Methylenedioxyphenyl Compounds Research Articles

Time-Dependent Inhibition of CYP1A2 by Stiripentol and Structurally Related Methylenedioxyphenyl Compounds via Metabolic Intermediate Complex Formation

Time-Dependent Inhibition of CYP1A2 by Stiripentol and Structurally Related Methylenedioxyphenyl Compounds via Metabolic Intermediate Complex Formation

Piperonyl butoxide, a synergist of pesticides can elicit male-mediated reproductive toxicity

A semi-synthetic methylenedioxyphenyl compound piperonyl butoxide (PBO) has been used as a ubiquitous synergist to increase the insecticidal effect of pesticides for agricultural and household use. Despite previously demonstrated effects of PBO, the detailed mechanism of PBO in spermatozoa and reproductive toxic effects on male germ cells have not been fully elucidated. Therefore, this study evaluated the effects of PBO on various sperm functions during capacitation and clarified the mechanisms of reproductive toxic effects on male fertility at different concentrations of PBO (0.1, 1, 10, and 100 μM). Sperm motility and kinematics were assessed using computer-assisted sperm analysis and the status of capacitation was evaluated using combined H33258/chlortetracycline (CTC) staining. Intracellular adenosine triphosphate (ATP) and cell viability levels were also measured. In addition, protein kinase A (PKA) activity and protein tyrosine phosphorylation were evaluated. In addition, in vitro fertilization was performed to determine the effects of PBO on cleavage and blastocyst formation rates. We found that PBO significantly decreased sperm motility, kinematics, and acrosome-reacted and capacitated spermatozoa. In addition, PBO suppressed the intracellular ATP levels and directly affected cell viability. Moreover, PBO detrimentally decreased the activation of PKA and altered the levels of tyrosine-phosphorylated proteins. Consequently, cleavage and blastocyst formation rates were significantly reduced in a dose-dependent manner. In line with our observations, the synergist of pesticides PBO may directly and/or indirectly cause disorder in male fertility. Hence, we suggest that careful attention is made to consider reproductive toxicity when using PBO as a synergist.

Effect of Piperonyl Butoxide on the Toxicity of Four Classes of Insecticides to Navel Orangeworm (Amyelois transitella) (Lepidoptera: Pyralidae).

Amyelois transitella (Walker) (Lepidoptera: Pyralidae), the navel orangeworm, is a highly polyphagous economic pest of almond, pistachio, and walnut crops in California. Increasing demand for these crops and their rising economic value has resulted in substantial increases of insecticide applications to reduce damage to acceptable levels. The effects of piperonyl butoxide (PBO), a methylenedioxyphenyl compound that can act as a synergist by inhibiting cytochrome P450-mediated detoxification on insecticide metabolism by A. transitella, were examined in a series of feeding bioassays with first-instar A. transitella larvae from a laboratory strain. PBO, however, can have a variety of effects on metabolism, including inhibition of glutathione-S-transferases and esterases and induction of P450s. In our study, PBO synergized the toxicity of acetamiprid, λ-cyhalothrin, and spinosad, suggesting possible involvement of P450s in their detoxification. In contrast, PBO interacted antagonistically with the organophosphate insecticide chlorpyrifos, reducing its toxicity, an effect consistent with inhibition of P450-mediated bioactivation of this pesticide. The toxicity of the anthranilic diamide insecticide chlorantraniliprole was not altered by PBO, suggestive of little or no involvement of P450-mediated metabolism in its detoxification. Because a population of navel orangeworm in Kern County, CA, has already acquired resistance to the pyrethroid insecticide bifenthrin through enhanced P450 activity, determining the effect of adding a synergist such as PBO on detoxification of all insecticide classes registered for use in navel orangeworm management can help to develop rotation practices that may delay resistance acquisition or to implement alternative management practices where resistance is likely to evolve.

