Effect of disposition on the relative delayed neurotoxicity potency among leptophos analogs

The oxons of leptophos, its desbromo analog, and its ethoxy analog all inhibit hen brain neurotoxic esterase (NTE) to a similar degree in vitro, but have large differences in minimum effective oral doses for organophosphorus-induced delayed neurotoxicity. The potencies of leptophos and ethoxyleptophos are increased 10-fold when administered to hens intravenously (iv), but the potency of desbromoleptophos is nearly the same whether administered iv or po. Leptophos distributes nearly five times more rapidly from the central compartment than does desbromoleptophos, and its attenuated oral potency may be due to slower net absorption and/or dilution by compartments other than nervous tissue. Intravenously administered phenylphosphonothionates are excreted rapidly into the gastrointestinal tract and a large proportion of the dose is eliminated in the first 48 hr. Indirect evidence indicates that ethoxyleptophos is more rapidly degraded and ethoxyleptophos-inhibited NTE recovers more rapidly than does NTE inhibited by leptophos or desbromomleptophos. It is proposed that differential aging of ethoxyleptophos chiral isomers as well as pharmacokinetic factors may contribute to the apparently anomalous behavior of these three analogs.     

Avian whole‐brain spheroid cultures: applications in pesticide toxicity

The adult hen is the species of choice when assessing the potential of organophosphate compounds (OPs) to evoke OP‐induced delayed neuropathy (OPIDN). OPIDN has been shown to depend on the irreversible covalent binding of certain OPs to the enzyme neuropathy target esterase (NTE). Levels of this enzyme in human brain are similar to those in avian species. We have previously shown that the hen embryo whole brain spheroids are histotypically similar to adult brain and express measurable levels of acetylcholine esterase (AChE) and NTE, the key enzymes in mechanisms of OP toxicity. Single‐cell suspensions were prepared from meninges‐free 7 day in ovo hen brains. Cells were grown in serum‐free media and incubated on an orbital shaker at 37 °C in 5% carbon dioxide humidified air. Spheroids can be maintained long‐term in culture and allow the effects of repeated dose and recovery studies to be conducted. Hen embryo brain spheroids were exposed to varying concentrations of tri‐o‐cresyl phosphate, leptophos and its metabolite leptophos oxon at day 14 in vitro and the effects on the activity of AChE and NTE measured. Whilst current legislation regarding toxicity testing of OPs requires the use of animals, our in vitro model provides a potential pre‐screen for novel chemical entities and commercial OP mixture variants. Such an approach should refine and reduce the number of animals used in development of new materials. © 2000 Society of Chemical Industry     

The neurotoxicity of O-(2,5-dichlorophenyl) O-methyl phenylphosphonothionate,an impurity and photoproduct of leptophos (phosvel) insecticide

The neurotoxic effects of leptophos and three degradation products of leptophos have been evaluated in white leghorn pullets. The data derived demonstrate that desbromo leptophos and leptophos oxon are approximately three and two times, respectively, more effective neurotoxic agents than leptophos in the white leghorn pullet.     

Interaction of some unsubstituted phosphoramidate analogs of methamidophos (O,S-dimethyl phosphorothioamidate) with acetylcholinesterase and neuropathy target esterase of hen brain

