Presence Of Piperonyl Butoxide Research Articles

Toxicity, transfer and metabolization of the pyrethroid insecticides cypermethrin and deltamethrin by reared black soldier fly larvae

Abstract Reared insects such as black soldier fly larvae (Hermetia illucens) are considered a potential alternative feed protein. However, dietary exposure to insecticide residues via the substrate could adversely affect performance indicators (yield/survival) and substance-transfer from substrate to larval biomass could result in non-compliance with low legal limits. Effects of pyrethroid insecticides cypermethrin and deltamethrin were tested at varying concentrations, with or without the synergist piperonyl butoxide (PBO). Concentration/response curves for yield were estimated and samples were analysed to determine concentrations of parent compounds and selected metabolites. Results suggest that deltamethrin is highly toxic to H. illucens larvae: the critical effect dose for 10% yield loss was estimated to be 0.04 mg/kg, compared to a legal limit in wheat of 2.0 mg/kg. Cypermethrin was comparatively less toxic, in line with prior studies, but may also cause significant adverse effects even for exposure levels below the legal limit – especially when combined with PBO. For both substances, transfer from substrate to larvae is a potential issue due to low limits, and transfer as well as toxicity are increased by presence of PBO. Some metabolites could be detected, but more research is needed to determine resistance mechanisms involved.

P450 gene family mediates allelochemical detoxification and tolerance to alkaloids in cactophilic Drosophila

AbstractCacti are characterized by the extensive production of a broad variety of toxic metabolites as an anti‐herbivore strategy but nevertheless cactophilic Drosophila spp. (Diptera: Drosophilidae) feed and breed on decaying cacti. The family of cytochrome P450 is a group of enzymes involved in the detoxification metabolism, which are critical for the adaptation of phytophagous insects, especially populations of Drosophila exploiting cacti. In this context, Drosophila buzzatii Patterson & Wheeler and Drosophila koepferae Fontdevila & Wasserman (Drosophilini), constitute an excellent model for the study of the genetic basis of tolerance to cacti allelochemicals. These closely related species allow testing to what extent a relatively small set of genes can explain host use patterns and their role in the divergent evolution of specialization. We evaluated the role of the P450 genes in the adaptation of these species to the use of one of their hosts, the columnar cactus Trichocereus terscheckii (Parm.) Friedrich & GD Rowley (Cactaceae). Experimentally evolved strains exhibiting different degrees of cactus specialization were exposed to artificial rearing media enriched with cactus allelochemicals and piperonyl butoxide (PBO), an inhibitor of P450 family activity. Our analyses showed a significant viability reduction in the presence of PBO, in the non‐tolerant strains of both species as well as in the strain historically subjected to mild concentrations of allelochemicals (the soft strain) of D. koepferae. We confirmed the key role of P450 genes for the evolution of cactus specialization but also found evidence for the evolution of other detoxification pathways.

The Effect of Synergistic Compounds on the Susceptibility of Euschistus heros (Hemiptera: Pentatomidae) and Chrysodeixis includens (Lepidoptera: Noctuidae) to Pyrethroids.

The Neotropical brown stink bug, Euschistus heros (F.), and the soybean looper, Chrysodeixis includens (Walker), are key pests of soybean in South America. Low susceptibility to pyrethroids has been reported for both species in Brazil. Here, we evaluate the addition of synergistic compounds piperonyl butoxide (PBO) and diethyl maleate (DEM) to manage E. heros and C. includens with resistance to λ-cyhalothrin and bifenthrin. The LD50 of technical grade and commercial products containing λ-cyhalothrin and bifenthrin decreased against field-collected E. heros exposed to PBO and DEM relative to unexposed insects; synergistic ratios up to 4.75-fold. The mortality also increased when E. heros were exposed to commercial formulations containing λ-cyhalothrin (from 4 to 44%) and bifenthrin (from 44 to 88%) in the presence of synergists. There was also a higher susceptibility of field-collected C. includens to technical grade λ-cyhalothrin when PBO was used; synergistic ratio of 5.50-fold. High lethally of technical grade λ-cyhalothrin was also verified in the presence of PBO, with mortality increasing from 6 to 57%. Our findings indicate the potential utility of synergists in reversing the resistance to λ-cyhalothrin and bifenthrin in E. heros and C. includens and suggest a significant role of metabolic mechanisms underlying the detoxification of both pyrethroids.