CYP2C19 Progress Curve Analysis and Mechanism-Based Inactivation by Three Methylenedioxyphenyl Compounds

Several in vitro criteria were used to assess whether three methylenedioxyphenyl (MDP) compounds, the isoquinoline alkaloids bulbocapnine, canadine, and protopine, are mechanism-based inactivators of CYP2C19. The recently reported fluorometric CYP2C19 progress curve analysis approach was applied first to determine whether these alkaloids demonstrate time-dependent inhibition. In this experiment, bulbocapnine, canadine, and protopine displayed time dependence and saturation in their inactivation kinetics with K(I) and k(inact) values of 72.4 ± 14.7 μM and 0.38 ± 0.036 min(-1), 2.1 ± 0.63 μM and 0.18 ± 0.015 min(-1), and 7.1 ± 2.3 μM and 0.24 ± 0.021 min(-1), respectively. Additional studies were performed to determine whether other specific criteria for mechanism-based inactivation were fulfilled: NADPH dependence, irreversibility, and involvement of a catalytic step in the enzyme inactivation. CYP2C19 activity was not significantly restored by dialysis when it had been inactivated by the alkaloids in the presence of a NADPH-regenerating system, and a metabolic-intermediate complex-associated increase in absorbance at approximately 455 nm was observed. In conclusion, the CYP2C19 progress curve analysis method revealed time-dependent inhibition by these alkaloids, and additional experiments confirmed its quasi-irreversible nature. This study revealed that the CYP2C19 progress curve analysis method is useful for identifying novel mechanism-based inactivators and yields a wealth of information in one run. The alkaloids bulbocapnine, canadine, and protopine, present in herbal medicines, are new mechanism-based inactivators and the first MDP compounds exhibiting quasi-irreversible inactivation of CYP2C19.

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm ( Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b 5 on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.

Metabolism of myristicin by Depressaria pastinacella CYP6AB3v2 and inhibition by its metabolite

Although methylenedioxyphenyl (MDP) compounds, such as myristicin, are useful in the management of insecticide-resistant insects, the molecular mechanisms for their action in mammals and insects have not been elucidated. In this study, GC–MS analyses of methanol extracts of foliage of wild parsnip ( Pastinaca sativa) have identified myristicin as a substrate for CYP6AB3v2, an imperatorin-metabolizing cytochrome P450 monooxygenase from Depressaria pastinacella (parsnip webworm). In contrast with its strong inhibitory effects on many mammalian P450s, myristicin is effectively metabolized by CYP6AB3v2 ( V max and K m of 97.9 pmol/min/pmol P450 and 17.9 μM, respectively) at a rate exceeding that recorded previously for imperatorin, the only other known substrate for this highly specialized enzyme. The myristicin metabolite of CYP6AB3v2 is 1-(3′,4′-methylenedioxy-5′-methoxyphenyl)-2,3-epoxypropane. Molecular dockings have indicated that, unlike other epoxide metabolites of furanocoumarins, this epoxide metabolite is likely to remain in the CYP6AB3v2 catalytic site due to its low binding energy (−31.0 kcal/mol). Inhibition assays indicate that myristicin acts as a mixed inhibitor of this insect P450 and suggest that the epoxide metabolite may be an intermediate involved in the formation of P450–methylenedioxyphenyl complexes.

Effects of Piperonyl Butoxide and Tetramethrin Combinations on Biological Activities of Selected Synthetic Pyrethroid Insecticides against Different Housefly (Musca domestica L., Diptera: Muscidae) Populations