At 37°C and pH 7.4–8.0, five higher O-alkyl analogs of methamidophos and four O-alkyl O-2,5-dichlorophenyl phosphoramidates all were more potent progressive inhibitors of hen brain AChE and neuropathy target esterase (NTE) than was methamidophos itself. For AChE, k_a increased from 7.2 × 10² to 1.0 × 10⁵ M⁻¹ min⁻¹ between methyl and n-hexyl S-methyl esters and from 9.3 × 10³ to 8.9 × 10⁵ M⁻¹ min⁻¹ between ethyl and n-hexyl dichlorophenyl analogs. For NTE, the ranges were from 16 to 7.9 × 10⁴ for S-methyl esters, and were 9.7 × 10⁴ to 7.8 × 10⁶ M⁻¹ min⁻¹ for dichlorophenyl. S-methyl esters were more active against AChE than against NTE and all the dichlorophenyl esters were more active against NTE than against AChE. Spontaneous reactivation of 75–100% activity without aging of AChE was found after 19 hr incubation at 37°C after inhibition by all nine straight-chain alkyl analogs. After inhibition by O-isopropyl S-methyl phosphorothioamidate, some spontaneous reactivation with complete aging of all remaining inhibited AChE occurred during 19 hr. No spontaneous reactivation or aging of inhibited NTE was detected. It was concluded that the molecular structures of the inhibited enzymes obtained from equivalent compounds in the two series of inhibitors were identical and that the leaving groups were, therefore, S-methyl and O-2,5-dichlorophenyl, respectively. Although hen brain NTE inhibited by methamidophos in vitro did not age, cases of delayed neuropathy in man have been reported and, presumably, require aging as well as inhibition of NTE. Possible explanations of this apparent discrepancy include (i) the fact that methamidophos consists of two chiral forms and that the form seen to be active in vitro may be disposed of preferentially in vivo, (ii) the possibility of activation in vivo to a different inhibitor, (iii) differences between conformation and ease of aging of inhibited NTE in vitro and in vivo, and (iv) species differences.     

Pyrethroid synergists suppress esterase-mediated resistance in Indian strains of the cotton bollworm, Helicoverpa armigera (Hübner)

The role of esterase in pyrethroid resistance was studied in the final larval instar of different strains of the cotton bollworm, Helicoverpa armigera. The resistant strains viz., Nagpur strain and the Delhi strain were found to have elevated midgut esterase activity in comparison to the susceptible strain. Nagpur strain and Delhi strain have 2.24 and 1.73-fold higher esterase activity, respectively, than that of the susceptible strain. The Native PAGE displayed important differences in the midgut esterase isozyme pattern between the susceptible and the pyrethroid-resistant strains. Out of the 10 esterase isozyme observed, susceptible strain lacked three bands, E2, E6 and E10 that were found in the resistant strains. The potency of the synergists piperonyl butoxide (PBO) and dihydrodillapiole (DDA) as esterase inhibitor were also studied both in vitro and in vivo. The in vitro results clearly show that both PBO and DDA inhibited esterase activity in the two resistant strains, while there was almost no esterase inhibition in the homogenate of the susceptible strain. The in vivo inhibition studies (topical application of PBO and DDA followed by biochemical analysis) illustrated that PBO- and DDA-esterase binding is rather slow and non permanent process. Esterase inhibition did not occur immediately after the synergist treatment but at 4 and 8 h post treatment in case of PBO and DDA, respectively. Native PAGE revealed that the in vivo esterase inhibition caused by both PBO and DDA was due to the binding of the synergist with the E6 isozyme which was not present in the susceptible strain.     

Oral administrations of methamidophos and TOCP have different effects on in vitro protein phosphorylation levels from subcellular fractions of hen brain

The effects of methamidophos and tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were studied in Beijing white laying hens during the early stage of delayed neurotoxicity. Phosphorylation of mitochondrial and synaptosomal proteins was assayed in vitro by using [γ-³²P]ATP as phosphate donor. Tri-o-cresyl phosphate (TOCP) administration enhanced the phosphorylation of synaptosomal proteins with molecular weight of 40 and 55 kDa by as much as 36% and 65%, respectively, and decreased the phosphorylation of mitochondrial protein (35 kDa) by 33%. A single dose of methamidophos enhanced the phosphorylation of 32-kDa synaptosomal protein by 44%; however, it had no effect on brain mitochondrial proteins. The activity of neuropathy target esterase (NTE) in dosed hens’ brain and spinal cord was assayed for the effects of methamidophos and TOCP. These results showed that methamidophos inhibited brain NTE by 41% compared with that of control after 7-day exposure, while TOCP inhibited brain NTE by 66%. Moreover, NTE activity from spinal cord in treated hens also exhibited a similar trend of activity inhibition. Together, these results suggested that methamidophos and TOCP induced changes of protein phosphorylation level from hen brain and resulted in different kinds of neurotoxicity.     

