Rifampicin synergizes the toxicity of insecticides against the green peach aphid, Myzus persicae.

Myzus persicae is an important pest that has developed resistance to nearly all currently used insecticidal products. The employment of insecticide synergists is one of the effective strategies that need to be developed for the management of this resistance. Our study showed that treatment with a combination of the antibiotic, rifampicin, with imidacloprid, cyantraniliprole, or clothianidin significantly increased their toxicities against M. persicae, by 2.72, 3.59, and 2.41 folds, respectively. Rifampicin treatment led to a noteworthy reduction in the activities of multifunctional oxidases (by 32.64%) and esterases (by 23.80%), along with a decrease in the expression of the CYP6CY3 gene (by 58.57%) in M. persicae. It also negatively impacted the fitness of the aphids, including weight, life span, number of offspring, and elongation of developmental duration. In addition, bioassays showed that the combination of rifampicin and a detoxification enzyme inhibitor, piperonyl butoxide, or dsRNA of CYP6CY3 further significantly improved the toxicity of imidacloprid against M. persicae, by 6.19- and 7.55-fold, respectively. The present study suggests that development of active ingredients such as rifampicin as candidate synergists, show promise to overcome metabolic resistance to insecticides in aphids.

Mechanisms of piperonyl butoxide cytotoxicity and its enhancement with imidacloprid and metals in Chinese hamster ovary cells.

The widespread use of chemicals and the presence of chemical and metal residues in various foods, beverages, and other consumables have raised concerns about the potential for enhanced toxicity. This study assessed the cytotoxic effects of Piperonyl butoxide (PBO) and its enhancement by combination with major contamination chemicals including Imidacloprid and metals, using different cytotoxic and genotoxic assays in Chinese hamster ovary (CHO) cells. PBO exhibited elevated cytotoxic effects in poly (ADP-ribose) polymerase (PARP) deficient CHO mutants but not in Glutathione S-transferase deficient CHO mutants. PBO cytotoxicity was enhanced by PARP inhibitor, Olaparib. PBO cytotoxicity was also enhanced with co-exposure to Imidacloprid, Lead Chloride, or Sodium Selenite. PBO induces γH2AX foci formation and apoptosis. The induction of DNA damage markers was elevated with PARP deficiency and co-exposure to Imidacloprid, Lead Chloride, or Sodium Selenite. Moreover, PBO triggers to form etch pits on plastic surfaces. These results revealed novel mechanisms of PBO cytotoxicity associated with PARP and synergistic effects with other environmental pollutants. The toxicological mechanisms underlying exposure to various combinations at different concentrations, including concentrations below the permitted limit of intake or the level of concern, require further study.

Utilization of piperonyl butoxide and 1-aminobenzotriazole for metabolic studies of toxic chemicals in Daphnia magna and Chironomus yoshimatsui.

Daphnids and chironomids have been used to assess the ecological effects of chemicals released into water bodies; however, the toxicity mechanisms in organisms are generally difficult to identify. Here, we developed a system capable of estimating the contribution of cytochrome P450 (CYP) to the metabolism of test substances in Daphnia magna and Chironomus yoshimatsui based on toxicity differences in the absence and presence of the CYP inhibitors piperonyl butoxide (PBO) and 1-aminobenzotriazole (ABT). The optimum concentrations of PBO and ABT that could effectively reduce the toxicity of diazinon, which is toxic after oxidative metabolism in vivo, were determined as 0.5 and 0.6 mg/L for D. magna, and 2.0 and 40.0 mg/L for C. yoshimatsui, respectively. Acute immobilization tests of 15 insecticides were conducted for D. magna and C. yoshimatsui, with and without the optimum concentrations of PBO or ABT. In the presence of either inhibitor, chlorpyrifos and chlorfenapyr toxicity was reduced in both organisms, whereas those of thiocyclam, nereistoxin, and silafluofen were enhanced in C. yoshimatsui. Liquid chromatography-mass spectrometry analysis of D. magna and C. yoshimatsui samples exposed to chlorfenapyr confirmed that the level of the active metabolite produced by CYP was decreased by PBO or ABT in both organisms. The system to which the test substance was co-exposed to PBO or ABT will be valuable for estimating the contribution of CYPs to metabolism and elucidating the toxicity mechanism in daphnids and chironomids.