Piperonyl butoxide (PBO), a methylenedioxyphenyl compound, is primarily used as a synergist in combination with space spray, residual and admixture products for the control of insect pests in or around domestic and commercial premises, especially food preparation areas. Also, tetramethrin is known as a knockdown agent on target organism and it is generally used with piperonyl butoxide. In this study, effects of piperonyl butoxide and tetramethrin combinations on biological activities of synthetic pyrethroids, cypermethrin, deltamethrin, and permethrin against different housefly (Musca domestica Linnaeus, 1758) populations were evaluated. In addition, the biological efficiency of the insecticides used in the study, insecticide + PBO and insecticide + PBO + tetramethrin combinations, against the WHO standard sensitive housefly population and housefly populations collected from different parts of Turkey were compared. Results showed that PBO extensively promoted the ratio of knockdown and killing effect values of the insecticides. The results also indicated that PBO and PBO + tetramethrin combinations moderately reduced the knockdown effect times of all formulation in all housefly populations. The knockdown effect times were more decreased by insecticide + PBO + tetramethrin combinations than insecticides that are used alone and insecticide + PBO combinations.

Metabolism of [14C]-5-chloro-1,3-benzodioxol-4-amine in male Wistar-derived rats following intraperitoneal administration

[14C]-5-chloro-1,3-benzodioxol-4-amine was administered intraperitoneally (i.p.) to bile duct-cannulated rats (Alpk:ApfSD, Wistar derived) at 25 mg kg−1 to determine the rates and routes of excretion of the compound and to investigate its metabolic fate. A total of 89.1% of the dose was excreted in the 48 h following administration, the majority being recovered in the urine during the first 12 h. The main metabolite in both urine and bile, detected by high-performance liquid chromatography (HPLC) with radioprofiling and mass spectrometry, was identified as a demethylenated monosulfate conjugate. Unchanged parent compound formed a major component of the radiolabel excreted in urine and, in addition to unchanged parent and demethylenated sulphate conjugate, a large number of minor metabolites were detected in urine and bile. The overall metabolic fate of 5-chloro-1,3-benzodioxol-4-amine in the rat was complex, with some similarities to previously studied methylenedioxyphenyl compounds.

Inhibition of CYP6B1-Mediated Detoxification of Xanthotoxin by Plant Allelochemicals in the Black Swallowtail (Papilio polyxenes)

The structural and biosynthetic diversity of allelochemicals in plants is thought to arise from selection for additive toxicity as a consequence of toxin mixture or for enhanced toxicity as a result of synergism. In order to understand how insects cope with this type of plant defense, we tested the effects of some allelochemicals in host plants of the black swallowtail Papilio polyxenes on the xanthotoxin-metabolic activity of CYP6B1, the principal enzyme responsible for the detoxification of furanocoumarins in this caterpillar. Additionally, the effects of some synthetic compounds not normally encountered by P. polyxenes on CYP6B1 were tested. These studies demonstrate that the integrity of furanocoumarin structure is important for competitive binding to the active site of CYP6B1, even though the carbonyl group on the pyranone ring apparently does not affect its inhibitory capacity, as in the case of furanochromones. Angular furanocoumarins are generally less phototoxic to many organisms than linear furanocoumarins due to their reduced capacity for cross-linking DNA strands, yet they are more toxic than linear furanocoumarins to black swallowtail larvae. This enhanced toxicity in vivo may be due to the ability of angular furanocoumarins to bind to the active site of CYP6B1 without being rapidly metabolized. This binding reduces the availability of CYP6B1 to metabolize other linear furanocoumarins. The structure-activity relationships for methylenedioxyphenyl compounds, flavonoids, imidazole, and imidazole derivatives are also discussed in light of their capacity to inhibit the xanthotoxin-metabolic activity of CYP6B1.

Altered CYP Expression and Function in Response to Dietary Factors: Potential Roles in Disease Pathogenesis