The effect of insect “Autoneurotoxin” on Periplaneta americana (L) and Schistocerca gregaria (Forskal) Malpighian tubules

The effect of insect “autoneurotoxin” upon Malpighian tubules of Periplaneta americana (L.) and Schistocerca gregaria (Forskal) was investigated in vitro. “Autoneurotoxin” collected under different stress conditions, caused an acceleration in the frequency of coiling. The increase of the observed coiling frequency was not solely due to 5HT.     

Studies on the chiral isomers of fonofos and fonofos oxon: II. In vitro metabolism

The in vitro metabolism of the chiral isomers of fonofos and fonofos oxon in the presence of mouse liver mixed-function oxidase and serum esterase was investigated. The metabolism of ³⁵S-labeled phenyl-(S)_P-fonofos mediated by mixed-function oxidase took place stereoselectively, resulting predominantly in (R)_P-fonofos oxon. Similarly, (R)_P-fonofos was converted to (S)_P-oxon. In each case, however, a significant amount of racemization occurred. Other products were diphenyl disulfide and diphenyl disulfide oxide. In addition to stereospecificity, the oxidative metabolism of (R)_P-fonofos proceeded at a rate faster than that of (S)_P-fonofos. Stereoselective rate differences also were observed in mouse or rat serum-catalzyed degradation of the fonofos oxon enantiomers, the (S)_P isomer being degraded about twofold faster than its enantiomer. The differences in toxicities of the isomers of fonofos and fonofos oxon were consistent with the in vitro metabolism data.     

Inhibition of neurotoxic esterase and acetylcholinesterase by organophosphorus compounds in selected ectothermic vertebrates

The sensitivity of brain acetylcholinesterase and neurotoxic esterase to inhibition by several organophosphorus compounds was studied in selected ectothermic vertebrates. These enzymes are associated with organophosphorus compound acute and delayed toxicity, respectively. In addition, the susceptibility of several of these species to delayed neurotoxicity induced by organophosphorus compounds was studied. Larvae of the gray treefrog, Southern leopard frog, and narrow-mouthed toad were exposed dermally to tri-o-tolyl phosphate or phenyl saliginen cyclic phosphate (PSCP); no symptoms of delayed neurotoxicity were observed in any of these animals up to 2 weeks after metamorphosis. No symptoms of delayed neurotoxicity were seen in juvenile bullfrogs exposed to multiple ip doses of PSCP. The specific activity of neurotoxic esterase was highest in the larval bullfrog, with juvenile channel catfish and adult mosquitofish demonstrating intermediate levels. The larval Southern leopard frog, adult Northern leopard frog, juvenile green treefrog, and adult marine toad exhibited extremely low activities. The specific activity of acetylcholinesterase was highest in the juvenile channel catfish. Neurotoxic esterase in the larval bullfrog was more sensitive to organophosphate inhibition than that in either fish. PSCP was a more potent neurotoxic esterase inhibitor than leptophos-oxon. The juvenile channel catfish had the acetylcholinesterase most sensitive to organophosphate inhibition. Under the conditions tested, no evidence of in vivo sensitivity to the organophosphate-induced delayed neurotoxicity phenomenon was observed.     

Studies on the chiral isomers of fonofos and fonofos oxon: I. Toxicity and antiesterase activities

The toxicity of the (R)_P and (S)_P chiral isomers and racemates of fonofos and fonofos oxon to insects and white mice were determined. (R)_P-Fonofos and (S)_P-fonofos oxon were 2- to 12-fold more toxic to house flies, mosquito larvae, and mice than were the corresponding enantiomers. The racemates were intermediate in toxicity. Stereoselectivity also was observed in the in vitro inhibition of house fly-head and bovine erythrocyte acetylcholinesterase, horse serum cholinesterase, chymotrypsin, trypsin, and a variety of esterases. In all cases the (S)_P-oxon was a more potent inhibitor than the (R)_P-oxon with k₁ ratios of $\text{(S)}{}_{\mathrm{<mtext>P</mtext>}}\text{(R)}{}_{\mathrm{<mtext>P</mtext>}}$ ranging from 4- to 60-fold. Further, differences in levels of house fly-head, mouse brain, and blood cholinesterase obtained from house flies and mice treated with the enantiomers and racemates of fonofos and fonofos oxon were observed. Differences in toxicity of the enantiomers and racemates to house flies and mice were more closely related to in vivo than to in vitro cholinesterase inhibition.     

