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 micro- somal enzymes. Consequently microsomal enzyme inhibitors, such as the methylenedioxy- phenyl 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 of different 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 and ecological hazards associated with pesticides in the environment, any material that could reduce the amount of an insecticide necessary for insect control would be expected to arouse considerable public and commercial interest. Insecticide syner- gists are materials of this type: although nontoxic per se at the dosage levels employed, they are able to enhance the efficacy of an insecticide chemical with which they are combined (Brooks, 1968; Casida, 1970; Metcalf, 1967, 1968; Wilkinson, 1968a, 1968b, 1971). In addition to the substantial econo- mic advantage that can result from the synergism of expensive materials, the insecticidal activity of a given chemical can often be greatly modified when applied in combination with a synergist. In some cases synergists can extend the effective- ness of certain insecticides by making possible the control of insect species or strains previously out- * Work conducted in this laboratory whose results are described herein was supported by grants from the US Public Health Service (No. ES-00400 and ES-00098) and from the Rockefeller Foundation. ' Department of Entomology, Cornell University, Ithaca, N.Y., USA. side the activity spectrum of the insecticide alone; or, conversely, they may allow a greater degree of selectivity to be achieved. As will be discussed, the latter is particularly important with regard to mammalian safety, but it is also likely that pre- viously unexpected cases of selective synergism will be found between different insect species, and these could have far-reaching consequences. It is probable, however, that the principal advantage of insecticide synergists lies in the control of insects that have gained some degree of resistance to insecticide chemicals (Wilkinson, 1968b). Several cases are known where the use of a synergist can- not only restore the susceptibility of a strain of insect resistant to an insecticide but also where the presence of a synergist during the selection period actually prevents the development of resistance to a given chemical. Under controlled laboratory conditions, the toxicity of several materials, representing most of the major groups of organic insecticides, can be substantially enhanced by combination with a suitable synergist. Relatively few combinations, however, are effective at low synergist: insecticide ratios, and combinations that show promise in 2625 - 171 - C. F. WILKINSON the laboratory frequently prove disappointing when evaluated under field conditions. Consequently, with the exception of synergized formulations of the expensive pyrethroid insecticides, the synergist concept remains an interesting though commercially unused idea. At present we are witnessing the rapid demise of persistent insecticides, typified by the chlorinated hydrocarbons, and as a result the success of insect control programmes in the immediate future will become more dependent on the carbamates and organophosphorus compounds that more readily undergo biological degradation. This paper will therefore concern itself with the effect of various synergists on the toxicity of these two important groups of insecticides, and will attempt to assess the potential advantages to be gained from their use in synergized formulations. SYNERGISTS AND THEIR MODE OF ACTION It is now generally accepted that insecticide syner- gists act by blocking the enzymes effecting insecti- cide detoxification and as a result they allow the insecticide to concentrate and exert its potential toxicity to a greater extent (Brooks, 1968; Casida, 1970; Metcalf, 1967, 1968; Wilkinson, 1968a, 1968b, 1971). The chemical structure of a synergist for a given insecticide therefore depends on the nature of the enzyme(s) responsible for the critical or rate-limiting reactions leading to its detoxification. Living organisms are able to metabolize to some extent all organic insecticides to which they are exposed (Lykken & Casida, 1969; Menzie, 1969; O'Brien, 1967; Smith, 1962) and do so by means of a large range of different enzymes catalysing such diverse reactions as ester hydrolysis, dehydro- chlorination, conjugation, methyl transfer, and oxidation. The latter deserves special mention as it is now clear that the numerous oxidative reactions catalysed by the mixed-function oxidase complex of mammalian liver (Brodie et al., 1958; Gillette et al., 1969; Parke, 1968; Shuster, 1964; Williams, 1959) and various insect tissues (Casida, 1969; Hodgson, 1968; Terriere, 1968a) are of extreme importance in the primary metabolism of lipophilic foreign compounds such as modern synthetic insecticides. The mixed-function oxidases are associated with the membranous endoplasmic reti- culum of the intact cell and on homogenization and differential centrifugation this yields the so- called microsomal fraction of the cell. In the presence of NADPH and molecular oxygen the enzymes will catalyse remarkably diverse bio- chemical transformations involving numerous func- tional groups, and their ability to accept a wide variety of substrates makes them an ideal biological safety mechanism. Both the carbamates and the organophosphates undergo extensive oxidative detoxification and consequently it is not surprising that many of the synergists with which we shall be concerned are inhibitors of the microsomal enzymes and are typified by compounds containing the methylene- dioxyphenyl (1,3-benzodioxole) ring. Despite the accumulation of a considerable amount of information in recent years the exact mechanism by which these synergists inhibit the microsomal enzymes is still a matter for some speculation (Casida, 1970; Wilkinson, 1968a, 1971). Three principal theories have been proposed, but they will only be briefly considered here. As a result of the fact that the methylenedioxy- phenyl compounds are themselves metabolized to the corresponding catechols by the microsomal enzyme complex (Casida, 1970; Casida et al., 1966; Esaac & Casida, 1969; Kamienski & Casida, 1970; Wilkinson & Hicks, 1969) it has been suggested that they act as alternative substrates and conse- quently competitively inhibit the metabolism of insecticides and other foreign compounds. Although it is likely that some inhibition could result from this mechanism, it seems equally probable that other factors are involved. Hennessy (1965, 1970) has suggested that inhibition could result from hydrogen-ion transfer from the methylene group of the ring and proposed that the electrophilic ion formed in this manner could interact by ligand displacement or addition at the haemochrome of cytochrome P-450, the terminal oxidase of the microsomal electron transport chain. More recently, Hansch (1968), on the basis of structure-activity relationships in a series of methylenedioxyphenyl compounds, has concluded that the synergists may act by a mechanism involving the formation of homolytic free radicals. This latter suggestion is of particular interest because, in addition to the suggested involvement of free radicals in the mecha- nism of microsomal hydroxylation (Staudinger et al., 1965), recent investigations have demonstrated that the methylenedioxyphenyl compounds and other types of synergist can interact with certain non- enzymatic free-radical-generating systems (Nakatsu- gawa & Dahm, 1965; Marshall & Wilkinson, 1970). 