Inhibitor Report for: Piperonyl-butoxide

The western flower thrips (WFT) Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), an important pest of various crops in the world, has invaded China since 2003. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in WFT, a resistant strain was selected under the laboratory conditions. Cross-resistance and the possible biochemical resistance mechanisms were investigated in this study. A 15.1-fold thiamethoxam-resistant WFT strain (TH-R) was established after selection for 55 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed extremely high level cross-resistance to imidaclothiz (392.1-fold) and low level cross-resistance to dinotefuran (5.7-fold), acetamiprid (2.9-fold) and emamectin benzoate (2.1-fold), respectively. No cross-resistance to other fourteen insecticides was detected. Synergism tests showed that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) produced a high synergism of thiamethoxam effects in the TH-R strain (2.6- and 2.6-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that mixed function oxidase (MFO) activities and carboxylesterase (CarE) activity of the TH-R strain were 2.8- and 1.5-fold higher than that of the TH-S strain, respectively. When compared with the TH-S strain, the TH-R strain had a relative fitness of 0.64. The results show that WFT develops resistance to thiamethoxam after continuous application and thiamethoxam resistance had considerable fitness costs in the WFT. It appears that enhanced metabolism mediated by cytochrome P450 monooxygenases and CarE was a major mechanism for thiamethoxam resistance in the WFT. The use of cross-resistance insecticides, including imidaclothiz and dinotefuran, should be avoided for sustainable resistance management.

BACKGROUND: Western flower thrips (WFT), Frankliniella occidentalis (Pergande), is among the most important crop pests in the south-eastern region of Spain. Its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. Use of synergists to inhibit the enzymes involved in insecticide detoxification is widely used to determine their responsibility for insecticide resistance. However, they do not always act as intended or expected, and caution must be exercised when interpreting synergist results. RESULTS: Laboratory-selected strains of WFT were used to analyse the effects of the synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and methiocarb on total esterase activity. Significant differences were found, indicating esterase activity inhibition by DEF, a lower effect for methiocarb and a small inhibition of the activity by PBO. Esterase isoenzyme inhibition by these compounds showed a similar result; this assay revealed an extreme sensitivity of Triplet A (resistance-associated esterases) to DEF. In an in vivo assay carried out with these compounds at different incubation times, only DEF caused posterior in vitro esterase activity inhibition, with a maximum effect 1 h after treatment. CONCLUSION: In this work, only DEF shows true synergistic inhibition of WFT esterases.

The effects of sub-lethal treatments (20 and 60% of 24-h LC(50)) with plant-derived molluscicides Annona squamosa, acetogenins, Argemone mexicana seed and protopine, in combination (1 + 5) with MGK-264 (ENT 8184) or piperonyl butoxide on the reproduction of Lymnaea acuminata has been studied. The plant-derived molluscicides and their active molluscicidal components, protopine and acetogenins, in combination with ENT 8184 or piperonyl butoxide caused a significant reduction in the fecundity, hatchability and survival of young snails. Combination of A squamosa seed powder with piperonyl butoxide was very effective as it caused a complete arrest of snail fecundity within 24 h of treatment. Removal of the snails to fresh water after the 96-h treatments caused a significant recovery in the fecundity of L acuminata.

