Metabolic detoxication and synergistic ratio of lindane analogs in house flies

Biodegradability of lindane analogs using house fly whole body, microsomes, and microsome supernatant fraction was examined. It decreased in the order of alkoxy ～ methylthio > methyl analogs > lindane in the whole body experiments, as well as with microsomes in the presence of NADPH. With the supernatant in the presence of glutathione, a different trend was observed. The synergistic effects of piperonyl butoxide when used together with lindane analogs were mostly explained in terms of the inhibition of the microsomal metabolism. Piperonyl butoxide was also shown to inhibit the penetration of compounds into the fly body and to make the central nervous system of the American cockroach less sensitive to the action of insecticides causing after and repetitive discharges. It was observed that the value of the percentage of metabolic disappearance of insecticides after a certain period decreases as the dose level initially applied in the whole body experiments increases. The synergistic ratio parallels the percentage of disappearance value after the insecticidal activity test period when a dose corresponding to the unsynergized LD₅₀ is initially applied. When quantitative comparisons are required for biodegradability of insecticides using house flies as the test insects, it should be on the basis of direct metabolism experiments using a fixed dose throughout the series of insecticides, but not on the basis of the synergistic ratio.     

Microsomal metabolism of juvenile hormone analogs in the house fly,Musca domestica L.

Of six juvenile hormone analogs of the alkyl 3,7,11-trimethyl-2,4-dodecadienate type, only the isopropyl ester was strongly morphogenic in the house fly, Musca domestica L. In vitro assays revealed that house fly microsomes contain B-esterases as well as oxidases which metabolize such analogs. However, these esterases did not hydrolyze the isopropyl ester, ZR-515. Enzymes prepared from larvae, pupae, and adults were all active and there was evidence that in the late larval stage the esterase activity was cyclic, showing a minimum in the early third instar and a maximum a few hours later. When microsomes from two susceptible and two resistant house fly strains were compared for metabolic activity against the juvenile hormone analogs, those from the resistant strains were 1.3 to 20 × higher in oxidase activity but there was no difference in esterase activity. The oxidative metabolism of two analogs ZR-515 and 512 was greatly enhanced when the flies were induced with phenobarbital but there was no enhancement in metabolism of three of the remaining analogs and only a slight enhancement of a fourth. It is concluded that the insecticidal action of ZR-515 is largely due to its stability in the presence of the house fly esterases.     

Structure and insecticidal activity of picrotoxinin analogs

Picrotoxinin, a convulsant in mammals, was nontoxic when topically applied on whole house flies. When synergized with piperonyl butoxide, the topical LD₅₀ of picrotoxinin on house flies was 6.50 μg/g or 260 times less toxic on house flies than the synergized insecticide, carbofuran, a carbamate cholinesterase inhibitor.When perfused on the isolated thoracic ganglion of house fly, picrotoxinin was less potent in producing convulsions than carbofuran; however, when assayed on the desheathed thoracic ganglion, picrotoxin was more potent than carbofuran. This suggested a substantial barrier to picrotoxinin preventing diffusion into the house fly central nervous system.Of several picrotoxinin analogs synthesized the 2-iodo,4-isopropyl, 6-methylenebromidecyclohexane-γ-lactone was the most potent convulsant.     

Comparative insecticidal activities and metabolic rates of lindane and its hexadeuterio-analog

Hexadeuterio-lindane(γ-BHC-d₆) was several times as toxic as lindane against the mosquito (Culex pipiens pallens), house fly (Musca domestica), German cockroach (Blattella germanica) and American cockroach (Periplaneta americana). The neuroexcitatory activity of these two compounds did not differ. Lindane was considerably synergized by piperonyl butoxide, but lindane-d₆ was not. A large isotope effect was observed in the in vivo breakdown of lindane-d₆. Thus, the intrinsic toxicities of both compounds are equivalent. The difference in insecticidal activity seems to be due to the different rates of detoxifying biodegradation caused by the kinetic deuterium isotope effect.     