Increasing evidence implicates dietary factors in the progression of diseases, including certain cancers, diabetes and obesity. Diet also regulates the expression and function of CYP genes, which impacts on drug elimination and may also significantly affect disease pathogenesis. Upregulation of CYPs 2E1 and 4A occurs after feeding of experimental diets that are high in fats or carbohydrates; these diets also promote hepatic lipid infiltration, which is a component of the metabolic syndrome that characterises obesity. Increased availability of lipid substrates for CYPs can enhance free radical production and exacerbate tissue injury. Similar processes may also occur in other models of experimental disease states that exhibit a component of altered nutrient utilization. Food-derived chemicals, including constituents of cruciferous vegetables and fruits, modulate CYP expression and the expression of genes that encode cytoprotective phase II enzymes. Certain dietary indoles and flavonoids activate CYP1A expression either by direct ligand interaction with the aryl hydrocarbon receptor (AhR) or by augmenting the interaction of the AhR with xenobiotic response elements in CYP1A1 and other target genes. Other dietary chemicals, including methylenedioxyphenyl (MDP) compounds and isothiocyanates also modulate CYP gene expression. Apart from altered CYP regulation, a number of dietary agents also inhibit CYP enzyme activity, leading to pharmacokinetic interactions with coadministered drugs. A well described example is that of grapefruit juice, which contains psoralens and possibly other chemicals, that inactivate intestinal CYP3A4. Decreased presystemic oxidation by this CYP increases the systemic bioavailability of drug substrates and the likelihood of drug toxicity. Dietary interactions may complicate drug therapy but inhibition of certain CYP reactions may also protect the individual against toxic metabolites and free radicals generated by CYPs. Chemicals in teas and cruciferous vegetables may also inhibit human CYP enzymes that have been implicated in the bioactivation of chemical carcinogens. Thus, food constituents modulate CYP expression and function by a range of mechanisms, with the potential for both deleterious and beneficial outcomes.

Metabolite-cytochrome P450 complex formation by methylenedioxyphenyl lignans of Piper cubeba: Mechanism-based inhibition

Five methylenedioxyphenyl lignans, (–)-clusin ( 1), (–)-dihydroclusin ( 2), (–)-yatein ( 3), (–)-hinokinin ( 4), and (–)-dihydrocubebin ( 5), were isolated from Piper cubeba as potent and selective inhibitors against cytochrome P450 3A4 (CYP3A4). In this study, we investigated the mechanism of inhibition of CYP3A4 by these lignans and the possibility of their mechanism-based inhibition. Using [ N-methyl- 14C]erythromycin as a substrate, all lignans appear to be showed mixed-type of inhibition with apparent K i of 1.96–4.07 μM. Furthermore, all lignans ( 1– 5) inhibited CYP3A4 in a time-, concentration-, and NADPH-dependent manners and thus appear to be the mechanism-based inhibitors of CYP3A4. The apparent inactivation parameter, K I for these compounds were in the range of 0.054–0.373 μM, whereas the k inact values were 0.225–0.320 min −1. Among them, (–)-clusin ( 1) and (–)-dihydroclusin ( 2) were found to be the most potent CYP3A4 inactivator with apparent K I and k inact values of 0.082, 0.054 μM and 0.253, 0.310 min −1, respectively. Spectral scanning of microsomes with these lignans yielded an absorbance at 455 nm, suggesting that all of them appear to inactivate the cytochrome P450 via the formation of a metabolite intermediate complex. This pattern is consistent with the metabolism of the methylenedioxyphenyl compounds. These results indicate that (–)-clusin ( 1), (–)-dihydroclusin ( 2), (–)-yatein ( 3), (–)-hinokinin ( 4), and (–)-dihydrocubebin ( 5) are effective mechanism-based inhibitors of CYP3A4.

Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole

The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC 50 order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC 50 values less than 20 μM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 μM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited.

Mechanisms of inhibitory and regulatory effects of methylenedioxyphenyl compounds on cytochrome P450-dependent drug oxidation.

