume of the aqueous solution into a known excess of 0.25 M H^sub 2^SO^sub 4^ and reading the absorbance at 401.5 nm where EUR = 423 000 M- ^sup 1^ cm- ^sup 1^ (11).

The tri(4-N-methyl-pyridyl)monophenyl-porphine tritosylate (TRP; F 273 000 M-^sup 1^ cm- ^sup 1^ at 417 nm in H20) was a product of Porphyrin Products.

The meso-di-(cis-4N-methyl-pyridyl)-cis-diphenyl-porphine ditosylate (DDP; EUR = 182000 M-^sup 1^ cm-^sup 1^ at 424 nm in H20) was synthesized by Midcentury (Posen, IL). The meso-substituted porphines showed a purity higher than 97% by HPLC analysis and were used as received. The EUR values refer to monomeric porphyrins in neutral aqueous solution.

Chemicals. Sucrose (crystalline, 99-100% pure) was purchased from Prolabo. The autolyzed yeast was obtained from Laboratorio Dottori Piccioni (Milano, Italy). Sodium dodecyl sulfate (SDS) was obtained from Merck (Darmstadt, Germany). Acetylcholinesterase and NADH were obtained from Sigma (St. Louis, MO). The determination of the protein content was per-formed by the bicinchoninic acid (BCA) assay (12) using a kit purchased from Pierce (Chicago, IL). All other chemicals were commercial products of at least analytical grade, unless otherwise stated.