Insecticidal and P450 mediate metabolism of fluralaner against red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae).

The red imported fire ant (Solenopsis invicta), a worldwide invasive and polyphagous pest, and often nests in residential areas. Finding an alternative pesticide that is both effective on S. invicta and environmentally friendly is urgent and crucial. Fluralaner, a novel isoxazoline insecticide, has been proven to possess selective toxicity for insects versus mammals and has been safe for mammals and non-target organisms, suggesting its potential in pest management. However, little toxicity information is available for the controlment of S. invicta. In this article, we studied the toxicity of fluralaner against S. invicta systematically, and the roles of metabolism-related enzymes in the metabolism process of fluralaner. The toxicity results showed that the topical application and feeding application were all effective for S. invicta. Moreover, fluralaner can be transmitted among workers by contacting and feeding which leads to a toxic reaction among nestmates. By exploring the biochemistry change, we found cytochrome P450 monooxygenase (P450) may be involved in the detoxification of fluralaner as well as carboxylesterase (CarE), but not glutathione S-transferase (GST). Synergism assays gave solid evidence in which piperonyl butoxide, an activity inhibitor of P450, increased the toxicity of fluralaner to S. invicta. Importantly, with the RNAi treatment, four of S.invicta P450 genes were significantly inhibited and showed more sensitivity to fluralaner at LC50 concentration. Our result indicated that fluralaner could be a potential alternative pesticide in S. invicta control. And CYP9AS16, CYP6AS161, CYP6SQ20, and CYP336A45 genes were closely associated with the metabolism process of fluralaner.

Developmental and reproduction toxicity of piperonyl butoxide part 3 two generation (two litter) reproduction study of piperonyl butoxide administered in the diet to CD ® (Sprague Dawley) rats.

Piperonyl butoxide (PBO) an insecticide synergist was evaluated in a guideline multigenerational toxicity study in rats. F0 and F1 adult generations consisted of groups of 26 male and 26 female CD (Sprague Dawley) rats that were exposed to PBO in the diet at concentrations of 0, 300, 1000 or 5000 ppm for 85 (F0) or 83 (F1) days prior to cohabitation and throughout two mating periods (F1a, F2a and F1b, F2b). Exposure to test diets continued through the mating, gestation, and lactation periods for the females. F2 generation pups were euthanized following weaning. There were no effects on survival, clinical observations, gross or histological findings, fertility, pup viability, lactation indices or sex ratio in adults or pups in any generation. All effects of PBO occurred in the 5000-ppm exposure group. These effects included reduced body weight gains for F0 and F1 males and females during pre-cohabitation resulting in reduced body weights during both gestation periods. Food consumption of the F1b group males was slightly or significantly less than control values from week 3 onward. F1a generation pup weights were reduced on days 4, 7, 14 and 21 postpartum. Pup weights in the F1 and F2 generations were significantly reduced on days 14 and 21 postpartum when diets were being consumed by pups. The no-observable-adverse-effect level (NOAEL) for general toxicity was 1000 ppm based on reductions in body weights (parental and offspring) at 5000 ppm; and the NOAEL for reproductive toxicity was 5000 ppm with no direct effects on reproduction.

Developmental and reproduction toxicity of piperonyl butoxide part 1 developmental safety of piperonyl butoxide in the CD® (Sprague Dawley) rat.

Developed as an insecticide synergist in the early 1940s, PBO increases the effectiveness of pyrethrins. Herein, the findings from a guideline developmental toxicity study in rat conducted in 1991 are reported. Timed-pregnant CD® (Sprague Dawley) rats were randomly assigned to a control and three treatment groups of 25 females each. A single daily dose of 200, 500 and 1000 mg/kg/day was administered orally (gavage) on days 6-15 of gestation. A vehicle group received deionized water. Cesarean sections were performed on all surviving females on gestation day 21 and fetuses were evaluated. All rats survived to GD 21 of gestation. Pregnancy rates in each group ranged from 88 % to 96 %. One dam in the 500 mg/kg/day dose had a single conceptus litter (one early resorption). Adverse clinical observations (urogenital wetness and staining) occurred in the 1000 mg/kg/day dose group. Maternal body weight decrease and food reductions occurred over the dosing period in the 500 and 1000 mg/kg/day groups. There were no treatment-related maternal necropsy findings. Terminal body weights and gravid uterine weights were comparable among the groups. Corrected body weight gain was decreased (>10 %) at 500 and 1000 mg/kg/day. Increased liver weights and relative liver weights were observed in the 1000 mg/kg/day dose group. There were no treatment-related effects on early resorptions, late resorptions, live fetuses per litter or sex ratio, or fetal weight per litter among the dose groups and no fetal malformations or variations attributed to PBO at any dose level.