172 SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES 173 It is possible that inhibition actually results from a combination of two or more different mechanisms and considerable work remains to be done before this is fully elucidated. CARBAMATE SYNERGISM As a result of intensive in vivo and in vitro studies during the last decade it has been clearly established that the methylcarbamates and dimethylcarbamates are metabolized almost exclusively by the NADPH- requiring microsomal enzymes of mammalian liver and insect tissues (Knaak, 1971; Lykken & Casida, 1969; Menzie, 1969; Metcalf et al., 1967; Oonithan & Casida, 1966, 1969; Tsukamoto & Casida, 1967). Although these enzymes are capable of oxidative demethylation of the methylcarbamoyl or dimethyl- carbamoyl moiety (Hansen & Hodgson, 1971; Hodgson & Casida, 1961) it appears that the most important sites of microsomal attack are associated with the aromatic ring and its substituents (Ooniithan & Casida, 1968). The diversity of the substituent groups that can be incorporated into this portion of the molecule is extremely broad and consequently almost all known types of microsomal enzyme activity have been shown to occur. Direct ring hydroxylation has been demonstrated with carbaryl (Dorough & Casida, 1964; Leeling & Casida, 1966) and propoxur (Shrivastava et al., 1969) and is probably of general significance with other carbamates. With propoxur, however, a main metabolic pathway involves initial hydroxyl- ation of the o-isopropoxy group with subsequent dealkylation to o-hydroxy-propoxur (Metcalf et al., 1967; Shrivastava et al., 1969). Hydroxylative at- tack at the isopropyl groups of in-isopropylphenyl methylcarbamate (UC 10854) and possibly 3,5- diisopropylphenyl methylcarbamate (HRS-1422) has also been demonstrated (Oonithan & Casida, 1968), as has hydroxylation of one of the ring methyl groups of Landrin t (Slade & Casida 1970). Similar aliphatic side chain oxidation is suggested for carba- mates containing longer or branched alkyl substi- tuents (Oonithan & Casida, 1968). Other types of microsomal reaction that have been demonstrated include N-dealkylation of the substituted amino groups of Zectran t (Oonithan & Casida, 1966, 1968) and aminocarb to yield the methylamino, ami- no, and formamido analogues, and oxidation of t Names against which this symbol appears are identified in the Glossary on pages 445-446. the thioether groups of methiocarb and aldicarb (Andrawes et al., 1967; Menzie, 1969; Oonithan & Casida, 1966, 1968) to the corresponding sulfoxides and sulfones. Synergists for the carbamate insecticides In view of the predominant role of the microsomal oxidases in carbamate metabolism it is not sur- prising that materials known to inhibit these en- zymes often have a dramatic synergistic effect on carbamate toxicity. The best known and most extensively studied group of synergists are the methylenedioxyphenyl compounds (Fig. la), which were initially developed Fig. 1 Synergists for the carbamate insecticides (see text for explanation) R-fIfC2 1 RtoXCO/ CH SCEN aCO(cH2)2N (a) RcJf C3H7 C2H5 (b) (c) (d) hca intia observation ci ci R-QINHcaH ci-c-ci ci-6-ci R N ~~~(e) CI-CH oNpCH-Ci (9) (h) as pyrethrin synergists. The initial observation by Moorefield (1958) that the toxicity of Isolan,t carbaryl, and m-tert-butylphenyl compounds, such as piperonyl butoxide, sulfoxide,t and propyl isome,t did much to stimulate interest in the concept of synergism, and carbamates have since been widely used in synergist studies. Synergism of the phenyl methylcarbamates and dimethylcarbamates is now firmly established as a general phenomenon and a vast number of data (Fukuto et al., 1962; Met- calf, 1967; Metcalf & Fukuto, 1965; Metcalf et al., 1966; Wilkinson, 1965; Wilkinson et al., 1966) show that, provided the carbamate possesses the structural requirements for inhibition of cholin- esterase, the degree of synergism observed is inversely related to the inherent toxicity of the carbamate. Data showing the degree of synergism of several commercially important carbamates with piperonyl butoxide are shown in Table 1. C. F. WILKINSON Table 1 Synergism of some commercially important phenyl methylcarbamates with piperonyl butoxide against susceptible (NAIDM) houseflies * Common name Topical LDso Methylcerbamate or ~~~~~(j&gper female fly) Degree ofMethylcarbamate | commercial synergism designation alone with P.B.(A) (B) (A/B) 1 -naphthyl carbaryl >100 0.25 >400 4-dimethylamino-3,5-xylyl Zectran 1.2 0.27 4.4 4-(methylthio)-3,5-xylyl methiocarb 0.48 0.25 1.9 4-dimethylamino-m-tolyl aminocarb 1.6 0.50 3.2 m-isopropylphenyl UC 10854 2.0 0.20 10.0 AC 5727 o-isopropylphenyl PPC-3 2.1 0.48 4.4 o-isopropoxyphenyl propoxur 0.47 0.14 3.4 2,3-dihydro-2,2-dimethyl-7-benzofuranyl carbofuran 0.13 0.05 2.6 2-methyl-2-(methylthio)-propionaldehyde aldicarb 0.11 0.067 1.6 0-oximyl 3,4,5-trimethylphenyl +2.3.5-trimethylphenyl Landrin 1.3 0.27 4.8 3,5-di-tert-butylphenyl butacarb 0.78 0.12 6.5 m-tert-butylphenyl RE 5030 >100 0.16 >625 3,4-xylyl Meobal 2.4 0.57 4.2 3-methyl-5-isopropyl (m-cym-5-yl) promecarb 0.58 0.11 5.3 Data from Georghiou & Metcalf (1961), Metcalf & Fukuto (1965), and Metcalf et al. (1967). Structure-activity relationships of the methylene- dioxyphenyl compounds have been thoroughly investigated (Metcalf, 1967, 1968; Metcalf et al., 1966; Moorefield & Weiden, 1964; Sacher et al., 1971; Wilkinson, 1965, 1967, 1968a; Wilkinson et al., 1966) and are now quite clearly defined. Maximum synergistic activity is closely associated with the intact methylenedioxyaryl (1,3-aryldioxole) ring and even slight structural modifications in this ring result in either a decrease or a complete loss of potency. Of critical importance are the oxygen atoms of the ring, though these can be replaced by sulfur with only slight loss of activity, and the unsubstituted methylene group. Compre- hensive discussions of structure-activity relation- ships are presented elsewhere (Metcalf, 1967, 1968; Metcalf et al., 1966; Wilkinson, 1968a, 1971) and the subject will not be further considered here. Almost any compound containing the methyl- enedioxyphenyl ring will inhibit microsomal oxi- dase activity in vitro (Lewis et al., 1967) and it is clear that the potential for synergism resides in this moiety. The extent to which this potential can be realized in vivo, however, is largely dependent on the substituents in the aryl ring (Wilkinson, 1967, 1968a). The unsubstituted parent 1,2-methyl- enedioxybenzene is itself largely devoid of synergis- tic activity, but ring substitution with a variety of simple groups results in a dramatic increase in synergistic potency (Wilkinson, 1967). In contrast, the unsubstituted methylenedioxynaphthalene (naph- tho-1,3-dioxole) is extremely active in combination with the carbamates (Metcalf, 1968; Sacher et al., 1971) and ring substitution in this case has little additional effect. It is probable that ring substitu- ents modify synergistic activity largely through their effect on the overall lipophilic character of the compound, a property that determines its ability to penetrate the insect cuticle and to translocate to the site of action. Large differences in the rate of cuticle penetration have been observed in com- pounds containing different ring substituents; and 174 SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES polar molecules, which are slow penetrators, show little or no activity following topical application to insects (Sacher et al., 1971; Wilkinson, 1967). If instead the synergistic activity of these polar materials is evaluated following their incorporation into carbaryl baits many show marked activity against houseflies (Weiden & Moorefield, 1965). This suggests that the physical properties of a synergist and the type of formulation in which it is incorporated could possibly constitute a means of obtaining some degree of selectivity. In addition to the methylenedioxyphenyl com- pounds, several other groups of materials (Fig. 1) have in recent years been shown to enhance the potency of the carbamate insecticides, and the relative potencies of some of these are shown in Table 2. Organothiocyanates such as isobornyl thiocyanato- acetate (Thanite t), dodecyl thiocyanate, and 1,2,3,4,- 7,7 - hexachloro -5 - isothiocyanatomethyl -5 -norborn- enel are all synergistically active in combination with several carbamates against both resistant and sus- ceptible strains of the housefly (El-Sebae et al., 1964; Hewlett, 1969) and subsequently a large number of benzyl thiocyanates (Fig. Ib) have been shown to have similar activity (Bakry et al., 1968; Metcalf, 1968). The drug-potentiating action in mammals of Table 2 Synergism of carbaryl against susceptible female houseflies by compounds representing different groups of synergists Topical LD5o of carbaryl in 1:5SyegsiSynergist ratio with ratio Ref. aynergist rai s(,Mg/g) None cs 900 - Methylenedioxyphenyl compounds piperonyl butoxide 12.5 72 4 sesamex t 8.24 109 5 1,2-methylenedioxynaphthalene 4.0 225 5 4-nitro-1,2-methylenedioxybenzene 16.4 54.5 5 Organothiocyanates 1 ,2,3,4,7,7-hexachloro-5-isothiocyanatomethyl- 5-norbornene 50.0 18.0 4 p-nitrobenzyl thiocyanate 77.5 11.6 1 Aryloxyalkylamines 2-[(4,6-dichloro-2-biphenylyl)oxy] triethylamine 106.0 8.5 4 (Lilly 18947) 2-(diethylamino)ethyl 2,2-diphenylpentanoate (SKF 525A) 58.5 15.4 4 Propynyl aryl ethers 2-propynyl 2,4,5-trichlorophenyl ether b 4.9 183.7 3 2-propynyl 4-chloro-2-nitrophenyl ether c 4.2 214.3 3 2-propynyl 1 -naphthyl ether d 15.5 58.1 6 Miscellaneous tri-o-tolyl phosphate (TOCP) 210 4.3 4 p-chlorophenyl p-chlorobenzenesulfonate 100 e > 7.5 2 a References: 1, Bakry