The mechanism of resistance to lindane and hexadeuterated lindane in the Third Yumenoshima strain of house fly

The factors which cause lindane resistance in the Third Yumenoshima strain, a strain of house flies highly resistant to insecticides, were studied using hexadeuterated lindane. Hexadeuterated lindane has the same physicochemical properties as lindane, but the former is much less biodegradable than the latter. The LD₅₀ ratio of lindane to hexadeuterated lindane in this strain, deuterium isotope effect on LD₅₀ values, was larger than that in S_NAIDM, a susceptible (nonresistant) strain. The penetration rates of labeled and nonlabeled lindane through the insect cuticle were about the same for both strains. Thus, penetration rate does not cause resistance. The metabolic degradation of lindane in the resistant strain in vivo occurred much faster than in the susceptible strain. This was also the case for lindane degradation processes in vitro such as microsomal oxidation and glutathione conjugation. In both strains, significant isotope effects were observed in the degradation rates in vitro of labeled and nonlabeled lindane. Therefore, principal biodegradation and detoxication pathways should include reactions which cleave the CH bonds. When the much less biodegradable d₆ counterpart of lindane was applied to both strains, the susceptible strain became much more highly intoxicated than the other within 20 to 30 min. This indicates that a combination of both greater degradability and probably lower sensitivity at the action site are the main factors underlying resistance in the Third Yumenoshima strain.     

Synergism of organophosphorus insecticides by diethyl maleate and related compounds in house flies

The toxicity of several organophosphorus and one carbamate insecticide for house flies is enhanced by simultaneous administration of diethyl maleate. Synergism factors vary from 2 to 116 and are strongly dependent on the combination of strain and insecticide studied. In general, it seems that thiono compounds are less synergized than their oxon analogs. Comparison of some analogs of maleic acid esters showed that the diethyl ester was not the most active compound. trans-Phenylbutenone was found to be a good alternative for diethyl maleate. Both diethyl maleate and trans-phenylbutenone deplete glutathione in the flies, but this depletion per se is certainly not the most important mode of action. In vitro experiments made clear that sufficient glutathione remained to permit a substantial insecticide degradation rate. Moreover, treatment of the flies with diethyl maleate 2–4 hr before the insecticide application resulted in a partial or complete disappearance of synergism in a period in which the glutathione concentration in the flies was still very low. Apart from this effect on the glutathione levels in the insect tissues it appeared from further experimental work in vitro that both compounds had a direct inhibitory effect on glutathione-dependent transferase(s) as well as on oxidative enzymes involved in insecticide degradation. The contribution of these reactions to the eventual synergism factor is discussed.     

Toxicity of spinosad to susceptible and resistant strains of house flies,Musca domestica

The toxicity of spinosad, a new insecticide derived from the bacterium Saccharopolyspora spinosa, was evaluated against susceptible and resistant strains of house fly (Musca domestica L.). Spinosad was highly toxic to house flies based on 72-h LD₅₀ values and the symptoms of poisoning were consistent with a neurotoxic mechanism of action. Spinosad was relatively slow acting, with the maximum toxicity noted at 72 h. Piperonyl butoxide and S,S,S,-tribu-tylphosphorotrithioate synergized the toxicity of spinosad by 3·0- and 1·8-fold, respectively, while diethyl maleate had no significant effect. These results suggest that there is a small degree of monooxygenase-mediated spinosad detoxification in house flies, while hydrolases may be only minimally important and glutathione transferases may have no role. There were no substantial levels of cross-resistance detected, except in the LPR strain where a low 4·3-fold cross-resistance was observed. The cyclodiene-resistant OCR strain was 2·7-fold more sensitive to spinosad than the susceptible strain (CS). These results suggest that cross-resistance may not be a limiting factor for the use of spinosad against house flies. © 1998 Society of Chemical Industry     

Insecticidal properties of monoterpenoid derivatives to the house fly (diptera: Muscidae) and red flour beetle (coleoptera: Tenebrionidae)

Monoterpenoid derivatives were synthesized and their insecticidal activities were evaluated against red flour beetles, Tribolium castaneum (Herbst), in fumigant bioassays and against house flies Musca domestica (L.) in topical, fumigant, and ovicidal bioassays. Acetate derivatives and haloacetate derivatives were compared with each other, and with the parent monoterpenoid to determine structure-activity relationships. Acetate derivatives were more active than the propionate derivatives of cyclic monoterpenoids in the topical, fumigant, and ovicidal bioassays. Pivalates were topically more insecticidal than acetates to adult house flies, while the acetates had the greater ovicidal activity. Acetates and pivalates were more effective than haloacetates in the topical, red flour beetle fumigation and ovicidal bioassays. Fluoroacetates of cyclic monoterpenoids were the most effective house fly fumigants, followed by acetates, and trichloroacetates. Several derivatives were produced that displayed enhanced activity relative to the parent alcohols or phenols.     