Physiological basis for isoxadifen-ethyl induction of nicosulfuron detoxification in maize hybrids.

Isoxadifen-ethyl can effectively alleviate nicosulfuron injury in the maize. However, the effects of safener isoxadifen-ethyl on detoxifying enzymes in maize is unknown. The individual and combined effects of the sulfonylurea herbicide nicosulfuron and the safener isoxadifen-ethyl on the growth and selected physiological processes of maize were evaluated. Bioassays showed that the EC50 values of nicosulfuron and nicosulfuron plus isoxadifen-ethyl for maize cultivar Zhengdan958 were 18.87 and 249.28 mg kg-1, respectively, and were 24.8 and 275.51 mg kg-1, respectively, for Zhenghuangnuo No. 2 cultivar. Evaluations of the target enzyme of acetolactate synthase showed that the I50 values of nicosulfuron and nicosulfuron plus isoxadifen-ethyl for the ALS of Zhengdan958 were 15.46 and 28.56 μmol L-1, respectively, and were 0.57 and 2.17 μmol L-1, respectively, for the acetolactate synthase of Zhenghuangnuo No. 2. The safener isoxadifen-ethyl significantly enhanced tolerance of maize to nicosulfuron. The enhanced tolerance of maize to nicosulfuron in the presence of the safener, coupled with the enhanced injury observed in the presence of piperonyl butoxide, 1-aminobenzotriazole, and malathion, suggested cytochrome P450 monooxygenases may be involved in metabolism of nicosulfuron. We proposed that isoxadifen-ethyl increases plant metabolism of nicosulfuron through non-P450-catalyzed routes or through P450 monooxygenases not inhibited by piperonyl butoxide, 1-aminobenzotriazole, and malathion. Isoxadifen-ethyl, at a rate of 33 mg kg-1, completely reversed the effects of all doses (37.5–300 mg kg-1) of nicosulfuron on both of the maize cultivars. When the two compounds were given simultaneously, isoxadifen-ethyl enhanced activity of glutathione S-transferases (GSTs) and acetolactate synthase activity in maize. The free acid 4,5-dihydro-5,5-diphenyl-1,2-oxazole-3-carboxylic was equally effective at inducing GSTs as the parent ester and appeared to be the active safener. GST induction in the maize Zhenghuangnuo No. 2 was faster than in Zhengdan 958.

Benzoylurea resistance in western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae): the presence of a point mutation in chitin synthase 1.

We examined the susceptibility of field strains (BO-1, BO-2, TO-1, and YH-1) and one laboratory strain (H-1) of the western flower thrip, Frankliniella occidentalis, to benzoylureas. LC50 values of novaluron were determined as 0.64 ppm against laboratory strain and 2.1-130 ppm against field strains. In the presence of piperonyl butoxide, a cytochrome P450 inhibitor, the insecticidal activity of novaluron tended to be enhanced. To examine whether point mutations in chitin synthase 1 (CHS1) discovered in an etoxazole-resistant strain of Tetranychus urticae and a benzoylurea-resistant strain of Plutella xylostella exist in F. occidentalis, the nucleotide sequence of CHS1 was analyzed. We found a nonsynonymous substitution that corresponded to the location of the mutations found in T. urticae and P. xylostella in the field strains of F. occidentalis but not in the laboratory strain, indicating that this point mutation might be associated with the benzoylurea resistance exhibited by the field strains.

