Effects of Synergists on the Metabolism and Toxicity of Anticholinesterases*

Bull. org. mond. Sante 1971, 4, 171-190 Bull Wld Hlth Org. J Effects of Synergists on the Metabolism and Toxicity of Anticholinesterases* C. F. WILKINSON 1 Insecticide synergists enhance insecticidal action through their ability to block the enzymatic detoxification of insecticides with which they are combined. The structure of the synergist is therefore determined by the nature of the insecticide and the critical biochemical pathway responsible for its degradation. Synergists can be broadly classified as either analogue synergists, whose structure closely resembles that of the insecticide they synergize, or inhibitors of microsomal oxidation. Metabolism of the phenyl methylcarbamates is effected largely by the microsomal enzymes. Consequently microsomal enzyme inhibitors, such as the methylenedioxyphenyl compounds, the aryloxyalkylamines, the thiocyanates, the propynyl aryl ethers, and the 1,2,3-benzothiadiazoles, are all effective carbamate synergists. The detoxification pathways of the organophosphates, however, are more complex and include hydrolysis, dealkylation, and carboxylesterase pathways as well as oxidation. Because phosphoro- thioates are activated by oxidation, their toxicity is often antagonized by oxidase inhibitors. The effectiveness ofdifferent synergists towards resistant strains of insects is likely to vary in a manner that reflects the critical metabolic pathway on which resistance depends. In view of recent concern over the possible human side the activity spectrum of the insecticide alone; and ecological hazards associated with pesticides or, conversely, they may allow a greater degree in the environment, any material that could reduce of selectivity to be achieved. As will be discussed, the amount of an insecticide necessary for insect the latter is particularly important with regard to control would be expected to arouse considerable mammalian safety, but it is also likely that pre- public and commercial interest. Insecticide syner- viously unexpected cases of selective synergism gists are materials of this type: although nontoxic will be found between different insect species, and per se at the dosage levels employed, they are able these could have far-reaching consequences. It is to enhance the efficacy of an insecticide chemical probable, however, that the principal advantage with which they are combined (Brooks, 1968; Casida, of insecticide synergists lies in the control of insects 1970; Metcalf, 1967, 1968; Wilkinson, 1968a, that have gained some degree of resistance to 1968b, 1971). In addition to the substantial econo- insecticide chemicals (Wilkinson, 1968b). Several mic advantage that can result from the synergism cases are known where the use of a synergist can- of expensive materials, the insecticidal activity not only restore the susceptibility of a strain of of a given chemical can often be greatly modified insect resistant to an insecticide but also where when applied in combination with a synergist. the presence of a synergist during the selection period In some cases synergists can extend the effective- actually prevents the development of resistance to ness of certain insecticides by making possible the a given chemical. control of insect species or strains previously out- Under controlled laboratory conditions, the toxicity of several materials, representing most of the major groups of organic insecticides, can be * Work conducted in this laboratory whose results are described herein was supported by grants from the US substantially enhanced by combination with a Public Health Service (No. ES-00400 and ES-00098) and suitable synergist. Relatively few combinations, from the Rockefeller Foundation. are at low synergist: insecticide ' Department of Entomology, Cornell University, Ithaca, however, effective N.Y., USA. ratios, and combinations that show promise in 2625 - 171 - 172 C. F. WILKINSON the laboratory frequently prove disappointing when presence of NADPH and molecular oxygen the evaluated under field conditions. Consequently, enzymes will catalyse remarkably diverse bio- with the exception of synergized formulations of chemical transformations involving numerous func- the expensive pyrethroid insecticides, the synergist tional groups, and their ability to accept a wide concept remains an interesting though commercially variety of substrates makes them an ideal biological unused idea. safety mechanism. At present we are witnessing the rapid demise of Both the carbamates and the organophosphates persistent insecticides, typified by the chlorinated undergo extensive oxidative detoxification and hydrocarbons, and as a result the success of insect consequently it is not surprising that many of the control programmes in the immediate future will synergists