et al. (1968); 2, Kato (1965); 3, Metcalf (1968) 4, Metcalf & Fukuto (1965); 5, Metcalf et al. (1 966); 6, Sacher et al. (1 968). b 2,4,5-trichloropropynyloxybenzene. c 4-chloro-2-nitropropynyloxybenzene. d 1 -propynyloxynaphthalene. e Data obtained with 1: 4 carbaryl: synergist ratio; the LD5o value for carbaryl alone was > 750 Mg/g. 13 175 176 C. F. WILKINSON several aryloxyalkylamines, such as 2-(diethylamino)- ethyl 2,2-diphenylpentanoate (SKF 525A) (Fig. Ic) and 2-[(4,6-dichloro-2-biphenylyl)oxy] triethylamine (Lilly 18947), has long been recognized to result from the ability of these compounds to inhibit the microsomal enzymes (Anders & Mannering, 1966). Moorefield & Tefft (1959) first demonstrated that in its free base form Lilly 18947 synergized carbaryl, and Fahmy & Gordon (1965) subsequently showed that a large number of structural analogues of this compound were potent synergists for several methylcarbamates and dimethylcarbamates against houseflies. SKF 525A has also been shown effective- ly to synergize carbaryl and m-isopropylphenyl methylcarbamate against susceptible houseflies, re- sulting in synergistic ratios (SR) of approximately 15 and 6, respectively (Metcalf & Fukuto, 1965). More recently considerable interest has been focused on several new types of carbamate synergist containing acetylenic bonds. The first of these to be discovered were the phenyl 2-propynyl ethers (Fig. Id) (Barnes & Fellig, 1969; Kooy, 1966), which appear to have a spectrum of activity and a mode of action similar to those of the methylene- dioxyphenyl compounds. Structure-activity investi- gations with the phenyl 2-propynyl ethers (Fellig et al., 1970) and a series of naphthyl propynyl ethers and related compounds (Sacher et al., 1968) have clearly established the importance of the acetylenic linkage. As with the methylenedioxyphenyl com- pounds, some activity is retained if the ethereal oxygen is replaced by sulfur and indeed the 12-fold synergism of carbaryl by 1-(2-propynyl)naphthalene suggests that the oxygen atom is not an absolute requirement for activity. Activity is not directly associated with the propynyl ether moiety per se, as studies with a series of longer-chain acetylenic ether derivatives of naphthalene have demonstrated that maximum activity with carbaryl is exhibited by the 3-butynyl-1-naphthyl ether (SR 176.5). The decrease in activity observed with the 4-pentynyl derivative (SR 75) indicates that the distance between the acetylenic linkage and the aryl ring may be important. Subsequent work has established that a high degree of synergistic potency is associated with other acetylenic compounds such as the oximino propynyl ethers (Fig. le) (Hennessy, 1970). Syner- gism of the carbamate carbofuran against several insect species has also been observed with several alkynyl phosphates such as NIA 16388 (propyl 1 1,2,3,4,7,7-hexachloro-5 -(isothiocyanatomethyl)bicyclo-[2.2. 11-5-heptene. 2-propynyl phenylphosphonate) and NIA 16824 (2-methylpropyl 2-propynyl phenylphosphonate) (Fig. If) (Knaak, J. B., personal communication, 1971). The toxicity of carbofuran was also syner- gized 4-8-fold against the two-spotted spider mite (Tetranychus telarinus), which is usually quite tole- rant of this toxicant. The most recent group of synergists to be dis- covered is the 1,2,3-benzothiadiazoles (Felton et al., 1970) (Fig. 1 g). The unsubstituted parent compound is an effective synergist with both Landrin t (SR 2.7) and Isolan t (SR 6.7), and activity is progressively increased in the 6-chloro and 5,6-dichloro deri- vatives. Synergism appears to be closely associated with the 1,2,3-benzothiadiazole ring structure, as several other closely related bicyclic compounds including 2,1,3-benzothiadiazoles, benzfurazans, in- doles, and 1H-benzotriazoles are all found to be inactive. In addition to the foregoing materials, which represent entire groups of compounds, carbamate synergism is also observed with several miscellane- ous compounds including bis(2,3,3,3-tetrachloro- propyl) ether (Georghiou & Metcalf, 1961a) (Fig. lh), aryl and alkyl boronic acids (Weiden & Moorefield, 1965), p-chlorophenylp-chlorobenzenesulfonate (Ka- to, 1965), tri-o-tolyl phosphate (TOCP) (Metcalf & Fukuto, 1965), and several noninsecticidal carba- mates (Plapp & Valega, 1967). There can be little doubt that the increase in carbamate toxicity in combination with a synergist results largely from inhibition of microsomal enzyme activity. Most of the groups of compounds that possess synergistic activity are established in- hibitors of microsomal oxidation in vitro (Casida, 1970) and many have been shown to stabilize various carbamates in vivo (Metcalf et al., 1967; Sacher et al., 1968; Shrivastava et al., 1969). Pre- treatment of both susceptible (SCRS) and resistant (RBaygon) houseflies with piperonyl butoxide mar- kedly impairs their ability to metabolize propoxur (Fig. 2) and this correlates well with the toxicity of synergized and unsynergized propoxur for these two strains (Shrivastava et al., 1969). Other types of synergist, including the organothiocyanates (El- Sebae et al., 1964), Lilly 18947 (Metcalf et al., 1967), and several of the propynyl ethers (Sacher et al., 1968) have been shown to have a similar stabilizing effect on propoxur, as measured by the liberation of 14C02 from the 14C-isopropoxy group (Table 3). These data confirm the results of earlier studies on the stabilization by piperonyl butoxide of im-iso- SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES Fig. 2 Effect of piperonyl butoxide on metabolism of propoxur in susceptible (SCRS) and resistant (RBaygon) houseflies 100 50 a 25 ' 10 CD 5 L 2.5 30 60 90 Time (min) 0 SCRS strain, propoxur alone (LD5o = 0.3 Mg/fly) * SCRS strain, propoxur + piperonyl butoxide (LD5o = 0.08 MLg/fly) o RBaygon strain, propoxur alone (LD5o = 1 5.0 ,ug/fly) * RBaygon strain, propoxur + piperonyl butoxide (LDso = 0.6 ug/fly) propylphenyl methylcarbamate and Zectran t (Georg- hiou & Metcalf, 1961b; Metcalf & Fukuto, 1965), in both susceptible (NAIDM) and resistant (RMIp) houseflies. The large variations observed in the degree to which different carbamates can be synergized by piperonyl butoxide suggest that the main micro- somal detoxification pathways are associated with the phenyl ring and its substituents rather than the methylcarbamoyl moiety, which is a common struc- tural feature (Metcalf, 1967, 1968; Metcalf et al., 1966; Wilkinson, 1968a). This view is supported by the relatively small amount of 14CO2 (1-2%) liberated from houseflies treated with propoxur- N14CH3 compared with that from iso-2-_4C-propoxy propoxur (30%) (Metcalf et al., 1967), as well as by the fact that synergistic ratios are not substan- tially changed when the -NHCH3 group is replaced by -NHCD3, -NHCF3, and -NH2, which cannot be dealkylated (Fahmy et al., 1966; Metcalf et al., 1966). Little is known regarding the role of the various substituent groups in determining the degree of synergism observed, though it appears that maxi- mum synergism (presumably maximum detoxifi- cation) is associated with longer and branched alkyl, alkoxy, and alkylthio groups, while minimum activity is found with the smaller groups (Metcalf et al., 1966). It is probable that the lipophilic characteristics of a carbamate also play a role in determining its rate of detoxification by the micro- somal enzymes and consequently the degree to which it can be synergized (Weiden, 1968). Although the relationship between the lipophilic character of a material and its ability to be accepted as a micro- somal substrate has received some attention with ble 3 Stabilization of 2-isoprop-1 4C-oxyphenyl methylcarbamate (propoxur) in R.Ip houseflies pretreated for 1.5 hours with 50 jg of synergist * Synergist Applied dose(Mg/female) Proportion (%) of applied dose absorbed 14Ch2 and (24 h) volatiles excreted (24 h) none 0.5 97.6 31.6 17.4 piperonyl butoxide 1.0 85.4 6.8 3.1 Lilly 18947 0.5 91.6 16.7 6.0 Thanite t 0.5 81.3 14.7 6.4 2,3-methylenedioxynaphthalene 1.0 94.2 15.0 10.8 2-propynyl 4-chloro-2-nitrophenyl ether 1.0 93.8 18.8 - 2-propynyl 1-naphthyl ether 1.0 96.8 9.5 7.2 * Data from Sacher et al. (1 968). 