Effects of alkyl groups attached to the carbamoyl nitrogen atom of an N-arylcarbamoylpyrazoline on insecticidal activity

N-Alkylated analogs (C₁–C₃) of an N-arylcarbamoylpyrazoline were prepared. The introduction of these alkyl groups completely changed the crystal structure in respect of the torsion angle of the amide C–N bond of the non-alkyl compound. The introduction of methyl and ethyl groups slightly, and that of an isopropyl group markedly, decreased insecticidal activity against American cockroaches and house flies. Conformational analyses of the compounds suggested that the insecticidally active conformer of this class of compounds is in the anti-position regarding the N′–C(=O) and N-aryl bonds in which the non-alkyl compound is energetically the most stable. The most stable conformers of the alkylated compounds are in the syn-position, and these compounds would interact with target sites in the less stable anti-form. © 1999 Society of Chemical Industry     

Metabolism of lindane in house flies: Metabolic desaturation,dehydrogenation and dehydrochlorination,and conjugation with glutathione

In lindane-treated house flies, a cis-dehydrogenated metabolite, $\text{(}\text{36}\text{45}\text{)-}\text{hexachlorocyclohexene}$, was identified by gas-liquid chromatography and mass spectrometry. The in vitro metabolism study showed that in the presence of NADPH the microsomal fraction of house flies converted lindane to three hexane-soluble metabolites. This conversion was inhibited by piperonyl butoxide, SKF-525A, and carbon monoxide. These metabolites were identified as $\text{(}\text{36}\text{45}\text{)-}\text{hexachlorocyclohexene}$, $\text{(}\text{36}\text{45}\text{)- and (}\text{346}\text{5}\text{)-pentachlorocyclohexene}$ (PCCHE) by gas-liquid chromatography. They, as well as lindane, were excellent substrates for the reaction with the postmicrosomal fraction in the presence of glutathione. While the reaction with lindane-d₆ showed a significant deuterium isotope effect (6.82), that of $\text{(}\text{36}\text{45}\text{)-}\text{PCCHE-d}{}_5$ did not (1.18). Enzymatic conjugation with glutathione probably occurs at the stage of PCCHE.     

Dehydrogenation: A previously unreported pathway of lindane metabolism in mammals

This study presents evidence for the dehydrogenation of lindane by a hepatic microsomal mixed-function oxidase system. Preliminary investigation established that the incubation of lindane with rat liver homogenates produces a chlorinated, nonpolar compound identified as hexachlorocyclohexene. Differential centrifugation resulted in the sedimentation of most of the dehydrogenase activity in the microsomal fraction. Optimum in vitro assay conditions were established and it was found that the dehydrogenase system required molecular oxygen and reduced pyridine nucleotide coenzyme for maximum activity. Inhibition by SKF 525-A and CO suggested that the enzyme was cytochrome P-450 dependent. Lack of inhibition by cyanide indicated that the cytochrome b₅ desaturase system was probably not involved. Pretreatment of rats with DDT, which stimulates lindane metabolism, also induced significantly higher dehydrogenase activity. Both the in vivo and in vitro metabolism of hexachlorocyclohexene produced previously identified lindane metabolites. The existence of a cytochrome P-450 dependent mixed-function oxidase which catalyzes the dehydrogenation of lindane has not previously been reported and may be of importance in the metabolism of other xenobiotics.     