Piperonyl butoxide (PBO)-synergized pyrethroid products are widely available for the control of pyrethroid-resistant mosquitoes. To date, no study has examined mosquito resistance after pre-exposure to PBO and subsequent enzymatic activity when exposed to PBO-synergized insecticides. We used Culex quinquefasciatus Say (Diptera: Culicidae), an important vector of arboviruses and lymphatic filariasis, as a model to examine the insecticide resistance mechanisms of mosquitoes to PBO-synergized pyrethroid using modified World Health Organization tube bioassays and biochemical analysis of metabolic enzyme expressions pre- and post-PBO exposure. Mosquito eggs and larvae were collected from three cities in Orange County in July 2020 and reared in insectary, and F0 adults were used in this study. A JHB susceptible strain was used as a control. Mosquito mortalities and metabolic enzyme expressions were examined in mosquitoes with/without pre-exposure to different PBO concentrations and exposure durations. Except for malathion, wild strain Cx quinquefasciatus mosquitoes were resistant to all insecticides tested, including PBO-synergized pyrethroids (mortality range 3.7 +/- 4.7% to 66.7 +/- 7.7%). Wild strain mosquitoes had elevated levels of carboxylesterase (COE, 3.8-fold) and monooxygenase (P450, 2.1-fold) but not glutathione S-transferase (GST) compared to susceptible mosquitoes. When wild strain mosquitoes were pre-exposed to 4% PBO, the 50% lethal concentration of deltamethrin was reduced from 0.22% to 0.10%, compared to 0.02% for a susceptible strain. The knockdown resistance gene mutation (L1014F) rate was 62% in wild strain mosquitoes. PBO pre-exposure suppressed P450 enzyme expression levels by 25~34% and GST by 11%, but had no impact on COE enzyme expression. Even with an optimal PBO concentration (7%) and exposure duration (3h), wild strain mosquitoes had significantly higher P450 enzyme expression levels after PBO exposure compared to the susceptible laboratory strain. These results further demonstrate other studies that PBO alone may not be enough to control highly pyrethroid-resistant mosquitoes due to multiple resistance mechanisms. Mosquito resistance to PBO-synergized insecticide should be closely monitored through a routine resistance management program for effective control of mosquitoes and the pathogens they transmit.

BACKGROUND: Piperonyl butoxide (PBO) is a well-known insecticide synergist capable of interacting with phase 1 metabolic enzymes, specifically esterases and cytochrome P450s. In this study, structure-activity relationship analyses were used to characterise the interaction of around 30 analogues of PBO with the esterase FE4 and the P450 CYP6CY3 from insecticide-resistant Myzus persicae (Sulzer), in order to predict the synthesis of more potent inhibitors.
RESULTS: Enzyme inhibition studies were performed against esterase and oxidase activities and, together with in silico modelling, key activity determinants of the analogues were identified and optimised. Novel analogues were then designed and synthesised, some of which showed greater inhibition against both enzymatic systems: specifically, dihydrobenzofuran moieties containing an alkynyl side chain and a butyl side chain against FE4, and benzodioxole derivatives with a propyl/butyl side chain and an alkynyl ether moiety for CYP6CY3.
CONCLUSIONS: In vitro assays identified potential candidate synergists with high inhibitory potency. The in vivo confirmation of such results will allow consideration for a possible use in agriculture. (c) 2016 Society of Chemical Industry.

BACKGROUND: Insecticide-based vector control, which comprises use of insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS), is the key method to malaria control in Madagascar. However, its effectiveness is threatened as vectors become resistant to insecticides. This study investigated the resistance status of malaria vectors in Madagascar to various insecticides recommended for use in ITNs and/or IRS.
METHODS: WHO tube and CDC bottle bioassays were performed on populations of Anopheles gambiae (s.l.), An. funestus and An. mascarensis. Adult female An. gambiae (s.l.) mosquitoes reared from field-collected larvae and pupae were tested for their resistance to DDT, permethrin, deltamethrin, alpha-cypermethrin, lambda-cyhalothrin, bendiocarb and pirimiphos-methyl. Resting An. funestus and An. mascarensis female mosquitoes collected from unsprayed surfaces were tested against permethrin, deltamethrin and pirimiphos-methyl. The effect on insecticide resistance of pre-exposure to the synergists piperonyl-butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) also was assessed. Molecular analyses were done to identify species and determine the presence of knock-down resistance (kdr) and acetylcholinesterase resistance (ace-1 R ) gene mutations.
RESULTS: Anopheles funestus and An. mascarensis were fully susceptible to permethrin, deltamethrin and pirimiphos-methyl. Anopheles gambiae (s.l.) was fully susceptible to bendiocarb and pirimiphos-methyl. Among the 17 An. gambiae (s.l.) populations tested for deltamethrin, no confirmed resistance was recorded, but suspected resistance was observed in two sites. Anopheles gambiae (s.l.) was resistant to permethrin in four out of 18 sites (mortality 68-89%) and to alpha-cypermethrin (89% mortality) and lambda-cyhalothrin (80% and 85%) in one of 17 sites, using one or both assay methods. Pre-exposure to PBO restored full susceptibility to all pyrethroids tested except in one site where only partial restoration to permethrin was observed. DEF fully suppressed resistance to deltamethrin and alpha-cypermethrin, while it partially restored susceptibility to permethrin in two of the three sites. Molecular analysis data suggest absence of kdr and ace-1 R gene mutations. CONCLUSION: This study suggests involvement of detoxifying enzymes in the phenotypic resistance of An. gambiae (s.l.) to pyrethroids. The absence of resistance in An. funestus and An. mascarensis to pirimiphos-methyl and pyrethroids and in An. gambiae (s.l.) to carbamates and organophosphates presents greater opportunity for managing resistance in Madagascar.