177 C. F. WILKINSON drugs (Gaudette & Brodie, 1959; Lien & Hansch, 1968) similar studies with insecticidal materials have not yet been carried out. A theoretically attractive way of utilizing the synergistic activity associated with certain groups is to incorporate them directly into the structure of the insecticide molecule. The success of this concept relies on the fact that the synergist grouping has no deleterious effect on the insecticidal pro- perties of the compound in which it is incorporated, and it is probably for this reason that attempts at self-synergism have not met with a great deal of success. In the case of the carbamates, however, inclusion of the methylenedioxy and 2-propynyl ether moieties in the phenyl ring yields potent non- synergizable insecticides, whose activity probably results from a self-synergizing mechanism (Metcalf et al., 1966) (Table 4), though the incorporation of Table 4 Toxicity of methylcarbamates containing synergistic groups, and the degree to which they can be synergized Topical LD5o Syner- Methylcarbamate (jug/g, female fly) gistic alone with P.B. ratio 3,4-methylenedioxyphenyl a 17.5 20.0 1.0 2-(2-propynyloxy)phenyl a 6.5 4.6 1.4 3- (2-propynyloxy)phenyl a 7.5 6.0 1.2 4-thiocyanatophenyl 1 000 b 1 000 b 1.0 6- methylcarbamoyl- 1,2,3-benzothiadiazole c - - Cl Data of Metcalf (1968). b LC50 values (ppm); data of Weiden & Moorefield (1965), measured by incorporation of piperonyl butoxide in bait. c Little or no activity (Kirby, personal communication, 1971). other synergistically active moieties, notably the thiocyanato (Weiden & Moorefield, 1965) and 1,2,3-thiadiazole groups (Kirby, personal communi- cation, 1971) has not been as successful. The insecticidal inactivity of 4-thiocyanatophenyl methyl- carbamate is somewhat surprising in view of its high anticholinesterase activity and suggests that the thiocyanato grouping is susceptible to rapid meta- bolism in vivo, possibly by glutathione S-transferase (Casida, 1970). Factors affecting carbamate synergism In addition to the chemical structure and physi- cal properties of both the carbamate and the syner- gist, the degree of synergism observed is dependent on the dose ratio of the synergist to the carbamate and on the enzymatic capability of the organism used in the test. From an economic viewpoint the efficiency of a synergist is an important consideration, and it can be gauged from the amount of the material required to produce a synergistic effect. This varies markedly between the different groups of synergists as well as between compounds of the same general structure. One of the most effective materials studied is 2,3-methylenedioxynaphthalene (naphtho- [2,3-d]-1,3-dioxole) (Table 5), which shows marked activity at low synergist: insecticide ratios and gives a synergistic ratio of 1.8 (Metcalf et al., 1966) at a concentration as low as 0.001 that of carbaryl. Several of the phenyl 2-propynyl ether synergists are also extremely effective at low ratios with carbaryl (Barnes & Fellig, 1969). Although most other materials are not as effec- tive as these, an increase in the synergist: insecti- cide ratio usually results in an increase in the degree of synergism obtained (Bakry et al., 1968; El- Sebae et al., 1964; Wilkinson, 1967) and shows an asymptotic approach to an upper limit (Hewlett & Wilkinson, 1967). Plots of the LDso of various carbaryl-synergist combinations (y) against the amount of synergist used in the combination (x) gave isobols approximating rectangular hyper- bolas, and of 26 synergists studied, 18 showed a reasonable straight-line relationship when y was plotted against 1/x. The extrapolated intercept on the y axis (when 1/x = zero) showed a mean value of 0.06 jug/fly for carbaryl. This is of interest because it represents the LD50 of carbaryl in the presence of an infinite amount of synergist and presumably therefore provides a measure of the ultimate toxicity of carbaryl when detoxification is fully suppressed (Hewlett & Wilkinson, 1967). The degree of synergism observed also varies inversely with the susceptibility of the insect to the unsynergized carbamate. This appears to be a direct reflection of variations in the insect's detoxi- fication capability, and differences between species and strains as well as those associated with physio- logical factors such as age and sex have been reported. The selective toxicity of the phenyl methyl- carbamates towards different insect species has 178 SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES Table 5 Effect of different insecticide: synergist ratios on degree of synergism * LDso (,ig/g, female flies) of carbaryl at the following insecticide: synergist ratios (w/w): Synergist 10:1 5:1 1:1 1: 5 1:10 4,5-dichloro-1 ,2-methylenedioxybenzene a' 4-nitro-1 ,2-methylenedioxybenzene a 2,3-methylenedioxynaphthalene b p-nitrobenzyl thiocyanate b LD5o SR LD5o SR LD5o SR LD5o SR LDso SR 28 89.3 1 3 192 10 > 250 16.5 1 51 8.5 300 5.5 >454 110 8.2 31 29 8 112 5 180 - - - - 125 7.2 85 10.6 40 22.5 * Data of Bakry et al. (1968), Metcalf et al. (1966), and Wilkinson (1967). It The synergistic ratios are based on an unsynergized LDso of > 50 ,ug/fly. b The synergistic ratios are based on an unsynergized LD5e of 900 jg/g. long been recognized (Metcalf & Fukuto, 1965) and is particularly well exemplified by the difference in toxicity of carbaryl to houseflies (LD50 900 ug/g) and honey-bees (Apis niellifera) (LD50 2.3 ,ig/g) (Metcalf et al., 1966). The corresponding LDde values of a 1: 5 carabaryl : piperonyl butoxide combination are 12.8 and 0.8 tig/g, giving synergistic ratios of about 72 and 2.9, respectively. This clearly indi- cates that the principal reason for the observed selectivity is differences in the level of microsomal enzyme activity between the two species. Brattsten & Metcalf (1970) have recently reported the results of a comprehensive survey of the degree of syner- gism of carbaryl by piperonyl butoxide against 54 insect species representing 8 orders and 37 families. These show an amazing 10 000-fold difference in the toxicity of carbaryl alone to these species and synergistic ratios varying by a factor of about 300. As a consequence, Brattsten & Metcalf (1970) suggest that synergistic ratios may provide a useful in vi4o indicator of microsomal enzyme activity in different insect species.. A recent investigation of the dependence of carbamate toxicity on various physiological factors such as age and sex shows similar results (El-Aziz et al., 1969). Thus, although the toxicity of six carbamates varied markedly with the age and sex of both resistant (R.1Ip) and susceptible (NAIDM) houseflies, the differences were almost completely obviated by synergists such as piperonyl butoxide, sesamex, and 1,2-methylenedioxynaphthalene (Table 6). Similar results have also been obtained using adult house crickets (Acheta lornesticus) of different Table 6 Effects of piperonyl butoxide on the toxicity of m-isopropylphenyl methylcarbamate against susceptible (NAIDM) flies of different age and sex * Carbamate alone Age (days) 2 3 4 5 6 7 males females LDso LDso (pg/g) (tg/g) 104.2 80.3 48.9 37.0 15.7 13.4 7.8 126.4 114.2 94.2 53.7 17.2 14.8 10.1 Carbamate plus 5 parts of piperonyl butoxide males females LDso LDso Si(Lg/g) S (Lg/g) 17.4 6.0 18.7 6. 1 5.3 5.2 18.8 6.' 9.8 5.0 17.1 5.! 9.6 3.8 1 3.7 3.! 9.6 1.6 9.2 1.! 8.0 1.7 7.4 2.1 6.8 1.1 6.7 1.' B 599 ) 5 - Data taken from El-Aziz et al. (1969). age and sex (Benke & Wilkinson, 1971). As shown in Fig. 3, the high susceptibility of newly moulted adults to carbaryl and the corresponding low degree of synergism with piperonyl butoxide correlate well with the low microsomal enzyme levels in tissues of the malpighian tubules at this stage of development. The subsequent increase in microsomal enzyme activity during the first week of life is accompanied by a decrease in susceptibility to 179 R C. F. WILKINSON Fig. 3 Relationship between microsomal enzyme activity in the malpighian tubules, susceptibility to carbaryl, and degree of synergism of carbaryl by piperonyl butoxide against adult crickets (Acheta domesticus) of different age and sex Syne fq ss |c Ra,o ID50 0~~~~~ L D 50 . / ug g 10 A .. ..... .... Spec f C * Ac y ~ ~ ~ ~ ~ A Ac t ~tvity / * nmo1 I0 s 1T(,I er X 101 A .... A AqA of AA. (^e As *- *females P....A males carbaryl and a concomitant increase in the degree of synergism that can be achieved. Of particular significance from a practical stand- point is the synergism of the carbamates against strains of insect that have developed resistance to these insecticides. It can be seen from Table 7 that strains of the housefly that have developed high levels of resistance to the carbamates as a result of either selection pressure with carbamates them- selves or through cross-resistance resulting from selection with other groups of insecticides usually remain relatively susceptible to a synergized carba- mate combination (Eldefrawi & Hoskins, 1961; Georghiou, 1962; Georghiou et al., 1961; Metcalf & Fukuto, 1965; Shrivastava