Developmental and reproduction toxicity of piperonyl butoxide part 2 developmental safety of piperonyl butoxide in the NZW rabbit.

Piperonyl butoxide (PBO) was developed in the 1940s. PBO increases the effectiveness of pyrethrins, thus it is called a synergist. Herein, the findings from a guideline developmental toxicity study in rabbits conducted in 1986 are reported. Inseminated New Zealand White rabbits were randomly assigned to a control and three treatment groups of 16 does each. Dose levels of 50, 100 and 200 mg/kg/day were selected based on a dosage-range study to avoid excessive maternal toxicity and administered orally (gavage) as a single daily dose on days 7-19 of gestation at a volume of 0.5 mL/kg. The control group received the vehicle only, Mazola® corn oil. Cesarean sections were performed on all surviving females on gestation day 29 and fetuses were evaluated. Survival for all study groups was 100%. Treatment-related maternotoxicity was manifested at the 100 and 200 mg/kg/day levels as decreased defecation and dose-related body weight losses during the treatment period (gestation days 7-13 and 7-19). The Cesarean section parameter values and fetal morphological observations of the treated groups did not differ significantly from the concurrent control group and were within the historical control range for this rabbit strain. No maternal or fetal adverse effects were seen at the 50 mg/kg/day dose level. Although maternal toxicity resulting from treatment was apparent at the 100 and 200 mg/kg/day dose levels, neither fetotoxicity nor teratogenicity were elicited in rabbits by piperonyl butoxide at dose levels as high as 200 mg/kg/day.

Biomarker development for neonicotinoid exposure in soil under interaction with the synergist piperonyl butoxide in Folsomia candida.

Pesticide toxicity is typically assessed by exposing model organisms to individual compounds and measuring effects on survival and reproduction. These tests are time-consuming, labor-intensive, and do not accurately capture the effect of pesticide mixtures. Moreover, it is unfeasible to screen the nearly infinite combinations of mixtures for synergistic effects on model organisms. Therefore, reliable molecular indicators of pesticide exposure have to be identified, i.e., biomarkers. These biomarkers can form the basis of rapid and economical screening procedures to assess the toxicity of pesticides even under synergistic interaction with other pollutants. In this study, we screened the expression patterns of eight genes for suitability as a biomarker for neonicotinoid exposure in the soil ecotoxicological model Folsomia candida (springtails). Springtails were exposed to the neonicotinoids imidacloprid and thiacloprid either alone or with various levels of piperonyl butoxide (PBO), which inhibits cytochrome P450 enzymes (CYPs): a common point of synergistic interaction between neonicotinoid and other pesticides. First, we confirmed PBO as a potency enhancer for neonicotinoid toxicity to springtail fecundity, and then used it as a tool to confirm biomarker robustness. We identified two genes that are reliably indicative for neonicotinoid exposure even under metabolic inhibition of CYPs by PBO, nicotinic acetylcholine receptor-subunit alpha 1 (nAchR) and sodium-coupled monocarboxylate transporter (SMCT). These results can form the basis for developing high-throughput screening procedures for neonicotinoid exposure in varying mixture compositions.

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.

Examining the developmental toxicity of piperonyl butoxide as a Sonic hedgehog pathway inhibitor.