The western flower thrips (WFT) Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), an important pest of various crops in the world, has invaded China since 2003. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in WFT, a resistant strain was selected under the laboratory conditions. Cross-resistance and the possible biochemical resistance mechanisms were investigated in this study. A 15.1-fold thiamethoxam-resistant WFT strain (TH-R) was established after selection for 55 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed extremely high level cross-resistance to imidaclothiz (392.1-fold) and low level cross-resistance to dinotefuran (5.7-fold), acetamiprid (2.9-fold) and emamectin benzoate (2.1-fold), respectively. No cross-resistance to other fourteen insecticides was detected. Synergism tests showed that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) produced a high synergism of thiamethoxam effects in the TH-R strain (2.6- and 2.6-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that mixed function oxidase (MFO) activities and carboxylesterase (CarE) activity of the TH-R strain were 2.8- and 1.5-fold higher than that of the TH-S strain, respectively. When compared with the TH-S strain, the TH-R strain had a relative fitness of 0.64. The results show that WFT develops resistance to thiamethoxam after continuous application and thiamethoxam resistance had considerable fitness costs in the WFT. It appears that enhanced metabolism mediated by cytochrome P450 monooxygenases and CarE was a major mechanism for thiamethoxam resistance in the WFT. The use of cross-resistance insecticides, including imidaclothiz and dinotefuran, should be avoided for sustainable resistance management.

Introduction The effects of piperonyl butoxide (PBO) on the toxicity of the organophosphate temephos (TE) and the role of esterases in the resistance of Aedes aegypti to this insecticide were evaluated. Methods A. aegypti L4 larvae susceptible and resistant to TE were pre-treated with PBO solutions in acetone at concentrations of 0.125, 0.25, 0.5, 1, and 2% for 24h and subsequently exposed to a diagnostic concentration of 0.02mg/L aqueous TE solution. The esterase activity of the larvae extracts pre-treated with varying PBO concentrations and exposed to TE for three time periods was determined. Results At concentrations of 0.25, 0.5, 1, and 2%, PBO showed a significant synergistic effect with TE toxicity. High levels of esterase activity were associated with the survival of A. aegypti L4 larvae exposed to TE only. Conclusions The results of the biochemical assays suggest that PBO has a significant inhibitory effect on the total esterase activity in A. aegypti larvae.

The present study was designed to understand the effects of piperonyl butoxide (PBO), modulator of cytochrome P450 (CYP 450), on the neurotoxicity of organophosphate pesticide fenthion in the brain of Oreochromis niloticus used as a model organism. Fish were exposed to one-fourth of the LC50 value of fenthion (0.567 mg/L) and 0.5 mg/L PBO concentration for 24 h, 96 h, and 15 days. Glutathione (GSH)-related antioxidant system, lipid peroxidation, stress proteins, and acetylcholinesterase (AchE) activity were investigated. Our results showed that PBO induced the neurotoxic effect of fenthion with increasing oxidative stress in long-term exposure. GSH-related antioxidant system might take a role in protecting the brain from these oxidative effects. PBO possibly inhibited the biotransformation of fenthion by inhibiting CYP 450; thereby preventing the brain from AChE inhibition in short-term exposure. Changes in parameters indicated that PBO caused biphasic response by affecting CYP 450 in the brain of O. niloticus.