Transcriptional analysis of four family 4 P450s in a Puerto Rico strain of Aedes aegypti (Diptera: Culicidae) compared with an Orlando strain and their possible functional roles in permethrin resistance.

A field strain of Aedes aegypti (L.) was collected from Puerto Rico in October 2008. Based on LD50 values by topical application, the Puerto Rico strain was 73-fold resistant to permethrin compared with a susceptible Orlando strain. In the presence of piperonyl butoxide, the resistance of Puerto Rico strain of Ae. aegypti was reduced to 15-fold, suggesting that cytochrome P450-mediated detoxification is involved in the resistance of the Puerto Rico strain to permethrin. To determine the cytochrome P450s that might play a role in the resistance to permethrin, the transcriptional levels of 164 cytochrome P450 genes in the Puerto Rico strain were compared with that in the Orlando strain. Of the 164 cytochrome P450s, 33 were significantly (P < 0.05) up-regulated, including cytochrome P450s in families four, six, and nine. Multiple studies have investigated the functionality of family six and nine cytochrome P450s, therefore, we focused on the up-regulated family 4 cytochrome P450s. To determine whether up-regulation of the four cytochrome P450s had any functional role in permethrin resistance, transgenic Drosophila melanogaster Meigen lines overexpressing the four family 4 P450 genes were generated, and their ability to survive exposure to permethrin was evaluated. When exposed to 5 microg per vial permethrin, transgenic D. melanogaster expressing CYP4D24, CYP4H29, CYP4J15v1, and CYP4H33 had a survival rate of 60.0 +/- 6.7, 29.0 +/- 4.4, 64.4 +/- 9.7, and 11.0 +/- 4.4%, respectively. However, none of the control flies survived the permethrin exposure at the same concentration. Similarly, none of the transgenic D. melanogaster expressing CYP4J15v1 or CYP4H33 ?5 survived when they were exposed to permethrin at 10 microg per vial. However, transgenic D. melanogaster expressing CYP4D24 and CYP4H29 had a survival rate of 37.8 +/- 4.4 and 2.2 +/- 2.2%, respectively. Taken together, our results suggest that CYP4D24 might play an important role in cytochrome P450-mediated resistance to permethrin.

The Importance of Oxidases in the Tolerance of Deciduous Leaf Infusions by Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae)

Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) larvae rely on oxidases to reduce toxicity of water soluble toxins from some senescent tree leaf infusions. The mortality of third instar Ae. aegypti larvae in live oak and pin oak leaf infusions increased significantly in the presence of piperonyl butoxide (PBO), a broad inhibitor of cytochrome P450s (CYPs). In contrast, PBO treatment did not increase mortality in water controls or infusions of northern red oak or sugar maple leaf infusions for Ae. aegypti larvae. A similar pattern was observed for Ae. albopictus larvae, that is, an increase in mortality when CYPs were inhibited in live oak leaf infusions and no increase in sugar maple leaf infusions or water controls. However, the fresh live oak leaf infusion (5 d old) was the most toxic infusion to Ae. aegypti, but appeared less toxic to Ae. albopictus than the older infusions. A direct comparison of survival between the two Aedes species revealed Ae. aegypti exhibited a greater mortality than Ae. albopictus in PBO-treated live oak leaf infusions. These findings suggest that toxic components of some leaf litter in larval habitats may impose cryptic energy costs (detoxification).

Neonicotinoid resistance and cDNA sequences of nicotinic acetylcholine receptor subunits of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae)