et al., 1969). It is also of considerable interest and importance that the development of resistance to the carbamates is often greatly reduced or almost completely prevented if the insect is placed under selection pressure with a carbamate-synergist combination (Georghiou, 1962; Georghiou et al., 1961; Moorefield, 1960). An association between insect resistance to the carbamates and increased titres of microsomal enzyme activity has been reported (Casida, 1970; Plapp & Casida, 1969; Shrivastava et al., 1969; Tsukamoto et al., 1968); this is further considered by Oppenoorth (1971). From the foregoing discussion there appear to be several possibilities for achieving some degree of selectivity with regard to the synergistic potency against different species. These possibilities have not yet been adequately explored and it is likely that in many cases they will be difficult to predict. In the case of selectivity between insects and mammals, however, two important factors exist that should warrant a certain degree of optimism. Firstly, although most comparative investigations on the nature of microsomal enzymes in insects and mammals have shown that apart from quanti- tative variations no fundamental biochemical diffe- rences exist (Casida, 1969; Hook et al., 1968; Terriere, 1968a, 1968b), almost all reports indicate that for some reason the insect system is consider- ably more susceptible to inhibition by various mate- rials (Brooks, 1968; Casida, 1970; Chakraborty & Smith, 1967; Lewis et al., 1967; Wilkinson, 1971; Wilkinson & Hicks, 1969). In addition, most of the synergists that have been studied have been found to be much more persistent in insects than in mammals (Casida et al., 1966; Sacher et al., 1968, 1969), a fact that, with methylenedioxyphenyl compounds and 2-propynyl ethers, undoubtedly reflects a greater resistance to attack by the micro- somal enzymes in insects (Casida, 1970; Casida et al., 1966; Esaac & Casida, 1969; Wilkinson, 1971; Wilkinson & Hicks, 1969). As a result of these two factors combinations of carbaryl with 2,3- methylenedioxynaphthalene (Sacher et al., 1969) and 1-propynyloxynaphthalene (Sacher et al., 1968), which are highly potent against houseflies, have little effect on mice. Indeed, most studies indicate that the acute synergism of drugs and insecticides against mammalian species occurs only at relatively high synergist doses and suggest that little human hazard is associated with present synergist usage (Casida, 1970). However, in view of the potential ability of the synergists to stabilize a large number of foreign compounds, including carcinogens, the future commercial development of new and perhaps more stable materials should be approached with some caution. SYNERGISM OF ORGANOPHOSPHORUS COMPOUNDS The metabolic interactions of the organophos- phate insecticides have been discussed in some detail 180 SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES Table 7 Synergism of four carbamates alone and in combination with 5 parts of piperonyl butoxide against susceptible and resistant strains of the housefly * Topical LDso (Mig per female fly) of the following methylcarbamates: Strain Susceptible NAIDM LAB SRSa SCRS b Resistant c MIP-1 d MIP-2 e Baygon Hokota r Resistant h Super Pollard i Stauffer Chlori Ronnel k m- isopropylphenyl o- isopropoxyphenyl naphthyl alone with P.B. alone with P.B. alone with P.B. 1.8 0.18 0.51 0.14 100 0.24 2.0 0.33 0.47 0.19 100 0.24 100 100 20 8.3 100 - 0.20 - 0.30 1.0 1.2 0.86 0.63 2.6 100 100 15 100 100 0.12 0.08 0.56 0.64 0.6 1.0 0.96 100 100 100 1.9 1 .9 31.0 * Data from Georgiou et al. (1961), Metcalf & Fukuto (1965), Metcalf et al. (1967), and Shrivastava et al. (1969). a Standard WHO reference strain. b Propagated from the SCR strain. c Selected with carbamate pressure. d Selected from the SLAB strain with m-isopropylphenyl methylcarbamate. e Selected from the SNAIDM strain with m-isopropylphenyl methylcarbamate. f Selected from the SCR strain with carbaryl and subsequently with propoxur. 'g Selected with propoxur. h Selected with insecticides other than carbamates. Selected with DDT and lindane. Selected with Chlorthion.t k Selected with Chlorthion t and subsequently with fenchlorphos. by Dauterman (1971) and for the purposes of the formation of an insecticidally inactive phosphodi- present discussion only a brief summary of this extremely complex area is necessary. The principal metabolic pathways that a typical phosphorothioate insecticide can follow are indicated in Fig. 4 and can be classified rather loosely into three main categories based on reaction type. These are oxi- dation of the phosphorothioate (P = S) to the corresponding phosphate (P = 0) (reaction 1); cleavage of the " anhydride " P-O-X linkage with subsequent liberation of the acidic or leaving group (reactions II and III); and cleavage of the P-0-R linkage or O-dealkylation (reactions IV and V). Reactions It. II. IV, and V all result in the ester and can therefore be considered to be detoxi- fication pathways, whereas reaction I, which is responsible for conversion of the inactive phosphoro- thioate to a highly potent phosphate anticholin- esterase, is an activation pathway. The latter reaction, often termed desulfuration, is catalysed by the microsomal mixed-function oxi- dase complex requiring NADPH and 02 and has been demonstrated in mammalian liver as well as several insect tissues (Dahm, 1970; Dahm & Nakatsugawa, 1968; Nakatsugawa & Dahm, 1965, 1967). As with the other microsomal enzymes it exhibits a high degree of nonspecificity and can 181 Isolan t alone 1.55 8.8 8.9 with P.B. 0.175 1.0 1.04 C. F. WILKINSON Fig. 4 Metabolic pathways of the organophosphorus insecticides RO S HO OX RO 0 HO/ OX RV O\ S RO OX II RO 0 V \R \ RO OX RO S RO OH RO 0 RO/ OH activate almost any phosphorothioate with which it comes into contact (Nakatsugawa et al., 1968). Reactions II and III have long been considered to be hydrolytic pathways catalysed by phosphatase enzymes (Heath, 1961; O'Brien, 1967). These are typified by " paraoxonase", which was initially classified by Aldridge (1953) as an A-esterase and which is widely distributed in mammalian sera, liver, and other tissues. Unfortunately we have little information on the range of substrates attacked by the A-esterases, although it appears that the structural features that determine whether or not an organophosphate will inhibit cholinesterase also determine its ability to be accepted as a sub- strate by the A-esterase (Heath, 1961). Consequently, good inhibitors of cholinesterase, mostly phosphates, are readily cleaved by the A-esterase, whereas poor inhibitors such as the phosphorothioates are usually not substrates for the enzyme. The situation is made even more complex by the fact that consider- able species differences occur with regard to sub- strate specificity (Heath, 1961) as well as by the probable existence of several isoenzymes in any one species (Kojima & O'Brien, 1968). In general it appears as though the A-esterases will cleave a large number of phosphate analogues (reaction III) but are not capable of direct cleavage of phos- phorothioates (reaction II). Many reports, however, indicate that dialkyl phosphorothioic acids are common metabolites of phosphorothioate insecti- cides (Menzie, 1969; O'Brien, 1967) and this indi- cates the direct cleavage of the P-O-X linkage of these materials. As a result there has been con- siderable speculation regarding the existence of phosphatases specific for P= S and P= 0 com- pounds. The problem has been clarified to a great extent by the excellent work of Nakatsugawa, Dahm, and their colleagues, who have shown that reaction II is catalysed not by the previously assumed ester- atic mechanism but by the NADPH-requiring oxi- dative enzymes associated with microsomes from both mammalian liver and insect tissues. Thus, parathion (Nakatsugawa & Dahm, 1967) and several of its analogues (Nakatsugawa et al., 1968) are effectively cleaved to the corresponding dialkyl phosphorothioic acids in this manner, as are diazi- non (Dahm, 1970; Nakatsugawa et al., 1969) and possibly malathion (Dahm, 1970). In contrast to the phosphatases, which attack primarily the phos- phate esters, the activity of the microsomal enzymes appears to be limited largely to cleavage of the aryl-phosphate bond of the phosphorothioates. Although some phenol production was observed in in vitro microsomal incubations of paraoxon and other closely related phosphates this was found to be independent of NADPH2 addition and was considered to result from phosphatase activity. One exception was noted-the