Methylparathion- and carbofuran-induced mitochondrial dysfunction and oxidative stress in Helicoverpa armigera (Noctuidae: Lepidoptera)

The cotton bollworm, Helicoverpa armigera is a polyphagous pest of several crops in Asia, Africa, and the Mediterranean Europe. Organophosphate and carbamate insecticides are used on a large-scale to control Helicoverpa. Therefore, we studied the effect of methylparathion and carbofuran, an organophosphate and carbamate insecticide, respectively, on oxidative phosphorylation and oxidative stress in H. armigera larvae to gain an understanding of the different target sites of these insecticides. It was observed that state III and state IV respiration, respiratory control index (RCI), and P/O ratios were inhibited in a dose-dependent manner by methylparathion and carbofuran under in vitro and in vivo conditions. Methylparathion and carbofuran inhibited complex II by ∼45% and 30%, respectively. Lipid peroxidation, H₂O₂ content, and lactate dehydrogenase (LDH) activity increased and glutathione reductase (GR) activity decreased in a time- and dose-dependent manner in insecticide-fed larvae. However, catalase activity was not affected in insecticide-fed larvae. Larval growth decreased by ∼64% and 67% in larvae fed on diets with 100 μM of methylparathion and carbofuran. The results suggested that both the insecticides impede the mitochondrial respiratory functions and induced lipid peroxidation, H₂O₂, and LDH leak, leading to oxidative stress in cells, which contribute to deleterious effects of these insecticides on the growth of H. armigera larvae, along with their neurotoxic effects.     

Inhibition of Penicillium duponti carboxylesterase by the fungicides captan and folpet

Captan, folpet, and perchloromethylmercaptan were effective inhibitors of Penicillium duponti p-nitrophenylpropionate esterase activity (I₅₀ = 0.5 – 2 μM) whereas α-naphthyl acetate esterase activity was not affected by the presence of these compounds. Captan and folpet are both equally effective at pH 7.3 and 8.3. The ionic composition of the medium had strong effects on the degree of inhibition produced by all inhibitors but did not alter esterase activity. Neither succinamide nor phthalimide caused inhibition of the p-nitrophenylpropionate esterase activity: The trichloromethylmercaptan portion of these fungicides appears to be responsible for the observed inhibition. The rapidity of captan and folpet inhibition of esterase activity (complete in < 1 min) compared to the rates of spontaneous decomposition ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{> 1}\text{min}$) and the insensitivity of captan and folpet inhibition to hydrogen ion concentration suggest that generation of spontaneous decomposition products is not required for inhibition. The results are consistent with a mechanism in which the entire fungicide molecule binds to the protein followed by enzyme-promoted reactions of captan and folpet which result in loss of esterase activity.     

Disposition and metabolism of [14C]chlorpyrifos in the black imported fire ant, Solenopsis richteri Forel

The short-term disposition and metabolism of topically administered [¹⁴C]chlorpyrifos was assessed in the black imported fire ant (Solenopsis richteri Forel) in the presence and absence of the mixed-function oxidase inhibitor piperonyl butoxide. Chlorpyrifos is readily absorbed into an internal organosoluble fraction which was quickly converted into a water-soluble fraction. The radioactivity was slowly excreted over a 24-hr period. Piperonyl butoxide slowed the conversion of the internal organosoluble radioactivity to the water-soluble fraction. Thin-layer chromatography indicated that piperonyl butoxide slowed the conversion of chlorpyrifos to material remaining at the origin, presumably water-soluble metabolites. The results of acid hydrolysis studies indicated that the water-soluble radioactivity was comprised mainly of conjugates. Although very little chlorpyrifos oxon was recovered in the metabolism experiments, in vitro studies on fire and head homogenates showed the compound to be an extremely potent anticholinesterase, with an I₅₀ of 4.6 × 10^?10M, while a major metabolite, 3,5,6-trichloropyridinol, was an ineffective acetylcholinesterase inhibitor.     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

Comparative effects of organophosphorus insecticides on the activities of acetylcholinesterase,diacylglycerol kinase,and phosphatidylinositol phosphodiesterase in rat brain microsomes