with which we shall be concerned are become more dependent on the carbamates and inhibitors of the microsomal enzymes and are organophosphorus compounds that more readily typified by compounds containing the methylene- undergo biological degradation. This paper will dioxyphenyl (1,3-benzodioxole) ring. therefore concern itself with the effect of various Despite the accumulation of a considerable synergists on the toxicity of these two important amount of information in recent years the exact groups of insecticides, and will attempt to assess mechanism by which these synergists inhibit the the potential advantages to be gained from their microsomal enzymes is still a matter for some use in synergized formulations. speculation (Casida, 1970; Wilkinson, 1968a, 1971). Three principal theories have been proposed, but SYNERGISTS AND THEIR MODE OF ACTION they will only be briefly considered here. As a result of the fact that the methylenedioxy- It is now generally accepted that insecticide syner- phenyl compounds are themselves metabolized gists act by blocking the enzymes effecting insecti- to the corresponding catechols by the microsomal cide detoxification and as a result they allow the enzyme complex (Casida, 1970; Casida et al., 1966; insecticide to concentrate and exert its potential Esaac & Casida, 1969; Kamienski & Casida, 1970; toxicity to a greater extent (Brooks, 1968; Casida, Wilkinson & Hicks, 1969) it has been suggested 1970; Metcalf, 1967, 1968; Wilkinson, 1968a, 1968b, that they act as alternative substrates and conse- 1971). The chemical structure of a synergist for a quently competitively inhibit the metabolism of given insecticide therefore depends on the nature insecticides and other foreign compounds. Although of the enzyme(s) responsible for the critical or it is likely that some inhibition could result from rate-limiting reactions leading to its detoxification. this mechanism, it seems equally probable that Living organisms are able to metabolize to some other factors are involved. Hennessy (1965, 1970) extent all organic insecticides to which they are has suggested that inhibition could result from exposed (Lykken & Casida, 1969; Menzie, 1969; hydrogen-ion transfer from the methylene group O'Brien, 1967; Smith, 1962) and do so by means of the ring and proposed that the electrophilic of a large range of different enzymes catalysing such ion formed in this manner could interact by ligand diverse reactions as ester hydrolysis, dehydro- displacement or addition at the haemochrome of chlorination, conjugation, methyl transfer, and cytochrome P-450, the terminal oxidase of the oxidation. The latter deserves special mention as microsomal electron transport chain. More recently, it is now clear that the numerous oxidative reactions Hansch (1968), on the basis of structure-activity catalysed by the mixed-function oxidase complex relationships in a series of methylenedioxyphenyl of mammalian liver (Brodie et al., 1958; Gillette compounds, has concluded that the synergists may et al., 1969; Parke, 1968; Shuster, 1964; Williams, act by a mechanism involving the formation of 1959) and various insect tissues (Casida, 1969; homolytic free radicals. This latter suggestion is of Hodgson, 1968; Terriere, 1968a) are of extreme particular interest because, in addition to the importance in the primary metabolism of lipophilic suggested involvement of free radicals in the mecha- foreign compounds such as modern synthetic nism of microsomal hydroxylation (Staudinger et al., insecticides. The mixed-function oxidases are 1965), recent investigations have demonstrated that associated with the membranous endoplasmic reti- the methylenedioxyphenyl compounds and other culum of the intact cell and on homogenization types of synergist can interact with certain non- and differential centrifugation this yields the so- enzymatic free-radical-generating systems (Nakatsu- called microsomal fraction of the cell. In the gawa & Dahm, 1965; Marshall & Wilkinson, 1970). SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES 173 It is possible that inhibition actually results the thioether groups of methiocarb and aldicarb from a combination of two or more different (Andrawes et al., 1967; Menzie, 1969; Oonithan & mechanisms and considerable work remains to be Casida, 1966, 1968) to the corresponding sulfoxides done before this is fully elucidated. and sulfones. Synergists for the carbamate insecticides CARBAMATE SYNERGISM In view of the predominant role of the microsomal As a result of intensive in vivo and in vitro studies oxidases in carbamate metabolism it is not sur- during the last decade it has been clearly established prising that materials known to inhibit these en- that the methylcarbamates and dimethylcarbamates zymes often have

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