diisopropyl homologue of paraoxon was cleaved oxidatively in a manner similar to the phosphorothioates (Nakatsugawa et al., 1968). The oxidative cleavage of EPN t indicates that substrate specificity is not limited to the esters of phosphoric acid (Nakatsugawa et al., 1968). The other principal metabolic pathway for the organophosphates is that of O-dealkylation (reac- tions IV and V) and this can apparently be effected by at least two enzyme systems, depending on the nature of the alkyl groups in the molecule. Of major importance in the metabolism of dimethyl phosphates is dealkylation by glutathione S- alkyl transferase (GSAT) (Fukami & Shishido, 1966; Hollingworth, 1969, 1970). Thus, glutathione- fortified mouse liver supernatant shows high activity with regard to the dealkylation of the dimethyl homologue of paraoxon, the oxygen analogue of fenitrothion (" Sumioxon "), and dichlorovs as well as dimethyl phosphorothioates such as fenitrothion (Hollingworth, 1969, 1970). The reaction results in the formation of S-methyl glutathione, which in vivo is further converted to volatile respirable com- pounds. A similar enzyme has been reported from several insect tissues (Fukunaga et al., 1969). Clea- vage apparently occurs at only one of the P-O- alkyl linkages and one report (Stenersen, 1969) indicating the simultaneous splitting of both methyl groups of bromophos by glutathione-dependent enzymes in mammalian liver and plants has subse- quently been retracted following discovery of an error in the reference materials employed. 182 SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES Diethyl compounds such as paraoxon, the di- ethyl homologue of dichlorvos, and chlorfenvinphos and compounds with larger alkyl groups are more refractory to cleavage by GSAT (Hollingworth, 1969, 1970) and with these materials dealkylation in both mammals (Donninger et al., 1967) and insects (Oppenoorth et al., 1971) appears to result from NADPH-dependent microsomal oxidation with production of the corresponding aldehyde. Deethylation ofparaoxon has also been demonstrated in vivo in susceptible and resistant houseflies (Nolan & O'Brien, 1970). In addition to the general pathways outlined in Fig. 4 important metabolic routes often result from direct enzymatic modification of the leaving group moiety without ester cleavage. These depend on the structural nature of the leaving group and will be referred to shortly with direct reference to the synergism of specific insecticides. The pathways in- clude both activation and degradation by micro- somal enzymes as well as those catalysed by more specific enzymes. Special mention should, however, be made of the carboxylesterases, which as their name suggests cleave the carboxy-ester groups of compounds such as malathion and are particular- ly important in determining the favourable (for mammals) selectivity observed with insecticides containing this group (O'Brien, 1967). Although the foregoing is a very brief and some- what simplified description of organophosphate metabolism it serves to emphasize the immense complexity involved. The principal metabolic path- way taken by any particular organophosphate in different mammalian or insect species will therefore depend on its structure as well as on the enzymatic constitution of the species involved. Both qualita- tive and quantitative variations in the latter can be expected to occur as a result of age, sex, and the development of resistance, so that it often becomes impossible truly to evaluate which pathway is critical or rate-limiting with regard to the death or survival of the organism. In view of these complexities it is not entirely surprising that the effects of different synergists on organophosphate metabolism and toxicity are often unpredictable and difficult to interpret. This is parti- cularly true with regard to combinations of the organophosphate insecticides with microsomal en- zyme inhibitors such as the methylenedioxyphenyl compounds. Combination of the latter with several organo- phosphates, particularly those containing substituted amino or amido groups, often results in substantial synergism (Sun & Johnson, 1960, 1969) and this immediately indicates the importance of the micro- somal enzymes in the degradative metabolism of these compounds. Thus, in combination with 1% sesamex t the toxicity of dicrotophos to houseflies is increased by a factor of 19.7 compared with fac- tors of 6.7 and 1.9, respectively, for monocrotophos (the corresponding mono-N-methyl analogue of dicrotophos) and the unsubstituted amide (Table 8). These, and similar values obtained by Menzer & Casida (1965), immediately suggest the impor- tance of the microsomal N-dealkylation of dicroto- phos and this is supported by the demonstration of this metabolic pathway both in vitro and in vivo (Hall & Sun, 1965, Menzer & Casida, 1965) and of its elimination in insects co-treated with sesamex (Hall & Sun, 1965). As shown in Table 8, however, when applied alone the unsubstituted amide has approximately 10 times the toxicity of dicrotophos itself, so that one would initially expect the dealkyl- ation reaction to result in an increase in toxicity and its inhibition by sesamex to antagonize rather than synergize the toxicity of dicrotophos. The explanation of this anomaly is not immediately obvious, though at least two possibilities are sug- gested. First, it could be argued that the inhibition of N-dealkylation by sesamex prevents the formation Table 8 Synergism of dicrotophos and analogues by sesamex against houseflies and the stability of dicrotophos analogues to alkaline hydrolysis * GENERAL STRUCTURE 0 0 R 11 11 //(CH30)2-P-OC = CHCN CH3 R' LCso of toxicant (%) Half-lifeSubstituents __________________ (min) of 0.1 % alone with synergistic solution in R R' (A) sesamex ratio 0.037N(B) (A/B) NaOH CH3 CH3 0.059 0.003 19.7 64 CH3 H 0.014 0.0021 6.7 42 H H 0.0037 0.00197 1.9 35 Toxicity data from Sun & Johnson (1960) and Sun et al.(1 967); alkaline hydrolysis data from Menzer & Casida (1 965). 183 C. F. WILKINSON of the unsubstituted amide, which may be more susceptible to enzymatic attack. The possibility of an increased susceptibility to phosphatase action is suggested by the decreasing stability of the -NHCH3 and -NH2 analogues to alkaline hydro- lysis (Menzer & Casida, 1965) (Table 8) and it is of interest in this connexion that Hall & Sun (1965) reported that, in addition to blocking N-dealkylation, sesamex t greatly reduced the hydrolytic products of dicrotophos in houseflies. However, the fact re- mains that the unsubstituted amide is itself a potent insecticide and this is not consistent with the fore- going explanation of dicrotophos synergism. An alternative possibility is that dicrotophos is suscep- tible to microsomal enzyme attack in at least two different sites on the molecule. One of these results in the observed N-dealkylation, which appears to make little difference to the potential toxicity of the molecule, whereas the other, which although unknown could conceivably be either microsomal O-dealkylation or microsomal attack at some other point on the leaving group, leads to a marked de- crease in insecticidal potency. If this is true then the pattern of synergism is readily explainable on the grounds that dicrotophos is a much better substrate for the degradation pathway than is the unsubstituted amide. This in turn could be a reflection of the decreased lipophilic character of the unsubstituted amide and its consequent failure to be accepted as a substrate by the microsomal enzymes. The latter explanation is further supported by the degree of synergism observed with a series of monocrotophos analogues in which the length of the carbon chain of the N-alkyl group is increased (Sun & Johnson, 1969) (Table 9). When applied alone to houseflies these analogues show a marked decrease in toxicity with increasing length of the alkyl chain, and there exists a 340-fold difference between the C8H17 analogue and the unsubstituted amide. In the presence of 1% sesamex, however, most of the series are almost equitoxic, indicating that the length of the alkyl chain is in some way associated with the relative ease with which each of the members of the series is detoxified. Although these materials represent an extension of the dicro- tophos series and the results show an identical trend, it is most doubtful whether the higher homo- logues of this series can be N-dealkylated to the unsubstituted amide. Consequently, it appears likely that because of the increase in lipophilic characteristics resulting from increasing length of the alkyl chain