Abstract Earlier research has evidenced the oxidative and neurotoxic potential of imidacloprid, a neonicotinoid insecticide, in different animal species. The primary aim of this study was to determine how metabolic modulators piperonyl butoxide and menadione affect imidacloprid's adverse action in the liver and kidney of Sprague-Dawley rats of both sexes. The animals were exposed to imidacloprid alone (170 mg kg-1) or in combination with piperonyl butoxide (100 mg kg-1) or menadione (25 mg kg-1) for 12 and 24 h. Their liver and kidney homogenates were analysed spectrophotometrically for glutathione peroxidase, glutathione S-transferase, catalase, total cholinesterase specific activities, total glutathione, total protein content, and lipid peroxidation levels. Imidacloprid displayed its prooxidative and neurotoxic effects predominantly in the kidney of male rats after 24 h of exposure. Our findings suggest that the observed differences in prooxidative and neurotoxic potential of imidacloprid could be related to differences in its metabolism between the sexes. Co-exposure (90-min pre-treatment) with piperonyl butoxide or menadione revealed tissue-specific effect of imidacloprid on total cholinesterase activity. Increased cholinesterase activity in the kidney could be an adaptive response to imidacloprid-induced oxidative stress. In the male rat liver, co-exposure with piperonyl butoxide or menadione exacerbated imidacloprid toxicity. In female rats, imidacloprid+menadione co-exposure caused prooxidative effects, while no such effects were observed with imidacloprid alone or menadione alone. In conclusion, sex-, tissue-, and duration-specific effects of imidacloprid are remarkable points in its toxicity.

BACKGROUND: Western flower thrips (WFT), Frankliniella occidentalis (Pergande), is among the most important crop pests in the south-eastern region of Spain. Its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. Use of synergists to inhibit the enzymes involved in insecticide detoxification is widely used to determine their responsibility for insecticide resistance. However, they do not always act as intended or expected, and caution must be exercised when interpreting synergist results. RESULTS: Laboratory-selected strains of WFT were used to analyse the effects of the synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and methiocarb on total esterase activity. Significant differences were found, indicating esterase activity inhibition by DEF, a lower effect for methiocarb and a small inhibition of the activity by PBO. Esterase isoenzyme inhibition by these compounds showed a similar result; this assay revealed an extreme sensitivity of Triplet A (resistance-associated esterases) to DEF. In an in vivo assay carried out with these compounds at different incubation times, only DEF caused posterior in vitro esterase activity inhibition, with a maximum effect 1 h after treatment. CONCLUSION: In this work, only DEF shows true synergistic inhibition of WFT esterases.

BACKGROUND Previous work has demonstrated that piperonyl butoxide (PBO) not only inhibits microsomal oxidases but also resistance-associated esterases. The ability to inhibit both major metabolic resistance enzymes makes it an ideal synergist to enhance xenobiotics but negates the ability to differentiate which enzyme group is responsible for conferring resistance. RESULTS: This study examines an analogue that retains the ability to inhibit esterases but is restricted in its ability to act on microsomal oxidases, thus allowing an informed decision on resistance enzymes to be made when used in conjunction with the parent molecule. CONCLUSION: Using examples of resistant insects with well-characterised resistance mechanisms, a combination of PBO and analogue allows identification of the metabolic mechanism responsible for conferring resistance. The relative potency of PBO as both an esterase inhibitor and an oxidase inhibitor is also discussed.

BACKGROUND: Conventional in vitro assays sometimes fail to reveal esterase inhibition by piperonyl butoxide (PBO), although synergism studies suggest loss of esterase-mediated sequestration of insecticide does take place. A new in vitro assay has been devised that routinely reveals binding between PBO and these esterases. RESULTS: The new 'esterase interference' assay detects the blockade of resistance-associated esterases in a species, Myzus persicae Slzer, where this has not previously been seen. The assay also demonstrates directly the protective effect esterases may confer to target sites of insecticides. CONCLUSION: The new assay reveals esterase blockade by PBO and thus has the potential to be used as a high-throughput screening method for other potential synergists.