Insect juvenile hormones: Induction of detoxifying enzymes in the housefly and detoxication by housefly enzymes

The cecropia juvenile hormone and three of its analogs were compared as inducers of microsomal epoxidase, O-demethylase, and DDT dehydrochlorinase in the housefly, Musca domestica L. The compounds were the cecropia juvenile hormone, methoprene, hydroprene, 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene, and piperonyl butoxide, a well known insecticide synergist. The compounds were administered by feeding at levels up to 1% in the diet for 3 days to 1-day-old female adults. Enzymes were then prepared and assayed for their activity using heptachlor, p-nitroanisole, and DDT as substrates.There was approximately a twofold increase in the microsomal oxidases and a 50% increase in DDT dehydrochlorinase after the treatment with the cecropia juvenile hormone, while methoprene had some activity as an inducer of the epoxidase (30% increase) but no activity in the case of the O-demethylase or the dehydrochlorinase. Hydroprene had no effect on any of the enzyme systems, while 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene was an inhibitor of the two microsomal oxidases. The latter compound and piperonyl butoxide were strong inducers of DDT dehydrochlorinase, causing approximately twofold increases in the activity of this enzyme.There was evidence that the microsomal preparations were able to metabolize and inactivate methoprene and hydroprene, the action being oxidative in the case of methoprene and both oxidative and hydrolytic in the case of hydroprene. The oxidative metabolism of the two juvenile hormone analogs by the microsomal preparations was inducible by the cecropia juvenile hormone and by phenobarbital and dieldrin.     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

CNS insensitivity to pyrethroids in the resistant kdr strain of house flies

Solutions of tetramethrin, RU 11679, or cismethrin caused uncoupled convulsions in 30–40 min in exposed thoracic ganglia from S_NAIDM house flies at concentrations down to 10^?10M: whereas these same compounds at 10^?6M concentrations failed to produce poisoning symptoms when perfused onto the exposed ganglia of the kdr strain of house fly. The pyrethroid analogs examined had a negative temperature coefficient of action on the exposed thoracic ganglia from S_NAIDM flies. DDT and GH-74 possessed positive temperature coefficients of action on the exposed thoracic ganglion of susceptible house flies. It is concluded that the central nervous system of the kdr strain of house fly is resistant to pyrethroid action; furthermore, the resistance appears to be widespread throughout the house fly nervous system, involving sensory, motor, and central neural elements.     

Laboratory studies on the mechanisms of resistance to permethrin in a field-selected strain of house flies

Enhanced oxidative metabolism appeared to be a major factor involved in resistance to permethrin in a field strain of house flies, selected with permethrin over 4 years. This was shown in the 7.8-fold synergism by piperonyl butoxide which reduced the resistance ratio from 97 to 15. The rate of permethrin detoxication was significantly higher (P=0.05) in the resistant flies compared with a susceptible strain or resistant flies pretreated with piperonyl butoxide. The esterase inhibitor S,S,S-tributyl phosphorotrithioate did not reduce the level of resistance to permethrin in the resistant strain, although some hydrolytic metabolism was apparent. Rates of penetration were similar in susceptible and resistant flies and in resistant flies pre-treated with piperonyl butoxide. A minor unidentified resistance factor, possibly reduced sensitivity of the nervous system, may also have been present in the resistant strain.     

Arguments against a fifth chromosomal factor in control of aldrin epoxidation and propoxur resistance in the Fc strain of the house fly

The DDT-resistant Fc strain of house flies, Musca domestica L., was analyzed genetically by means of crosses with a susceptible strain carrying a recessive mutant marker for each of the five autosomes. Progeny (substrains) retaining combinations of two, three, or four chromosomes of the resistant parent were selected for measurement of their microsomal aldrin expoidase activity and its correlation with chromosomal makeup and level of resistance to DDT and propoxur. There was no evidence that microsomal epoxidation of aldrin or resistance to propoxur, is associated with chromosome V in the Fc strain as has been reported. Instead, the well-known oxidase regulating factor on chromosome II was of major importance in the strain's microsomal oxidation of aldrin. There was also evidence, though not conclusive, that a factor on chromosome I has an influence on the oxidative metabolism of insecticides in this strain, possibly through an interaction with the factor on chromosome II. The reasons for the conflicting reports on the genetic control of microsomal oxidation in the Fc strain are discussed.     