The activities of acetylcholinesterase, diacylglycerol kinase, and phosphatidylinositol phosphodiesterase in rat brain microsomes were measured in the presence and absence of the organophosphorus insecticides, parathion and diazinon, and their respective oxon analogs, paraoxon and diazoxon. Marked inhibition of acetylcholinesterase (by 45–99%) was observed in the presence of paraoxon (10^?2–10^?6M) and diazoxon (10^?2–10^?4M). Reduction of acetylcholinesterase activity (by 22–33%) was achieved with the parent insecticides at high concentrations only (10^?2M). In most cases, diacylglycerol kinase was insensitive to the pesticides. Marked stimulation of phosphatidylinositol phosphodiesterase (by 10–57%) was observed in the presence of all pesticides (10^?2–10^?3M). The phosphodiesterase exhibited slightly greater sensitivity to the parent compounds compared to the oxon derivatives. Stimulation of the phosphodiesterase by the insecticides was not correlated with acetylcholinesterase inhibition. Accordingly, the increase in phosphodiesterase activity was judged not to be acetylcholine mediated, but rather represented a direct effect of the pesticides on the enzyme or its microenvironment. Based on the present in vitro observations, it is proposed that certain organophosphorus pesticides may interfere with the normal process of synaptic transmission through both the inhibition of acetylcholinesterase and the stimulation of phosphatidylinositol phosphodiesterase. In view of the high concentrations of pesticides required to elicit the latter effect, interpretation of its physiological significance must await results from further studies performed in vivo.     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Precocene II metabolism: Comparative in vivo studies among several species of insects,and structure elucidation of two major metabolites

Several species of insects, exhibiting varying responsiveness to the juvenile hormone antagonist precocene II (6,7-dimethoxy-2,2-dimethylchromene), were challenged topically with a tritiated preparation of the title compound. Metabolism of [³H]precocene II was subsequently examined by withdrawing hemolymph samples from treated animals at appropriate time intervals and characterizing the extractable radiolabel chromatographically. Quantitative (or qualitative) differences observed between the respective metabolic profiles were found not correlative with specimen sensitivity to precocene. Production of two heretofore unreported metabolites, identified by spectral and chemical means as O-β-glucosides of 6- and 7-monodemethylated precocene II, was demonstrated in both sensitive and insensitive species. No evidence for the presence of a hemolymphborne, biologically effective “activated metabolite” produced in vivo by precocene-susceptible insects could be found. The latter finding may well argue for in situ bioactivation of precocene at the target tissue(s) by these sensitive insects.     

The apparent in vivo neuromuscular effects of the δ-endotoxin of Bacillus thuringiensis var. israelensis in mice and insects of four orders

Alkaline-dissolved crystal δ-endotoxin from Bacillus thuringiensis var. israelensis (serovar H 14) was injected into mice and seven species of insects representing the orders Lepidoptera, Orthoptera, Coleoptera, Hemiptera, and Diptera. High in vivo toxicity, at 1 to 5 ppm (μg toxin/g body wet wt), was observed with mice and some insects, including some that are not sensitive to the toxin when administered orally. Neuromuscular effects were observed when the toxin was injected directly into the body cavity of the test animals. Biochemical studies suggested that different protein fragments within the crystal δ-endotoxin may be responsible for the majority of the mosquito larvacidal activity and the neurotoxic symptoms observed in larvae of Trichoplusia ni.     

Inhibition of permethrin-hydrolyzing esterases from Wiseana cervinata larvae

Inhibition of permethrin-hydrolyzing enzymes from larvae of the porina moth Wiseana cervinata has been examined in vivo and in vitro. Significant inhibition was shown by carbaryl and pirimiphos-methyl. 1-Dodecylimidazole substantially inhibited permethrin hydrolysis only in liver insects. The triphenylmethane dye tetrabromophenolphthalein was a moderate inhibitor only in vitro. TMDM (bis(N-dimethyl-4-aminophenyl)methane) had little effect on hydrolysis. These observations extend the range of species and substrates for which the triphenylmethane dyes and 1-dodecylimidazole are useful inhibitors of insecticide metabolism.