The western flower thrips, Frankliniella occidentalis (Pergande), is difficult to control because of high insecticide resistance. In this study, susceptibility to major insecticides was examined in two Japanese strains (H-1 and KC) and a Chinese strain (BJ) using a leaf-dipping method. All three strains were resistant to permethrin and acetamiprid at agriculturally recommended doses. The median lethal concentration (LC50) for acetamiprid was 1720 ppm in strain H-1, 4780 ppm in strain KC and >6680 ppm in strain BJ. In the presence of piperonyl butoxide, an inhibitor of cytochrome P450 monooxygenases, the LC50 for acetamiprid was 312 ppm in strain H-1, 837 ppm in strain KC and 1250 ppm in strain BJ. These results suggested that metabolism by cytochrome P450 monooxygenases is involved in acetamiprid resistance in these strains, though other factors also seem to play a role. Furthermore, cDNA cloning of the nicotinic acetylcholine receptor (nAChR) subunits was performed using degenerate primers, and the presence or absence of a point mutation in nAChR β1 was confirmed. The R81T mutation that had been reported in Myzus persicae (Sulzer) nAChR β1 was not found in F. occidentalis strains tested in this study.

Neurotoxic effects of lambda-cyhalothrin modulated by piperonyl butoxide in the brain ofOreochromis niloticus

The objective of this research was to investigate the neurotoxic effects of pyrethroid pesticide lambda-cyhalothrin by the modulation of cytochrome P450 with piperonyl butoxide in the brain of juvenile Oreochromis niloticus. The fish were exposed to 0.48 μg L(-1) (1/6 of the 96-h LC50 ) lambda-cyhalothrin and 10 μg L(-1) piperonyl butoxide for 96 h and 15 days. tGSH, GSSG, TBARS contents, GPx, GR, GST, and AChE enzymes activities were determined by spectrophotometrical methods and Hsp70 content was analyzed by ELISA technique. Lambda-cyhalothrin had no significant effect on the components of GSH redox system, lipid peroxidation and Hsp70 levels but inhibited AChE activity. In the presence of piperonyl butoxide, lambda-cyhalothrin caused increases in tGSH, GSSG, TBARS and Hsp70 contents, GST activity, and decrease in AChE activity. Present results showed that in the presence of piperonyl butoxide, lambda-cyhalothrin caused neurotoxic effects by increasing oxidative stress. Adaptation to its oxidative stress effects may be supplied by GSH-related antioxidant system. Piperonyl butoxide revealed neurotoxic effect of lambda-cyhalothrin.

Oxidative and apoptotic effects of lambda-cyhalothrin modulated by piperonyl butoxide in the liver of Oreochromis niloticus

The aim of this study was to investigate the toxic effects of pyrethroid pesticide lambda-cyhalothrin in the presence of piperonyl butoxide as a modulator in the liver of juvenile Oreochromis niloticus. LC50 (96h) value of lambda-cyhalothrin was determined as 2.901μg/L for O. niloticus. The fish were exposed to 0.48μg/L (1/6 of the 96-h LC50) lambda-cyhalothrin and 10μg/L piperonyl butoxide for 96-h and 15-d. tGSH, GSH, GSSG, Hsp70 and TBARS contents, GPx, GR, GST and caspase-3 enzymes activities were determined. Lambda-cyhalothrin caused increases in tGSH, GSH, TBARS contents, and GST activity. Piperonyl butoxide treatment with lambda-cyhalothrin caused significant increases in tGSH GSH, Hsp70, TBARS contents, and GPx and GST activities while caspase-3 activity was decreased. The results of the present study revealed that lambda-cyhalothrin caused oxidative stress which upregulated GSH and GSH-related enzymes. Piperonyl butoxide increased the oxidative stress potential and apoptotic effects of lambda-cyhalothrin.

Metabolic Mechanisms Only Partially Explain Resistance to Pyrethroids in Australian Broiler House Populations of Lesser Mealworm (Coleoptera: Tenebrionidae)