Metabolism of carbosulfan [2,3-dihydro-2,2-dimethylbenzofuran-7-yl (di-n-butylaminothio)methylcarbamate] in the rat and house fly

The metabolism of carbosulfan, 2,3-dihydro-2,2-dimethylbenzofuran-7-yl (di-n-butylaminothio)-methylcarbamate, was studied in the rat and house fly. Carbosulfan was metabolized via at least two major pathways in the rat, by initial oxidation of the sulfur atom and by NS bond cleavage. The principal rat metabolites were the conjugated keto-phenol from the ring-labeled carbo-sulfan, carbon dioxide from the carbonyl-labeled carbosulfan, and dibutylamine from the dibutylamino-labeled carbosulfan. In house flies, carbosulfan was converted primarily into carbofuran and related oxidation products. The lower mammalian toxicity of carbosulfan compared to its insecticidal activity is explained on the basis of differences in routes and rates of metabolism of carbosulfan in mammals and insects.     

Behavioural and electrophysiological investigation of 1-(7-ethoxygeranyl)-2-methylbenzimidazole on the house fly Musca domestica

The insecticidal properties of 1-(7-ethoxygeranyl)-2-methylbenzimidazole (EGMB) were investigated on larval and adult house flies. Unsynergised EGMB gave topical LD₅₀ values of 0.53 μg per female fly on NAIDM strain house flies. When flies were pretreated with 5.2 μg piperonyl butoxide, susceptibility was increased (LD₅₀ 0.12 μg per female fly). House fly larvae were less susceptible to EGMB (LD₅₀ 2.2 μg). Poisoning with EGMB resulted in a rapid reduction in locomotor activity of both larval and adult house flies. This reduction in locomotion was progressive and led to complete paralysis. Various parameters of larval nervous system function were investigated in larvae during these early phases of poisoning. As early as 15 min after dosing larvae with LD₉₅ doses of EGMB, sensory nerves were less responsive. Over a somewhat longer time (2–4 h), neurally evoked contractures were adversely affected by EGMB. In some cases, this effect appeared to be due to reduced postsynaptic potential amplitude; in other instances, it appeared to be due to an effect independent of neuromuscular transmission. The close temporal correlation between behavioural and electrophysiological observations suggests that the nervous and muscular systems are important sites of action of EGMB.     

Quantitative structure-activity studies of substituted benzyl chrysanthemates: 4. Physicochemical properties and the rate of progress of the knockdown symptom induced in house flies

A procedure to evaluate the knockdown activity of pyrethroids against house flies in which metabolic factors could be eliminated as far as possible was established. With piperonyl butoxide and NIA 16388 as the inhibitors of oxidative and hydrolytic metabolism, respectively, the “intrinsic” knockdown potencies of 22 substituted benzyl (1R)-trans-chrysanthemates and related compounds were determined 2.5–3 hr after topical application to house flies. From the intrinsic knockdown potency and the rate of progress of the knockdown symptom from the earliest stage of intoxication, a “penetration” rate constant was estimated by first-order kinetics using a two-compartment model. The rate constant was correlated quantitatively with the hydrophobic parameter of the molecule. The lower the hydrophobicity, the higher the rate constant within the range of compounds used in this study.     

Acetylcholinesterase inhibition by nootkatone and carvacrol in arthropods

The essential oils from many botanicals have been screened for insecticidal activity. Two constituents of the Alaskan yellow cedar tree, the monoterpenoid carvacrol and the sesquiterpenoid nootkatone, both are toxic against several arthropods. The mode of action through which nootkatone and carvacrol exert their insecticidal activity remains uncertain. It has been hypothesized that they may inhibit acetylcholinesterase enzyme activity. The degree of acetylcholinesterase inhibition of carvacrol and nootkatone was compared to that of carbaryl, a known acetylcholinesterase inhibitor, in the house fly (Musca domestica), yellow fever mosquito (Aedes aegypti), American dog tick (Dermacentor variabilis) and American cockroach (Periplaneta americana). The concentration of carbaryl, at which 50% of the acetylcholinesterase activity was inhibited (IC₅₀), was less than 2 μM in all four arthropod models. Carvacrol was observed to cause slight inhibition of the acetylcholinesterase enzyme in house flies, ticks and cockroaches, but it did not inhibit the mosquito acetylcholinesterase enzyme. Nootkatone did not inhibit the acetylcholinesterase enzyme in any of the four arthropod models tested. From this study, we conclude that the acetylcholinesterase inhibition is not likely the primary mode of action for insecticidal activity by nootkatone or carvacrol.