Piperonyl butoxide (PBO) is a semisynthetic chemical present in hundreds of pesticide formulations used in agricultural, commercial, and residential settings. PBO acts as a pesticide synergist by inhibiting insect cytochrome P450 enzymes and is often present at much higher concentrations than active insecticidal ingredients. PBO was recently discovered to also inhibit Sonic hedgehog (Shh) signaling, a key molecular pathway in embryonic development and in brain and face morphogenesis. Recent animal model studies have shown that in utero PBO exposure can cause overt craniofacial malformations or more subtle neurodevelopmental abnormalities. Related adverse developmental outcomes in humans are etiologically heterogeneous, and, while studies are limited, PBO exposure during pregnancy has been linked to neurodevelopmental deficits. Contextualized in PBO's newly recognized mechanism as a Shh signaling inhibitor, these findings support more rigorous examination of the developmental toxicity of PBO and its potential contribution to etiologically complex human birth defects. In this review, we highlight environmental sources of human PBO exposure and summarize existing animal studies examining the developmental impact of prenatal PBO exposure. Also presented are critical knowledge gaps in our understanding of PBO's pharmacokinetics and potential role in gene-environment and environment-environment interactions that should be addressed to better understand the human health impact of environmental PBO exposure.

Resistance to insecticides and synergism by enzyme inhibitors in Aedes albopictus from Punjab, Pakistan.

The widespread use of insecticides has ecological consequences such as emergence of insecticide resistance and environmental pollution. Aedes albopictus is a major vector of dengue virus in the Punjab province, Pakistan. Control of Ae. albopictus with insecticides along with source eradication is critical in the prevention and control of dengue fever but is threatened by the development of insecticide resistance. Here, field strains of Ae. albopictus from eight cities of Punjab were evaluated for resistance against temephos, deltamethrin and permethrin. For temephos, high resistance (RRLC50 > tenfold) was found in larvae of the Rawalpindi strain, moderate resistance (RRLC50 = five- to tenfold) in Multan, Faisalabad, Sialkot, Lahore and Sheikhupura strains, and low resistance (RRLC50 < fivefold) in Kasur and Sahiwal strains. In the case of deltamethrin, high resistance was seen in adults of the strain from Faisalabad, moderate resistance in the strains from Sialkot, Sheikhupura, Lahore and Kasur, and low resistance in Sahiwal, Multan and Rawalpindi strains. For permethrin, adults of all the field strains exhibited high levels of resistance. In synergism bioassays, toxicity of all the insecticides in the field strains significantly enhanced when tested in combination with piperonyl butoxide or S,S,S-tributylphosphorotrithioate, suggesting the probability of metabolic-based mechanisms of resistance. In conclusion, field strains of Ae. albopictus from Punjab exhibit resistance to temephos, deltamethrin and permethrin, which might be associated with metabolic mechanisms of resistance.

Piperonyl butoxide: Mode of action analysis for mouse liver tumour formation and human relevance.

In a 79 week bioassay the pesticide synergist piperonyl butoxide (PBO) was shown to significantly increase the incidence of hepatocellular adenoma (but not hepatocellular carcinoma) in male CD-1 mice at dietary levels of 100 and 300 mg/kg/day PBO and in female mice at a dietary level of 300 mg/kg/day. As PBO is not a genotoxic agent, a series of investigative studies were undertaken to elucidate the mode of action (MOA) for PBO-induced mouse liver tumour formation. Male CD-1 mice were fed diets to provide intakes of 0 (control), 30, 100 and 300 mg/kg/day PBO and for purposes of comparison 500 ppm sodium phenobarbital (NaPB), a known constitutive androstane receptor (CAR) activator, for 7 and 14 days. Treatment with 100 and 300 mg/kg/day PBO and 500 ppm NaPB increased relative liver weight which was associated with hepatocyte hypertrophy, with hepatocyte replicative DNA synthesis (RDS) being increased after 7 days treatment. The treatment of CD-1 mice with 30-300 mg/kg/day PBO for 14 days resulted in significant dose-dependent increases in hepatic microsomal cytochrome P450 (CYP) content and 7-pentoxyresorufin O-depentylase (PROD) activity and in hepatic Cyp2b10 mRNA levels. In contrast, PBO produced a biphasic effect on markers of activation of the peroxisome proliferator-activated receptor alpha (PPARα), with small increases in microsomal lauric acid 12-hydroxylase activity and hepatic Cyp4a10 mRNA levels being observed in mice given 100 mg/kg/day with PBO, with either no increase or a significant inhibition being observed in mice given 300 mg/kg/day PBO. The hepatic effects of PBO in male CD-1 mice were generally similar to those produced by NaPB and were reversible after the cessation of treatment for 28 days. Studies were also performed in male C57BL/6J (wild type) mice and in hepatic CAR and pregnane X receptor (PXR) knockout mice (CAR KO/PXR KO mice), where in the CAR KO/PXR KO mice PBO had little effect on markers of CAR activation, but produced some increases in markers of PPARα activation. The treatment of male CD-1 mouse hepatocytes for 4 days with 5-50 µM PBO, 10-1000 µM NaPB and 25 ng/mL epidermal growth factor (EGF) resulted in significant increases in hepatocyte RDS. While treatment of hepatocytes from one male and one female human donor with 5-500 µM PBO and 10-1000 µM NaPB for 4 days had no effect on hepatocyte RDS, treatment with EGF resulted in significant increases in RDS in both human hepatocyte preparations. In summary, PBO is predominantly a hepatic CAR activator at carcinogenic dose levels in CD-1 mice, with activation of hepatic CAR resulting in a suppression of the effect of PBO on hepatic PPARα. A robust MOA for PBO-induced mouse liver tumour formation has been established, this MOA being similar to that previously identified for NaPB and some other rodent liver CAR activators. Based on the lack of effect of PBO on RDS in human hepatocytes, it is considered that the MOA for PBO-induced mouse liver tumour formation is qualitatively not plausible for humans.