The susceptibility of six Australian broiler house populations and an insecticide susceptible population of lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), to cyfluthrin, beta-cyfluthrin, gamma-cyhalothrin, and deltamethrin was investigated. One broiler house population had equivalent susceptibility to the susceptible to beta-cyfluthrin and beta-cyhalothrin, with higher susceptibility to cyfluthrin and deltamethrin. The remaining five populations demonstrated strong resistance to cyfluthrin (19-37-fold), the insecticide used most widely for management of A. diaperinus in Australia. Each cyfluthrin-resistant population demonstrated reduced susceptibility to beta-cyfluthrin (resistance ratios were 8-17-fold), deltamethrin (2.5-8-fold), and gamma-cyhalothrin (6-12-fold) compared with the laboratory population, but cross-resistance patterns varied considerably between populations. Adding piperonyl butoxide (PBO) had no effect on the susceptibility of the susceptible population to any of the insecticides, but it increased the susceptibility of each of the five cyfluthrin-resistant populations: to cyfluthrin (synergism ratio range, 1.9-5.0-fold), beta-cyfluthrin (1.6-4.1-fold), and y-cyhalothrin (1.7-2.0-fold). PBO had a more variable effect on susceptibility to deltamethrin, with three of the cyfluthrin-resistant populations being more susceptible to deltamethrin in the presence of PBO, but susceptibility of the remaining two populations was unaffected by adding PBO (synergism ratio range, 0.9-2.5-fold). Overall, the addition of PBO to the four pyrethroids had variable effects on their susceptibility. This variability indicated the presence of other resistance mechanisms in beetle populations apart from metabolic resistance. In addition, the relative importance of metabolic resistance in each beetle population varied widely between pyrethroids. Thus, it cannot be assumed that PBO will reliably synergize pyrethroids against cyfluthrin-resistant lesser mealworm populations when using it to mitigate insecticide resistance.

Metabolism of Methoxychlor by the P450‐Monooxygenase CYP6G1 Involved in Insecticide Resistance of Drosophila melanogaster after Expression in Cell Cultures of Nicotiana tabacum

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

Piperonyl butoxide enhances the bioconcentration and photoinduced toxicity of fluoranthene and benzo[ a]pyrene to larvae of the grass shrimp ( Palaemonetes pugio)

Piperonyl butoxide (PBO) is a commonly used synergist in many pyrethroid formulations due to its ability to interfere with cytochrome P450 (CYP) monooxygenases. Because PBO can co-occur in the estuarine environment with polycyclic aromatic hydrocarbons (PAHs), a class of compounds metabolized by CYP isozymes, the overall objective of this study was to investigate the influence of PBO on the bioconcentration and photoinduced toxicity of two common PAH contaminants, fluoranthene (FLU) and benzo[ a]pyrene (BaP), on the larvae of the grass shrimp ( Palaemonetes pugio). PBO alone was not particularly toxic to grass shrimp larvae. In dark exposures and under simulated sunlight (UV-A = 211.0 ± 7.0 μW/cm 2, UV-B = 9.8 ± 2.4 μW/cm 2), 96-h LC 50 values were similar (814.4 and 888.6 μg/L, respectively), suggesting that PBO toxicity is not enhanced in the presence of sunlight. The presence of sublethal concentrations of PBO in single PAH toxicity tests increased the bioconcentration of the two tested PAHs, and these increases were greatest at the lowest tested PAH concentrations. Mean bioconcentration factors (BCF) at the three lowest FLU and BaP treatments increased 14.3- and 7.1-fold, respectively, in the low PBO (127 μg/L) exposure compared to that of the no PBO exposure. Under simulated sunlight, PBO exposure also increased the photoinduced toxicity of the two tested PAHs, and this increase occurred in a PBO concentration-dependent fashion. For FLU, 96-h LC 50 values decreased from 2.35 μg/L in the absence of PBO to 0.76 μg/L in the high PBO (256 μg/L) exposure. For BaP, 96-h LC 50 values similarly decreased from 1.02 μg/L in the absence of PBO to 0.30 μg/L in the high PBO exposure. The presence of PBO also influenced the PAH tissue residue–response relationship, but in different ways for FLU and BaP. For FLU, slopes of the tissue residue–response relationship decreased in the presence of PBO, and for BaP, there was a trend towards increased slopes in the presence of PBO. These results demonstrate that sublethal levels of PBO increase the bioconcentration and photoinduced toxicity of certain PAH in grass shrimp larvae, and underscore the need to consider the potential for PBO to synergize the toxicity of co-occurring environmental contaminants in future risk assessments.