the higher homologues become Table 9 Synergism of monocrotophos homologues by sesamex against houseflies * GENERAL STRUCTURE 0 H R 11 11(CH30)2-P-OC = CHCN CH3 R Topical LDso (,g/female fly) R I 1 Synerqistic substituent alone tsesamex rat/o(A) ~~(B) H 0.018 0.0098 1.8 CH3 0.065 0.0105 6.2 C2H5 0.123 0.0173 7.1 C31H-7 0.66 0.0172 38.4 C4H9 0.64 0.0159 40.3 C61H-13 3.5 0.292 120.0 C81H-17 6.1 0.083 73.5 Data from Sun & Johnson (1 969). better microsomal enzyme substrates. It is probable that the critical microsomal pathway leading to detoxification is common to each member of the series and does not directly involve the N-alkyl moiety per se, although the nature of this pathway has not yet been established. The possible importance of the overall lipo- philic characteristics of a molecule in determining its acceptability as a microsomal enzyme substrate is again suggested by the synergistic ratios obtained with a series of 1-thiovinyl phosphates (Sun et al., 1967). Thus, the data in Table 10 indicate that increasing the size of the alkyl substituent from CH3 to C4H9 is associated with an increase in the degree of synergism with sesamex. It is probable that one of the major metabolic pathways for these materials involves microsomal oxidation of the thioether linkage to the corresponding sulfoxides and sulfones, which, as shown in Table 10, results in some loss of toxicity. However, the 7.8-fold syner- gism obtained with sesamex in combination with diethyl-1-phenylsulfonyl vinyl phosphate indicates that additional oxidative metabolism is involved in the detoxification of this compound. Interaction of the phosphorothioate insecticides with inhibitors of microsomal oxidation presents 184 SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES Table 10 Synergism of some 1-thiovinyl phosphates by sesamex against houseflies* GENERAL STRUCTURE 0 CH2 (R0)2-P OCS_ Table 11 Synergism and antagonism of some phosphorothioates by sesamex against houseflies * Insecticide LCso (% w/v) Synergistic parathion-methyl alone with 1 % ratio parathion(Alon sesamex (A/B3)(A) __ (B) Chlorthion t diazinon 0.023 0.041 0.113 0.21 0.046 0.094 0.0028 0.0026 0.0066 0.0098 8.2 15.8 17.1 21.0 0.0042 11.0 0.012 7.8 phorate demeton disulfoton alor (A 0.0( 0.0( 0.0! 0.0! LC50 (% W/V) with 1 % (B) )53 0.013 )41 0.0065 58 0.198 53 0.0137 0.043 0.13 0.13 0.0052 0.0036 0.0036 Synergistic ratio (A/B) 0.41 0.63 0.30 3.9 8.3 36.1 36.1 *_ Data from Sun & Johnson (1960) and Sun et al. (1 967). * Data from Sun et al. (1967). additional problems owing to the fact that these materials are subject to both activative and degra- dative metabolism by the microsomal enzymes (Dahm & Nakatsugawa, 1968). The antagonistic action of sesamex t and other methylenedioxyphenyl compounds on the toxicity of several phosphoro- thioates (Table 11) (Sun & Johnson, 1960) is there- fore readily explained in terms of inhibition of the P=StoP=O conversion, although an explanation of the marked synergism observed with other ma- terials of this type is not immediately forthcoming. Thus, sesamex causes a 36-fold synergism of demeton and disulfoton against houseflies and a lower though significant degree of synergism of other phosphoro- thioates such as diazinon (Sun et al., 1967) (Table 11). As diethyl phosphorothioates these materials will presumably be susceptible to microsomal degradation through cleavage of both the leaving group (Dahm, 1970) and the P-O-alkyl linkage (Donninger et al., 1967; Oppenoorth et al., 1971), and the presence of either the thioether group or the substituted pyrimidine ring provides additional sites at which microsomal oxidation takes place (Dahm & Nakatsugawa, 1968). The net result of synergist interaction (antagonism or synergism) will therefore depend on a metabolic balance between the critical pathways responsible for activation and degradation and on the degree to which each of these pathways is inhibited by the synergist. In compounds containing several groups that are potential sites for microsomal metabolism we have as yet no information that enables us to pre- dict those sites that are likely to be preferentially attacked. Nor do we have data that indicate which types of microsomal reaction are most susceptible to inhibition by the synergist. It should be kept in mind that inhibition of any of the microsomal reactions is by no means complete, so that all we can conclude in those cases where synergism is observed is that the net balance of the synergist interaction favours a biological stabilization of the insecticide, which allows a greater realization of its potential toxicity. Species and strain variations can also be expected markedly to influence the observed effects, and in many cases where insect resistance is associated with an increased microsomal enzyme capability (El- Bashir & Oppenoorth, 1969; Plapp, 1970) synergists can often exhibit a relatively greater effect. Thus the 10-fold resistance of the F, strain of the house- fly to diazinon can be almost completely reversed by co-treatment with sesamex (Oppenoorth, 1965). Few studies have been undertaken to investi- gate the synergistic activity of organophosphate combinations with oxidative inhibitors other than the methylenedioxyphenyl compounds, although it is likely that similar results would be obtained. R substituent CH3 C2H5 iso-C3H7 n-C4Hs C2H5 (sulfoxide) C2H5 (sulfone) 185 C. F. WILKINSON MGK 264,t WARF Antiresistant,t and SKF 525A t have been shown to effectively block phosphoro- thioate oxidation in in vitr-o preparations from insects (Nakatsugawa & Dahm, 1965) and mammals (Murphy & DuBois, 1957; Murphy, 1969) and the latter compound antagonizes the toxicity of schra- dan, azinphos-methyl (O'Brien & Davison, 1958), and dimethoate to mammals. In houseflies, how- ever, azinphos-methyl was synergized about 2-fold in the presence of SKF 525A (O'Brien, 1961). The same synergist is also reported to stabilize paraoxon in vivo in both mice and cockroaches (O'Brien, 1961), a fact that might reflect inhibition of oxidative O-deethylation. Inhibition of the microsomal detoxification pathways of organophosphates and the resulting synergism of the insecticides against insects is not restricted to better known inhibitors of the micro- somal enzymes, such as the methylenedioxyphenyl compounds and SKF 525A. Thus, it has recently been demonstrated that several phosphorothioates, including parathion, diazinon, and the nontoxic O,O-diethyl O-phenyl phosphorothioate (SV,) are more effective than sesamex in inhibiting the micro- somal dealkylation of paraoxon in in vitro prepara- tions from several strains of the housefly and are also very effective synergists for several organo- phosphates in vivo (Oppenoorth et al., 1971). These data are fully described by Oppenoorth (1971). The effect is apparently similar to the inhibition of micro- somal oxidation of cis-mevinphos by its thiono ana- logue in mice (Morello et al., 1968) and it is pos- sible that synergism by phosphorothioates is a gene- ral phenomenon that should be further investigated. It is probable that the phosphorothioates are acting as alternative substrates for the microsomes, as presumably they are themselves susceptible to both desulfuration and/or ester cleavage. In view of the fact that the diisopropyl homologue of paraoxon is also a microsomal enzyme substrate (Nakatsugawa et al., 1968), it is possible that the 2-fold and 10-fold synergism of diazinon by this compound against a susceptible and a 36-fold diazinon-resistant strain of the housefly, respectively (Oppenoorth & Van Asperen, 1961), results from a similar mechanism. It was initially suggested that the synergism observed with the diisopropyl homo- logue of paraoxon resulted from a selective inhi- bition of phosphatase action, though the evidence for this was not convincing. Perhaps the most thoroughly studied and best known examples of organophosphate synergism are those involving combinations of several materials with malathion. In mammals and resistant (though not susceptible) strains of insect, malathion is detoxi- fied largely by the action of carboxylesterase (Heath, 1961; O'Brien, 1967), which has been identi- fied with " aliesterase" in rat and human liver by Main & Braid (1962) and can be classified as a B-esterase according to Aldridge (1953). As such, the enzyme is susceptible to inhibition by a number of phosphates (O'Brien, 1967) and the synergism of malathion against both mammals (Murphy, 1969) and resistant insects by these compounds has long been associated with this mechanism. Two of the best known malathion synergists are EPN t and tri-o-tolyl phosphate, although in recent years a large number of other materials have been eval- uated for synergistic activity. In a 1: 1 ratio with malathion a variety of com- pounds, mostly trisubstituted aliphatic and aro- matic phosphate esters, are found to be excellent synergists against strains of housefly and mosquito larvae (Culex tarsalis) that show approximately 100-fold resistance to malathion (Plapp et al., 1963; Plapp & Tong, 1966). As shown in Table 12, the resistance factor is completely reversed by some of these materials, though none were found to be effective in synergizing malathion against Table 12 Synergism of malathion against malathion-resistant houseflies and mosquito larvae (Cu/ex tarsalis) by some noninsecticidal phosphate esters * 24-hour toxicity of malathion + synergist (1:1 ratio) for Synergist _ None (C4H9S)3P=o (DEFt) (C4HaS)3P (C6H5O)3P=o (C2H5s)3P=S (iso-C3H7S)3P=S (C3H7S)3P=S houseflies mosquito larvae LD5o(jig/jar) SR LC50 (%) SR(Ag/ a ) 'i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1 800 a 25 40 80 85 85 20 72 45 22.5 21.2 21.2 90 2.4 b 0.03 0.025 0.024 0.90 0.10 0.10 80 96 100 2.7 24 24 * Data from Plapp & Tong (1966) and Plapp et al. (1963). a LD5o for susceptible flies = 1 7 jig/jar b LC5o for susceptible larvae = 0.025 %. 