The genome of the marine rotifer Brachionus koreanus sheds light on the antioxidative defense system in response to 2-ethyl-phenanthrene and piperonyl butoxide.

BRACHIONUS: spp. (Rotifera: Monogononta) have been introduced as ecotoxicological model-organisms that are widely distributed in aquatic environments. Among the Brachionus spp., the monogonont rotifer Brachionus koreanus has been widely used for ecology, ecotoxicology, and evolution, thus, providing the whole genome data of B. koreanus is important for further understandings of in-depth molecular mechanisms. In this study, the completed assembly and characterization of the B. koreanus genome resulted in a total length of 85.7 Mb with 14,975 annotated genes. The final number of scaffolds was 567 with an N50 value and a GC content of 1.86 Mb and 24.35 %, respectively. Based on the fully constructed genome database, a total of 24 CYPs, 23 GSTs, two SODs, and a single CAT genes were identified and analyzed antioxidant activities (CAT, SOD, and GST), and transcriptional regulation of the entire CYPs, GSTs, SODs, and CAT in response to 2-ethyl-phenanthrene (2-ethyl-PHE) and piperonyl butoxide (PBO), to demonstrate the usefulness of the whole genome library of B. koreanus in response xenobiotic-induced oxidative stress. The assembled B. koreanus genome will provide a better understanding on the molecular ecotoxicology in the view of molecular mechanisms underlying toxicological responses, particularly on xenobiotic detoxification processes in the rotifer B. koreanus.

Developmental Toxicity Assessment of Piperonyl Butoxide Exposure Targeting Sonic Hedgehog Signaling and Forebrain and Face Morphogenesis in the Mouse: An in Vitro and in Vivo Study.

BACKGROUND: Piperonyl butoxide (PBO) is a pesticide synergist used in residential, commercial, and agricultural settings. PBO was recently found to inhibit Sonic hedgehog (Shh) signaling, a key developmental regulatory pathway. Disruption of Shh signaling is linked to birth defects, including holoprosencephaly (HPE), a malformation of the forebrain and face thought to result from complex gene-environment interactions. OBJECTIVES: The impact of PBO on Shh signaling in vitro and forebrain and face development in vivo was examined. METHODS: The influence of PBO on Shh pathway transduction was assayed in mouse and human cell lines. To examine its teratogenic potential, a single dose of PBO (22-1,800mg/kg) was administered by oral gavage to C57BL/6J mice at gestational day 7.75, targeting the critical period for HPE. Gene-environment interactions were investigated using Shh+/- mice, which model human HPE-associated genetic mutations. RESULTS: PBO attenuated Shh signaling in vitro through a mechanism similar to that of the known teratogen cyclopamine. In utero PBO exposure caused characteristic HPE facial dysmorphology including dose-dependent midface hypoplasia and hypotelorism, with a lowest observable effect level of 67mg/kg. Median forebrain deficiency characteristic of HPE was observed in severely affected animals, whereas all effective doses disrupted development of Shh-dependent transient forebrain structures that generate cortical interneurons. Normally silent heterozygous Shh null mutations exacerbated PBO teratogenicity at all doses tested, including 33mg/kg. DISCUSSION: These findings demonstrate that prenatal PBO exposure can cause overt forebrain and face malformations or neurodevelopmental disruptions with subtle or no craniofacial dysmorphology in mice. By targeting Shh signaling as a sensitive mechanism of action and examining gene-environment interactions, this study defined a lowest observable effect level for PBO developmental toxicity in mice more than 30-fold lower than previously recognized. Human exposure to PBO and its potential contribution to etiologically complex birth defects should be rigorously examined. https://doi.org/10.1289/EHP5260.