Resistance of the generalist moth Trichoplusia ni (Noctuidae) to a novel chemical defense in the invasive plant Conium maculatum

Conium maculatum is an apiaceous species native to Eurasia that is highly toxic to vertebrates due to the presence of piperidine alkaloids, including coniine and γ-coniceine. More than 200 years after invading the United States this species remains mostly free from generalist insect herbivores. The presence of novel chemical defenses in the introduced range could provide invasive species with a competitive advantage relative to native plants. The cabbage looper (Trichoplusia ni) is a generalist lepidopteran found throughout the US that occasionally feeds on C. maculatum. We evaluated the toxicity of piperidine alkaloids to T. ni and determined putative resistance mechanisms, both behavioral and physiological, that allows this insect to develop successfully on C. maculatum foliage. T. ni larvae raised on diets enriched with coniine and γ-coniceine showed a decrease in consumption and longer development time, but no effects on growth were found at any alkaloid concentration. In a diet choice experiment T. ni larvae showed no avoidance of alkaloid-enriched diets, suggesting that the deterrence produced by alkaloids was related to a post-ingestive metabolic response. The ability of T. ni to consume diets high in alkaloid content could be due to at least three different mechanisms: 1) a decreased consumption rate, 2) efficient excretion of at least 1/3 of ingested alkaloids unmetabolized in frass, and 3) partial detoxification of alkaloids by cytochrome P450 s, as shown by the decreased larval growth in the presence of piperonyl butoxide, a P450 inhibitor. Even though T. ni tolerates C. maculatum alkaloids, the use of this species as a host plant could be ecologically disadvantageous due to prolonged larval growth and thus increased exposure to predators. Novel plant secondary compounds do not guarantee increased resistance to generalist herbivores.

Toxicity of Aflatoxin B1 to Helicoverpa zea and Bioactivation by Cytochrome P450 Monooxygenases

Infestation of corn (Zea mays) by corn earworm (Helicoverpa zea) predisposes the plant to infection by Aspergillus fungi and concomitant contamination with the carcinogenic mycotoxin aflatoxin B1 (AFB1). Although effects of ingesting AFB1 are well documented in livestock and humans, the effects on insects that naturally encounter this mycotoxin are not as well defined. Toxicity of AFB1 to different stages of H. zea (first, third, and fifth instars) was evaluated with artificial diets containing varying concentrations. Although not acutely toxic at low concentrations (1-20 ng/g), AFB1 had significant chronic effects, including protracted development, increased mortality, decreased pupation rate, and reduced pupal weight. Sensitivity varied with developmental stage; whereas intermediate concentrations (200 ng/g) caused complete mortality in first instars, this same concentration had no detectable adverse effects on larvae encountering AFB1 in fifth instar. Fifth instars consuming AFB1 at higher concentrations (1 microg/g), however, displayed morphological deformities at pupation. That cytochrome P450 monooxygenases (P450s) are involved in the bioactivation of aflatoxin in this species is evidenced by the effects of piperonyl butoxide (PBO), a known P450 inhibitor, on toxicity; whereas no fourth instars pupated in the presence of 1 mug/g AFB1 in the diet, the presence of 0.1% PBO increased the pupation rate to 71.7%. Pupation rates of both fourth and fifth instars on diets containing 1 mug/g AFB1 also increased significantly in the presence of PBO. Effects of phenobarbital, a P450 inducer, on AFB1 toxicity were less dramatic than those of PBO. Collectively, these findings indicate that, as in many other vertebrates and invertebrates, toxicity of AFB1 to H. zea results from P450-mediated metabolic bioactivation.