Cytochrome P450 (CYP) enzymes catalyse the oxidative conversion of drugs and other lipophilic compounds to hydrophilic metabolites. Thus, CYPs play a dominant role in the elimination of drugs from the body. Inhibitory interactions occur when drugs compete for oxidation by specific CYPs whereas certain drugs increase the capacity for oxidative biotransformation by inducing the synthesis of new CYPs. Methylenedioxyphenyl (MDP) compounds have been widely employed as commercially important pesticide synergists and a number of derivatives are found in oils and spices. MDP compounds are of considerable toxicological significance because of their capacity to inhibit and induce CYP enzymes in mammals; some derivatives produce neurotoxic and hepatotoxic effects. Although there are relatively few therapeutic agents of present clinical importance that possess the MDP structural feature, the synthesis and preclinical evaluation of such agents appears to be increasing. In the context of the existing literature surrounding MDP compounds it is noteworthy that these potential drugs also elicit significant modulatory effects on CYP activities in rat and human liver. These developments indicate the importance of understanding the chemical mechanisms by which MDPs interact with CYPs. Thus, the presence of the MDP structure may undermine the potential clinical value of new drugs.

Isoform selective inhibition and inactivation of human cytochrome P450s by methylenedioxyphenyl compounds

1. A series of methylenedioxyphenyl compounds were evaluated for their inhibitory and inactivation effects on nine human cytochrome P450 (CYP) activities using microsomes from human B-lymphoblast cells expressing specific human CYP isoforms. 2. Methylenedioxyphenyl compounds which possess a bulky structure such as 1,4- benzothiazine showed substantial inhibition of S-warfarin 7-hydroxylation catalysed by CYP2C9, S-mephenytoin 4'-hydroxylation by CYP2C19, bufuralol 1-hydroxylation by CYP2D6, and testosterone 6β-hydroxylation by CYP3A4. Regarding ethoxyresorufin O-deethylation catalysed by CYP1A1 and benzyloxyresorufin O-dealkylation by CYP2B6, the subtle change of a substitution of the 1,4-benzothiazine structure affected the inhibition selectivity. Ethoxyresorufin O-deethylation by CYP1A2, coumarin 7-hydroxylation by CYP2A6, and chlorzoxazone 6-hydroxylation by CYP2E1 were not inhibited by almost any of the methylenedioxyphenyl compounds. The inhibitory effects of methylenedioxyphenyl compounds that possess a short chain amino group on the human CYP isoforms were not significant. 3. The methylenedioxyphenyl compounds inactivated CYP1A1 (kinact=0.034 min-1 and Ki=0.81 μM), CYP2C9 (kinact=0.041 and 0.042 min-1 and Ki=0.56 and 0.15 μM), CYP2D6 (kinact=0.044-0.339 min-1 and Ki=0.21-19.88 μM), and CYP3A4 (kinact=0.076-0.251 min-1 and Ki=0.25-0.69 μM). These results suggested that the methylenedioxyphenyl compounds investigated in this study would be potent mechanismbased inactivators of these human CYP isoforms. In contrast, CYP2B6 and CYP2C19 were not inactivated. 4. The present study suggested that the selectivity of inhibition or inactivation of human CYP isoforms by methylenedioxyphenyl compounds may vary according to the structure of the side chain.

Biological Activity of Pyrethroid Analogs in Pyrethroid-Susceptible and -Resistant Tobacco Budworms, Heliothis virescens (F.)

The phenoxybenzyl moiety of conventional pyrethroids is a major site of oxidative metabolism in resistant tobacco budworms, Heliothis virescens (F.). In this study, this group was replaced with known P450 monooxygenase-inhibiting or oxidatively blocked groups. A variety of isomers (1R/1S, cis/trans) of the resulting chrysanthemates were tested as insecticides or synergists against tobacco budworms that were insecticide-susceptible (LSU) or that expressed metabolic resistance to cypermethrin (Pyr-R). A number of compounds with pentafluorophenyl, methylenedioxyphenyl, and propargyloxyphenyl groups were insecticidal, and activity was dependent on both geometric and stereochemical configuration of the acid moiety. Both trans and cis isomers of 1(R)-fenfluthrin, which contains a pentafluorophenyl group, suppressed resistance to cypermethrin in Pyr-R insects, confirming that oxidative metabolism of the phenoxybenzyl moiety is a major mechanism of resistance in this strain. Of the methylenedioxyphenyl compounds,...