Effects of piperonyl butoxide on exploratory behaviour in female mice.

Previous studies reported that piperonyl butoxide (PBO) induces adverse effects on exploratory behaviour in male mice. However, no consistent effects of PBO treatment were observed in female mice. This study aimed to evaluate PBO's neurobehavioral effects in female mice. Female mice were exposed to PBO through diet to provide levels of 0 (control), 0.025%, 0.1%, and 0.4% from 5 to 12 weeks of age, and selected behavioural parameters were measured. The average female body weight showed no significant effect from PBO treatment through the experimental periods. Regarding multiple-T water maze performance at 10 weeks of age, no significant effect caused by PBO treatment was observed. Exploratory behaviour examination of 8-week-old female mice indicated that the average speed declined in a significant dose-related manner, and the longitudinal pattern indicated a significant difference between the control and high-dose groups. For exploratory behaviour examination at 11 weeks of age, the total exploration distance shortened in a significant dose-related manner, and the average speed declined similarly. These longitudinal patterns showed significant differences between the control and high-dose groups. The PBO dose levels in this study produced several adverse effects on exploratory behaviour in female mice.

Knockdown of cytochrome P450 CYP6 family genes increases susceptibility to carbamates and pyrethroids in the migratory locust, Locusta migratoria.

Insect cytochrome P450 monooxygenase (CYP) plays a key role in the detoxification of insecticides. In this study, four cDNA sequences of CYP6 genes were identified and characterized. Transcription levels of LmCYP6HC1 and LmCYP6HCL1 were high in first- and fourth-instar nymph stages, respectively. LmCYP6HN1 was primarily expressed in the egg to third-instar nymph stages, while LmCYP6HQ1 was predominantly expressed in the stages from fourth-instar nymph to the adult. The four CYP6 genes were predominantly distributed in the antenna, brain, fat body, integument, and hemolymph. Piperonyl butoxide exposure inhibited total CYP activity and synergized the toxicity of carbamates and pyrethroids. Knockdown of LmCYP6HL1, LmCYP6HN1, and LmCYP6HQ1 increased nymph mortality following exposure to carbaryl, and silencing of LmCYP6HC1, LmCYP6HL1, LmCYP6HN1, and LmCYP6HQ1 comprehensively raised nymph mortality following exposure to fluvalinate. Knockdown of LmCYP6HL1 or LmCYP6HN1 significantly increased nymph mortality following exposure to cypermethrin or fenvalerate, respectively. These results suggest that the CYP6 family plays a key role in determining the susceptibility of Locusta migratoria to both carbamates and pyrethroids.

Integrated analysis of in vitro data and the adverse outcome pathway framework for prioritization and regulatory applications: An exploratory case study using publicly available data on piperonyl butoxide and liver models.

The integration of existing knowledge to support the risk assessment of chemicals is an ongoing challenge for scientists, risk assessors and risk managers. In addition, European Union regulations limiting the use of new animal testing in cosmetics makes already existing information even more valuable. Applying a previous SEURAT-1 program framework to derive predictions of in vivo toxicity responses for a compound, we selected piperonyl butoxide (PBO) as a case study for identification of knowledge and methodology gaps in understanding a compound's effects on the human liver. This is investigated through integration of data from human in vitro transcriptomics studies, biological pathway analysis, chemical and disease associations, and adverse outcome pathway (AOP) information. The outcomes of the analysis are used to generate AOPs of liver-related endpoints, identifying areas of concern for risk assessors and regulators. We demonstrate that integration of data through already existing and publicly available tools can produce outcomes comparable to those that may be found through more conventional time- and resource-intensive methods. It is also expected that, with more refinement, this approach could in the future provide evidence to support chemical risk assessment, while also identifying data gaps for which additional testing may be needed.