Synergism between insecticides permethrin and propoxur occurs through activation of presynaptic muscarinic negative feedback of acetylcholine release in the insect central nervous system

Although synergism between pesticides has been widely documented, the physiological mechanisms by which an insecticide synergizes another remains unclear. Toxicological and electrophysiological studies were carried out on two susceptible pest species (the mosquito Culex quinquefasciatus and the cockroach Periplaneta americana) to understand better the physiological process involved in pyrethroid and carbamate interactions. Larval bioassays were conducted with the susceptible reference strain SLAB of C. quinquefasciatus to assess the implication of multi-function oxidases and non-specific esterases in insecticide detoxification and synergism. Results showed that the general theory of synergism (competition between pesticides for a common detoxification enzyme) was unlikely to occur in the SLAB strain since the level of synergy recorded between permethrin and propoxur was unchanged in the presence of piperonyl butoxide and tribufos, two inhibitors of oxidases and esterases, respectively (synergism ratios were similar with and without synergists). We also showed that addition of a sub-lethal concentration of nicotine significantly increased the toxicity of permethrin and propoxur at the lower range of the dose–mortality regression lines, suggesting the manifestation of important physiological disruptions at synaptic level. The effects of both permethrin and propoxur were studied on the cercal-afferent giant-interneuron synapses in the terminal abdominal ganglion of the cockroach P. americana using the single-fibre oil-gap method. We demonstrated that permethrin and propoxur increased drastically the ACh concentration within the synaptic cleft, which thereby stimulated a negative feedback of ACh release. Atropine, a muscarinic receptor antagonist, reversed the effect of permethrin and propoxur mixtures. This demonstrates the implication of the presynaptic muscarinic receptors in the negative feedback regulation process and in synergism. Based on these findings, we propose a cascade of molecular events explaining the occurrence of synergistic effects between pyrethroid and carbamate on many susceptible insects including C. quinquefasciatus, a mosquito of medical importance.

Synergism of rotenone by piperonyl butoxide in Haemonchus contortus and Trichostrongylus colubriformis in vitro: Potential for drug-synergism through inhibition of nematode oxidative detoxification pathways

The anthelmintic properties of rotenone and its activity in combination with the cytochrome P450 inhibitor piperonyl butoxide, were examined in in vitro assays with adults and larvae of Haemonchus contortus and larvae of Trichostrongylus colubriformis. Rotenone was toxic to larvae of both species, with LC 50 values in larval development assays of 0.54 and 0.64 μg/ml for H. contortus and T. colubriformis, respectively. The compound also caused complete cessation of movement in adult H. contortus after 72 h at a concentration of 20 μg/ml. Toxicity of rotenone towards the larvae of both species was increased in the presence of piperonyl butoxide (synergism ratios of 3–4-fold at the LC 50) and the activity against adult H. contortus was also significantly enhanced following pre-treatment with piperonyl butoxide. This significant synergism suggests that these nematode species are able to utilize a cytochrome P450 enzyme system to detoxify rotenone and indicates that a role may exist for cytochrome P450 inhibitors to act as synergists for other anthelmintics which are susceptible to oxidative metabolism within the nematode.

Effect of piperonyl butoxide on diazinon resistance in field strains of the sheep blowfly, Lucilia cuprina (Diptera: Calliphoridae), in New Zealand

AbstractPretreatment of first instar larvae of 28 resistant strains of Lucilia cuprina(Wiedemann) with the inhibitor of microsomal oxidases, piperonyl butoxide, resulted in a biphasic response to the phosphorothioate insecticide diazinon. Analysis of the data revealed a complex response in which both synergist-dependent and independent effects occurred. The responses varied markedly from strain to strain. A laboratory susceptible strain and field strains with resistance factors of less than 20-fold exhibited, in the presence of piperonyl butoxide, an increased LC50 with respect to diazinon whereas those strains with &gt; 20-fold resistance were synergized by the compound. We conclude tentatively that microsomal mixed-function oxidases play a contributory role in the development of resistance and that the variation in synergist effect from strain to strain may be attributed, at least in part, to the two-fold effect of these enzymes on phosphorothioate insecticides such as diazinon.

Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity

Iron plays a role in free radical-mediated tissue injury, including cisplatin-induced nephrotoxicity. However, the source of iron (catalyzing free radical reactions) is not known. We examined the role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity both in vivo and in vitro. Cisplatin-induced acute renal failure was produced in rats by intraperitoneal injection of cisplatin (10 mg/kg body wt). Piperonyl butoxide, a cytochrome P-450 inhibitor, was administered intraperitoneally (400 mg/kg body wt twice at 48-hr intervals) prior to cisplatin injection. The effects of cisplatin in the absence or presence of piperonyl butoxide on the belomycin-detectable iron, cytochrome P-450 content in the kidney, and renal functional and histological changes were evaluated. In an in vitro study, the effect of cytochrome P-450 inhibitors, cimetidine or piperonyl butoxide, on cisplatin-induced cytotoxicity and catalytic iron release from LLC-PK1 cells was examined. In cisplatin-treated rats, there was a marked decrease in the cytochrome P-450 content specifically in the kidney, accompanied by increased bleomycin-detectable iron content in the kidney. Piperonyl butoxide prevented cisplatin-induced loss of cytochrome P-450 as well as the increase of bleomycin-detectable iron in the kidney, along with both functional and histological protection. Both cimetidine and piperonyl butoxide prevented cisplatin-induced increase in bleomycin-detectable iron and cytotoxicity in LLC-PK1 cells. Treatment of cimetidine did not affect cellular uptake of cisplatin. Cytochrome P-450, a group of heme proteins, may serve as a significant source of catalytic iron in cisplatin-induced nephrotoxicity.

Toxicity of Imidacloprid in the German Cockroach (Dictyoptera: Blattellidae), and the Synergism Between Imidacloprid and Metarhizium anisopliae (Imperfect Fungi: Hyphomycetes)

German cockroaches, Blattella germanica (L.), orally exposed to solid imidacloprid baits exhibited toxic symptoms rapidly (within 30 min). However, cockroaches recovered from this toxicity. The percentage of knockdown was positively correlated and the rate of recovery was negatively correlated with concentration. In the liquid bait tests, imidacloprid produced rapid moribundity (within minutes) in all exposed cockroaches, but this moribundity was transient. Topically applied imidacloprid also produced rapid but transient moribundity in both in the laboratory-snsceptible (JWax-S) and the field-collected resistant (Muncie'86) strains. In the topical application bioassay, recovery time was dependent on cockroach strain and presence of piperonyl butoxide (PBO). Resistant cockroaches recovered faster than susceptible cockroaches; cockroaches pretreated with PBO recovered faster than those not pretreated. The JWax and Lafayette (field-collected) strains were killed significantly faster with The entomopathogenic fungus, Metarhizium anisopliae alone, or Metarhizium + solid imidacloprid bait than imidacloprid bait alone in both the choice and no-choice bioassays. Combinations of direct contact to the conidia of Metarhizium and feeding on imidacloprid bait indicated synergy when compared with the activity of the individual components. We found no differences in mortality when the JWax cockroaches fed on either 500 or 1,000 ppm imidacloprid baits. The presence of alternate food slowed the lethal time for imidacloprid against the JWax and Lafayette strains. Both strains were killed significantly faster when cockroaches lind on imidacloprid after a topical application of spore suspensions of Metarhizium than when insects fed on imidacloprid bait alone, indicating possible synergistic interaction (i.e., greater than expected mortality) between imidacloprid bait and Metarhizium The mortality of cockroaches inoculated topically with spore suspensions of Metarhizium increased significantly with an increase in the number of fungal spores. No significant differences in daily cockroach mortality were detected among imidacloprid concentrations for each Metarhizium dose. Imidacloprid; d bait alone has a limited potential for use in controlling cockroaches, but it may have a potential if used in combination with a fungal pathogen such as Metarhizium.