186 SYNERGISTS AND METABOLISM AND TOXICITY OF ANTICHOLINESTERASES susceptible houseflies. Similarly, triphenyl phos- phate, which has been shown to effect a 189-fold synergism of malathion against a resistant strain of flour beetle, Tribolium castaneum, has no effect on a susceptible strain (Dyte & Rowlands, 1967). DEF t and EPN t have also been reported as synergists for dicrotophos, dimethoate, and phorate against boll weevils (Anthonomnus grandis) (Bull et al., 1965), which suggests that these synergists are able to inhibit enzymes other than carboxylesterase. Dimethoate is also synergized by EPN in mice (Uchida et al., 1966) and it was suggested that this resulted from inhibition of the enzyme carboxya- midase, which effects direct cleavage of the carbox- yamide group (O'Brien, 1967). The significance of this enzyme appears in some doubt in the light of more recent work on microsomal metabolism of substituted amides such as dicrotophos and dimetho- ate. It is of interest in this regard that EPN is a micro- somal enzyme substrate (Nakatsugawa et al., 1968) and the fact that both activation and degradation of dimethoate (Lucier & Menzer, 1970) and mala- thion (Dahm, 1970) have been associated with the microsomes may suggest that their synergism by EPN results from an interaction with these enzymes as well as carboxylesterase or carboxyami- dase. Plapp & Valega (1967) have recently tested a large number of noninsecticidal carbamates as synergists for malathion against a resistant strain of the housefly. Several of these, particularly the aromatic esters of dibutylcarbamic acid, are found to be as effective as DEF (Table 13) in reversing malathion resistance when applied in a 1: 1 ratio with the insecticide. A novel case of organophosphate synergism has recently been demonstrated to occur in mammals between methyl iodide and several dimethyl phos- phates which undergo P-O-alkyl cleavage by glutathione S-alkyl transferase (Hollingworth, 1969, 1970). As a result of the finding that treatment of mice with methyl iodide causes a transient depression in free glutathione levels it is suggested that syner- gism by this compound results from a competition with respect to the donation of methyl groups to glutathione. Consequently, in the presence of methyl iodide less free glutathione is available to accept the methyl groups from the organophosphate. The toxicity of fenitrothion is also substantially synergized by diethyl maleate, which also reduces liver glutathione levels (Hollingworth, 1970). These Table 13 Synergistic activity of noninsecticidal carbamates against malathion-resistant houseflies (M. domestica) * GENERAL STRUCTURE C4H9 0 (S) NC-0-R C4H9 24-hour toxicity of R malathion + synergist Synergistic substituent (1:1 ratio) ratio (LD5o, Mg/jar) None 3 000 a- phenyl 150 20 thio, S-phenyl 30 100 o-tolyl 50 60 thio, S-o-tolyl 45 67 o-chlorophenyl 25 120 m-chlorophenyl 30 100 * Data from Plapp & Valega (1967). a LDso for susceptible flies = 8 Mg/jar. examples represent the only ones known where synergism appears to result directly from synergist interaction with a cofactor of the detoxification enzyme. CONCLUSIONS Although in some cases it is clear that synergists have the potential to substantially enhance insecti- cidal potency it would be wrong to suggest that their immediate commercial development would lead to some kind of universal pest control revolution. It would also be unrealistic to suggest that widespread agricultural use of insecticide synergists is likely to occur in the near future. On the contrary, the facts that synergistic activity has been recognized for over 30 years and that during this period it has been successfully applied only with the pyre- throids, cannot help but cause a certain amount of pessimism. In spite of some commercial interest in synergism the argument often used by those in the chemical industry is that as long as adequate insect control can be achieved with a single active material there is little or no justification for using a combination 187 188 C. F. WILKINSON of two. It is usually considered, with some justi- fication, that such combinations cause additional formulation problems and the high cost of most existing synergists is at present an additional barrier to their use on anything but a limited scale. For the last two decades chemists have provided a continuous flow of new and potent insecticides. Throughout the world, however, the chemical industry is at present taking a long hard look at the future and in many cases reevaluating and modifying its direction of emphasis. There is little doubt that new compounds will continue to be discovered and developed for commercial use, but it is also clear that these will differ somewhat from those to which we are accustomed. Emphasis will be placed on more selective, more biodegradable materials with lower toxicity for mammals and, as these will be more difficult to develop, their cost will of necessity increase substantially. In view of this factor and of the growing spectre of insect resistance to insecticides it seems possible that the synergist concept may gain increasing favour in commercial circles. 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(1968a) In: Hodgson, E., ed., Enzymatic oxidation of toxicants, Raleigh, North Carolina State University Press, pp. 113-150 Wilkinson, C. F. (1968b) Wld Rev. Pest Control, 7, 155-168 Wilkinson, C. F. (1971) In: Proceedings of the Second International Congress of Pesticide Chemistry, Tel- Aviv, Israel, 21-27 February 1971, Ottawa, Canada, IUPAC, Commission on Terminal Pesticide Residues (in press) Wilkinson, C. F. & Hicks, L. J. (1969) J. agric. Food Chem., 17, 829-836 Wilkinson, C. F., Metcalf, R. L. & Fukuto, T. R. (1966) J. agric. Food Chem., 14, 73-79 Williams, R. T. (1959) Detoxication mechanisms, New York, Wiley DISCUSSION DAUTERMAN: Is the synergism of carboxyamide-contain- ing organophosphorus compounds with sesamex f strictly the result of the inhibition of the mixed-function oxidases or are the carboxyamidases also inhibited? WILKINSON: Unfortunately, we have little information on the ability of sesamex to inhibit enzymes other than the mixed-function oxidases, and consequently I should not like to hazard a guess as to the effect of sesamex on carboxyamidases. The effect of synergists on different enzyme systems should be given further consideration. HOLLINGWORTH: With respect to the relationship of synergism to toxicity, it should be pointed out that it is sometimes dangerous to assume that synergists act specifically on a single detoxification mechanism, as pointed out by Dr Wilkinson. Dr Murphy's group have shown that the binding of toxicants to proteins plays a role in determining the toxicity of organophosphorus compounds, and that synergists-e.g., tri-o-tolyl phos- phate-may prevent this, thus decreasing toxicity. I have found that inhibitors of microsomal mixed-function oxidase may also inhibit nonoxidative enzymes in vitro. Piperonyl butoxide is not a potent inhibitor in this respect, but SKF-525A t is quite active. This, of course, com- plicates any analysis of the basis of synergism, and I am sure that many other examples of multiple interactions of synergists could be brought forward, such as the effects of synergists on the penetration of toxicants into insects.