Eight Latin American strains of Aedes aegypti were evaluated for resistance to 6 organophosphates (temephos, malathion, fenthion, pirimiphos-methyl, fenitrothion, and chlorpirifos) and 4 pyrethroids (deltamethrin, lambdacyhalothrin, betacypermethrin, and cyfluthrin) under laboratory conditions. In larval bioassays, temephos resistance was high (resistance ratio [RR50], > or =10X) in the majority of the strains, except for the Nicaragua and Venezuela strains, which showed moderate resistance (RR50, between 5 and 10X). The majority of the strains were susceptible to malathion, fenthion, and fenitrothion. However, resistance to pirimiphos-methyl ranged from moderate to high in most of the strains. Larvae from Havana City were resistant to 3 of the pyrethroids tested and moderately resistant to cyfluthrin. The Santiago de Cuba strain showed high resistance to deltamethrin and moderate resistance to the other pyrethroids (lambdacyhalothrin, betacypermethrin, and cyfluthrin). The rest of the strains were susceptible to pyrethroids, except for the Jamaica and Costa Rica strains, which showed moderate resistance to cyfluthrin, and Peru and Venezuela, which showed resistance to deltamethrin. Adult bioassays showed that all the strains were resistant to dichlorodiphenyl-trichloroethane and to the majority of pyrethroids evaluated. The use of the synergists S,S,S,-tributyl phosphorotrithioate and piperonil butoxide showed that esterase and monooxygenases played an important role in the temephos, pirimiphos-methyl, and chlorpirifos resistance in some strains. Biochemical tests showed high frequencies of esterase and glutathione-S-transferase activity; however, the frequency of altered acetylcholinesterase mechanism was low. The polyacrylamide electrophoresis gel detected the presence of a strong band called Est-A4. Insecticide resistance in Ae. aegypti is a serious problem facing control operations, and integrated control strategies are recommended to help prevent or delay the temephos resistance in larvae and pyrethroids resistance in adults.

The cotton bollworm, Helicoverpa armigera, is a cosmopolitan, insecticide-resistant insect pest of food and fiber (Gunning et al., 1992). Acetylcholinesterase (AChE) is the insect target site for carbamate insecticides, and H. armigera has developed an insensitive form of AChE as a resistance mechanism (Gunning et al., 1996). Insensitive AChE is normally considered an intractable resistance mechanism in pests. The methenedioxphenyl compound, piperonyl butoxide (PBO), has a long history as an insecticide synergist in the control of resistant arthropod pests; it is known to inhibit mono-oxygenases and nonspecific esterases (Gunning et al., 1998). This work discusses PBO inhibition of AChE in H. armigera and explores synergism.

The present study was undertaken to investigate the significance of monooxygenases in bioactivation of DEF to a more effective anticholinesterase in fish. Channel catfish were exposed via the water column for 20 h to piperonyl butoxide (PBO) followed by a 4-h exposure to the organophosphate defolient DEF (concurrent with the PBO). Acetylcholinesterase (AChE) and aliesterases (ALiEs) activities were determined at 0 and 12 h after the exposure period. Inhibition of brain, liver, and plasma AChE activity by DEF was antagonized by PBO; muscle AChE was not inhibited by DEF. Piperonyl butoxide did not antagonize the inhibition of liver or plasma ALiEs by DEF. These results suggest that PBO retards the formation of the metabolite(s) of DEF that inhibit AChE, and that DEF is an effective inhibitor of ALiEs without metabolic activation.

Pyrethroid resistance in B-type Bemisia tabaci Gennadius and Australian Helicoverpa armigera Hubner field populations is primarily conferred by esterase isoenzymes which metabolise and sequester pyrethroid insecticides. It has been shown previously that pyrethroid resistance-associated esterases in H. armigera are inhibited by the insecticide synergist piperonyl butoxide (PBO) over a 22-h period. It is demonstrated here that similar inhibition can be obtained against B-type B. tabaci. Small-scale field trials showed excellent levels of pyrethroid control when insects were pretreated with PBO and then dosed with pyrethroid during the time of maximum esterase inhibition. These results demonstrate that PBO can restore pyrethroid efficacy in the field against both B-type B. tabaci and resistant H. armigera.