Cytogenetic effects of piperonyl butoxide and safrole in CHO-K1 cells

Recently, hepatocarcinogenicity in rats and mice was reported with regard to the methylenedioxyphenyl compound, piperonyl butoxide (PB), which is used as a synergist for pyrethrins and related insecticides. Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs) due to PB were investigated using CHO-K1 cells with or without rat liver S9 fraction (S9); at the same time, the effects of safrole (SF), a methylenedioxyphenyl compound and a weak hepatocarcinogen, were also examined. PB (0.25 and 0.3 mM) and SF (0.8 mM) caused a slight but significant increase in SCEs followed by a cell-cycle delay in the 3-h treatment without S9. In the presence of S9 (4.5%), the cytotoxicity of PB or SF was weakened greatly or slightly, the top dose capable of cell division was raised to 0.6 mM (2-fold) or 1 mM, respectively. PB with S9 induced SCE at doses of 0.4 and 0.5 mM, and caused endoreduplications (ERDs, 7%) at a dose of 0.6 mM, while SF caused a dose-related significant increase in SCE at all doses used (0.4–1 mM) with S9. Genotoxicity of the metabolites of PB or SF was cleared by changing the dose of S9 (1.5–9%) while holding the dose of each chemical constant. In the case of SF (0.6 mM), induction of SCE, ERD and cell-cycle delay intensified almost in a dose-effect relationship, and CAs and a high level of ERD (14%) were caused by a 9% dose of S9. The concentration of unchanged SF in the incubated medium was certainly in inverse proportion to the dose of S9. This strongly suggests that the metabolites of SF are genotoxic. In the case of PB (0.3 mM), no positive responses were produced in the cultures, even with a high level of S9, though the amount of unchanged PB left in the incubated medium was very slight. This indicates that the metabolites of PB may not be genotoxic. In conclusion, PB and SF are possible to somewhat induce SCE at high dose(s) in the absence of S9, and the genotoxic effects of SF are more intensified in the presence of S9 than in its absence, while PB is probably no genotoxic in the presence of sufficient metabolic activation.

The Biochemical Disposition of Methylenedioxyphenyl Compounds

Abstract: This review summarizes the interactions of the methylene­ dioxyphenyl or benzodioxole function with cytochrome P450 and their toxicological and pharmacokinetic implications. The methylenedioxy group is a common group in natural and synth,etic medicinal compounds and provides an electronegative function that •is relatively unreactive and non polar. The function is oxidized by cytochrome P450 to form a catechol and T formate or carbon monooxide or alternatively, forms a complex with the heme iron of cytochrome P450. This complex, characterized by its absorption in the 455 nm range, can be very stable and inhibits the catalytic cycle of the enzyme. This inhibitory action has been used to enhance the actions of insecticides and has been the basis for pharmacokinetic drug interactions, The catechol product of benzodioxole ring cleavage is redox active and can participate in quinone­ reactive oxygen based toxicity. The chemical basis for these interactions are proposed together with experimental ob ervations reported in the recent literature. Examples of pharmacokinetic drug interactions are also included.