Molecular Pathological Differences in Global Gene Expression between Two Sustained Proliferative Lesions, Nodular Regenerative Hepatocellular Hyperplasia and Hepatocellular Adenoma, in Mice.

Long-term exposure to piperonyl butoxide (PBO) induces multiple nodular masses along with hepatocellular tumors in the liver of mice. The histopathological features of the nodules led to our diagnosis of nodular regenerative hepatocellular hyperplasia (NRH). However, because of the lack of data on the biological characteristics of NRH, whether this lesion is truly nonneoplastic remains unknown. In this study, the molecular characteristics of NRH were compared with those of hepatocellular adenoma (HCA) by global gene expression analysis. Six-week-old male ICR mice were fed a diet containing 6,000 ppm PBO for 43 weeks to induce NRH and HCA development. Complementary DNA microarray analysis was performed using messenger RNA extracted from NRH and HCA frozen sections collected by laser microdissection. Hierarchical cluster analysis showed that all NRH samples clustered together but were separate from the HCA cluster. Pathway analysis revealed activation of the cell cycle and Delta-Notch signaling in both lesions, but the latter was more upregulated in HCA. Downregulation of cytochrome p450 enzymes was observed in NRH, but not in HCA. These results imply that NRH differs from HCA in terms of not only morphological but also molecular characteristics.

Toxicity and sublethal effects of fluralaner on Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

The increasing occurrence of resistance to chemical insecticides in insect pest populations is a serious threat to the integrity of current pest management strategies, and exploring new alternative chemistries is one important way to overcome this obstacle. Fluralaner, as a novel isoxazoline insecticide, has broad spectrum activity against a variety of insect pests, but little data is available about its effect on Lepidopterans. The effects of fluralaner on Spodoptera litura Fabricius, a widespread and polyphagous pest, were evaluated in the present study. Our results showed younger larvae were more susceptible to fluralaner treatment, but feeding and topical applications were similarly effective in 3rd instar larvae. Synergism assays indicated that piperonyl butoxide (PBO) could increase the toxicity of fluralaner to S. litura to a certain degree and P450 may be involved in the detoxification of fluralaner in vivo. Sublethal developmental effects included reduced larval body weight, decreased pupation and emergence, and notched wings in adults, accompanied by changes in the transcript levels of chitinase 5 (CHT5) and juvenile hormone acid methyltransferase (Jhamt), genes vital for insect development. Above results manifested that fluralaner is highly toxic to S. litura larvae via either topical or oral application and provide an indication of how this insecticide is metabolized in vivo. Further, our results provided a foundation for further development of fluralaner as a new tool in insect pest management.

Tracking pyrethroid toxicity in surface water samples: Exposure dynamics and toxicity identification tools for laboratory tests with Hyalella azteca (Amphipoda).

Pyrethroid insecticides are commonly used in pest control and are present at toxic concentrations in surface waters of agricultural and urban areas worldwide. Monitoring is challenging as a result of their high hydrophobicity and low toxicity thresholds, which often fall below the analytical methods detection limits (MDLs). Standard daphnid bioassays used in surface water monitoring are not sensitive enough to protect more susceptible invertebrate species such as the amphipod Hyalella azteca and chemical loss during toxicity testing is of concern. In the present study, we quantified toxicity loss during storage and testing, using both natural and synthetic water, and presented a tool to enhance toxic signal strength for improved sensitivity of H. azteca toxicity tests. The average half-life during storage in low-density polyethylene (LDPE) cubitainers (Fisher Scientific) at 4 °C of 5 pyrethroids (permethrin, bifenthrin, lambda-cyhalothrin, cyfluthrin, and esfenvalerate) and one organophosphate (chlorpyrifos; used as reference) was 1.4 d, and piperonyl butoxide (PBO) proved an effective tool to potentiate toxicity. We conclude that toxicity tests on ambient water samples containing these hydrophobic insecticides are likely to underestimate toxicity present in the field, and mimic short pulse rather than continuous exposures. Where these chemicals are of concern, the addition of PBO during testing can yield valuable information on their presence or absence. Environ Toxicol Chem 2018;37:462-472. © 2017 SETAC.