The effects of sub-lethal treatments (20 and 60% of 24-h LC(50)) with plant-derived molluscicides Annona squamosa, acetogenins, Argemone mexicana seed and protopine, in combination (1 + 5) with MGK-264 (ENT 8184) or piperonyl butoxide on the reproduction of Lymnaea acuminata has been studied. The plant-derived molluscicides and their active molluscicidal components, protopine and acetogenins, in combination with ENT 8184 or piperonyl butoxide caused a significant reduction in the fecundity, hatchability and survival of young snails. Combination of A squamosa seed powder with piperonyl butoxide was very effective as it caused a complete arrest of snail fecundity within 24 h of treatment. Removal of the snails to fresh water after the 96-h treatments caused a significant recovery in the fecundity of L acuminata.

Pyrethroid resistance in field populations of Australian Helicoverpa armigera (Hbner) is primarily a consequence of the overproduction of esterase isoenzymes which metabolise and sequester pyrethroid insecticides. Biochemical studies have shown that pyrethroid-resistance-associated esterases in H armigera are inhibited by the insecticide synergist piperonyl butoxide (PBO). Esterase inhibition by PBO did not occur immediately after dosing, but exhibited maximum inhibition 3-4 h after dosage. Esterase activity subsequently recovered until full activity was restored by 24 h. Topical bioassays using a pre-treatment of PBO showed that maximum H armigera mortality was achieved with pre-treatment times corresponding to maximum esterase inhibition. These results demonstrated that, with correct temporal application, PBO can restore pyrethroid efficacy against H armigera. It would also be expected that restoration of efficacy with other conventional insecticides, currently compromised by esterase-based resistance mechanisms, would occur.

The use of chemical inhibitors/inducers is one of the strategies employed to determine whether a particular metabolic pathway is involved in the metabolism of a xenobiotic. The objective of this study was to assess the role of piperonyl butoxide (PBO) on the toxicity of an organophosphorus insecticide, chlorpyrifos (CPF) to two species, Ceriodaphnia dubia (waterflea) and Xenopus laevis (South African clawed frog). Chlorpyrifos was highly toxic to C. dubia (48-h LC50: 0.05 microg/L) in comparison with X. laevis (96-h LC50: 2410 microg/L). Piperonyl butoxide at 200 microg/L reduced the toxicity of chlorpyrifos to C. dubia by a factor of 6. Piperonyl butoxide at 3000 microg/L also reduced the toxicity of CPF to X. laevis with respect to mortality and malformations. Acetylcholinesterase (AChE) activity was used as a biomarker to further assess the role of PBO in chlorpyrifos toxicity. X. laevis exposed to CPF and PBO exhibited a biphasic response in terms of AChE activity with an initial increase in the AChE activity followed by a drastic decrease. The results from the present study indicate that C. dubia and X. laevis have the capability to metabolize chlorpyrifos via cytochromes P450 mediated reactions. The results also indicate that the use of the biomarker AChE is useful in determining metabolic processes of organophosphorus insecticides, which require metabolic activation.

Synergism of an oil of Azadirachta indica, a powdered extract of Allium sativum bulbs and an oleoresin of Zingiber officinale rhizomes by piperonyl butoxide and MGK-264 was studied against the snails Lymnaea acuminata and Indoplanorbis exustus. The active components of these plant-derived molluscicides, respectively azadirachtin, allicin and [6]gingerol, were also combined with these synergists. Both piperonyl butoxide and MGK-264 enhanced the toxicity of all of the test compounds. The response of snails to the synergised mixtures was both time- and dose-dependent.

The toxicity of diazinon and diazoxon to fourth instar Pieris brassicae larvae was determined, with or without modifying chemicals. Piperonyl butoxide and sesamex antagonised diazinon but synergised diazoxon. The penetration, excretion and internal concentration of diazinon were measured following topical application. Penetration of diazinon followed first order kinetics and was considerably slower after pretreatment with sesamex and piperonyl butoxide. Pretreatment with piperonyl butoxide increased the internal concentration of diazinon, but the onset of symptoms of poisoning was delayed, presumably because of inhibition of diazinon activation.