Section 9. Pyrethroid resistance: synergists

SummarySixty-five synergists and seven solvents were tested on larvae of a pure breeding pyrethroid resistant strain of Helicoverpa armigera (homozygous for a metabolic detoxification mechanism fully suppressible by piperonyl butoxide (Pbo), presumably via a microsomal monooxygenase system). The methylenedioxyphenyl and acetylenic compounds were the most effective synergists with moderate activity from some organophosphate compounds, particularly the N-alkyl phthalimidomethyl phosphorodithioate, phosmet. All the other compounds tested were either ineffective or only marginally effective including the majority of organophosphates (12 out of 18 tested), pyrethroid analogues, N-alkyls, esterase and glutathione transferase inhibitors, various nitrogen heterocycles, juvenile hormone and analogues, formamidines, organochlorines, anti-oxidants and kojic acid. The most promising synergists indicated for further evaluation were synthetic analogues of Pbo, phosmet, propargite and possibly also fenthion, phosalone, azinphos-ethyl, pyrazophos and kinoprene. All solvents (except dodecane) had no impact on improving kill, indicating that the mode of action of Pbo and the other synergists in this study is principally true biochemical inhibition and not quasi-synergism (improved penetration). Dodecane marginally improved kill in the resistant but not the susceptible strain, indicating some preferential penetration synergism in the resistant strain.A number of trials with Pbo were designed to facilitate the development of this synergist for commercial field use within the Australian IRM strategy. From these, it was found that: 1. A set rate of Pbo should be used, irrespective of the activity of the accompanying pyrethroid. 2. Residual activity of Pbo is poor (less than 50% of initial activity by two days post spray) but that this could be partially overcome by increasing the rate. 3. Straight Pbo applied onto a weathered pyrethroid deposit could restore control but only temporarily and would probably be of little practical field use. 4. No difference in residual activity could be found between the four formulations of Pbo tested. The synthetic analogue PBX was slightly less intrinsically active than the natural product (Pbo) but when tested in the field against Pbo at the same rate, was equivalent in efficacy to both the standard commercial and research Pbo formulations.In order to preserve the long-term effectiveness of Pbo as a pyrethroid synergist within the Australian IRM strategy, an optimal use strategy (based on synergist rotation within the present insecticide rotation scheme) is proposed.

Differences in Induction of Three P450 Isozymes by Piperonyl Butoxide, Sesamex, Safrole, and Isosafrole

The induction of the P450 isozymes 1a-1, 1a-2, and 2b-10 by four methylenedioxyphenyl (MDP) compounds, safrole (SAF), isosafrole (ISO), piperonyl butoxide (PBO), and sesamex (SES), and by the nonmethylenedioxyphenyl analog of SAF, allyl benzene (AB), was studied using male C57BL/6N mice. P450 1a-1 was not detected in control animals, and was induced by SES and PBO, with SES inducing more 1a-1 protein than PBO. P450 1a-2 mRNA was detected in the livers of control animals and was also increased by all MDP compounds (SES > PBO ≅ ISO > SAF), although SAF treatment did not increase 1a-2 protein. P450 2b-10 mRNA and protein, not detected in untreated animals, were also increased by all MDP compounds (PBO > SES ≅ ISO > SAF). AB treatment did not induce detectable levels of 1a-1, 1a-2, or 2b-10, suggesting that, in this series of compounds, the methylenedioxyphenyl moiety is important in induction.

Regulation of cytochrome P-450 isozymes by methylenedioxyphenyl compounds

Regulation of cytochrome P-450 isozymes 1a-1, 1a-2, and 2b-10 by methylenedioxyphenyl compounds was studied by measuring levels of mRNA, protein, and enzyme activity in hepatic tissue from C57BL/6 (Ah +) and DBA/2 (Ah-) mice dosed with isosafrole (ISO) or piperonyl butoxide (PBO). Increases in 1a-2 and 2b-10 protein were observed for ISO and PBO in both strains of mice, suggesting an Ah receptor-independent mechanism for induction of these isozymes; 1a-1 induction, however, was seen only in C57 mice. Piperonyl butoxide was the more potent inducing agent in both strains. In C57 mice treated with five dose levels of PBO, induction of 1a-1 mRNA, protein, and enzyme activity were seen at doses equal to or greater than 104 mg/kg, but were not detected at lower doses. With isosafrole, induction of 1a-1 mRNA was observed only at the highest dose tested (400 mg/kg); however, neither 1a-1 protein nor increased enzymatic activity was seen at this dose. Dose-response studies showed maximum inducible levels for 1a-2 and 2b-10 protein, beyond which the mRNAs continued to increase while the protein levels remained constant.