Project description:We examined how Raoultella ornithinolytica-ZK4 degraded pyrethroid pesticides within soil sediment from an abandoned pesticide plant. Lambda-cypermethrin and deltamethrin are two pyrethroid insecticides with high insecticidal activity and a wide range of applications. However, their increased use has raised concerns regarding toxicity and accumulation. We isolated a strain of ZK4 (Raoultella ornithinolytica-ZK4) from soil taken from a channel that surrounded a pesticide plant. We used enzyme localization to study degrading bacteria ZK4. The ZK4 strain underwent intracellular enzyme degradation. The degradation rates of lambda-cyhalothrin and deltamethrin were 55% and 53%, respectively. The optimum pH of the two kinds of pyrethroids in ZK4 was 6.5, and their optimum temperature was 37 °C. The intracellular degradation of the crude enzyme produced by the ZK4 strain had a pH of 6.0-8.0 and a temperature of 20-42 °C. The ZK4 strain genome contained 5310 genes with a total length of 4,864,494 bp. Sugar metabolism and exogenous chemical metabolism accounted for the largest proportion of metabolic activities. We used the clusters of orthologous groups (COG) alignment and found numbers for 4686 protein sequences, accounting for 88.25% of the total predicted protein. ZK4 degraded lambda-cyhalothrin and deltamethrin, and may serve as a reference for the preparation of future degrading microbial agents to assist with environmental restoration efforts.
Project description:Attention-deficit hyperactivity disorder (ADHD) is estimated to affect 8-12% of school-age children worldwide. ADHD is a complex disorder with significant genetic contributions. However, no single gene has been linked to a significant percentage of cases, suggesting that environmental factors may contribute to ADHD. Here, we used behavioral, molecular, and neurochemical techniques to characterize the effects of developmental exposure to the pyrethroid pesticide deltamethrin. We also used epidemiologic methods to determine whether there is an association between pyrethroid exposure and diagnosis of ADHD. Mice exposed to the pyrethroid pesticide deltamethrin during development exhibit several features reminiscent of ADHD, including elevated dopamine transporter (DAT) levels, hyperactivity, working memory and attention deficits, and impulsive-like behavior. Increased DAT and D1 dopamine receptor levels appear to be responsible for the behavioral deficits. Epidemiologic data reveal that children aged 6-15 with detectable levels of pyrethroid metabolites in their urine were more than twice as likely to be diagnosed with ADHD. Our epidemiologic finding, combined with the recapitulation of ADHD behavior in pesticide-treated mice, provides a mechanistic basis to suggest that developmental pyrethroid exposure is a risk factor for ADHD.
Project description:Recently, we demonstrated that the oligosaccharide portion of ganglioside GM1 is responsible, via direct interaction and activation of the TrkA pathway, for the ability of GM1 to promote neuritogenesis and to confer neuroprotection in Neuro2a mouse neuroblastoma cells. Recalling the knowledge that ganglioside GM1 modulates calcium channels activity, thus regulating the cytosolic calcium concentration necessary for neuronal functions, we investigated if the GM1-oligosaccharide would be able to overlap the GM1 properties in the regulation of calcium signaling, excluding a specific role played by the ceramide moiety inserted into the external layer of plasma membrane. We observed, by calcium imaging, that GM1-oligosaccharide administration to undifferentiated Neuro2a cells resulted in an increased calcium influx, which turned out to be mediated by the activation of TrkA receptor. The biochemical analysis demonstrated that PLC? and PKC activation follows the TrkA stimulation by GM1-oligosaccharide, leading to the opening of calcium channels both on the plasma membrane and on intracellular storages, as confirmed by calcium imaging experiments performed with IP3 receptor inhibitor. Subsequently, we found that neurite elongation in Neuro2a cells was blocked by subtoxic administration of extracellular and intracellular calcium chelators, suggesting that the increase of intracellular calcium is responsible of GM1-oligosaccharide mediated differentiation. These results suggest that GM1-oligosaccharide is responsible for the regulation of calcium signaling and homeostasis at the base of the neuronal functions mediated by plasma membrane GM1.
Project description:Application of pyrethroid insecticides in residential settings may result in children's exposures to these chemicals and possible adverse health effects. Household dust is a recognized reservoir for pyrethroids and a potential medium for multi-route pyrethroid exposure. Young children move and play in a manner that resuspends dust, and since their breathing zone is close to the floor, they will have higher inhalation exposure to pesticide-laden dust than other age groups. Directly measuring a toddler's exposure to household dust presents many logistic challenges. We simulated the dust resuspension induced by a toddler using a robot, which also served as a platform to collect air samples at the toddler's breathing zone height. We performed simulated pyrethroid residential spray and dust resuspension experiments on vinyl and carpeted floors. The mean pyrethroid airborne concentrations in the stationary and mobile samples were 0.065??g/m3 and 0.143??g/m3 for the vinyl floor with 1?g/m2 dust loading, and 0.034??g/m3 and 0.061??g/m3 for the carpeted floor with 10?g/m2 dust loading, respectively. Pyrethroids concentrations in the settled dust samples were significantly lower than that measured in the stationary and mobile samples in the carpeted floor experiments. Thus, the use of stationary samples and settled dust samples may underestimate a toddler's personal inhalation exposure to pyrethroids in residential houses.
Project description:Pyrethroids are a class of neurotoxic insecticides, and some studies have used single-time wiping of hard surface flooring to estimate indoor pyrethroid concentrations. Considering that human activities may affect concentrations, knowledge of temporal variability is needed to reduce the uncertainty of exposure estimates that are calculated using wipe sampling of pyrethroids in occupied housing. During weeks one, two, and six of a 6-week study, two wipe samples of hard surface kitchen flooring were collected in each of 50 occupied residences and used to estimate the temporal variability of eight pyrethroids and six pyrethroid degradation products. Beginning 1 month prior to sample collection, the participants kept pesticide use diaries. All pyrethroids were widely distributed among the houses, and co-occurrence of multiple pyrethroids was common structured. Application diaries and detection frequencies appeared unconnected, but the applications were correlated with measurable changes in pyrethroid concentrations. In general, degradation products were detected less frequently and at lower concentrations than their parent pyrethroids. Estimates of the intraclass correlation coefficient (ICC) for individual pyrethroids ranged from 0.55 (bifenthrin) to 0.80 (deltamethrin), and two sampling events at each residence would have been sufficient to estimate the mean concentration of most pyrethroids with an ICC of 0.80.
Project description:Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools.
Project description:Pyrethroid insecticides exert their insecticidal and toxicological effects primarily by disrupting voltage-gated sodium channel (VGSC) function, resulting in altered neuronal excitability. Numerous studies of individual pyrethroids have characterized effects on mammalian VGSC function and neuronal excitability, yet studies examining effects of complex pyrethroid mixtures in mammalian neurons, especially in environmentally relevant mixture ratios, are limited. In the present study, concentration-response functions were characterized for five pyrethroids (permethrin, deltamethrin, cypermethrin, ?-cyfluthrin and esfenvalerate) in an in vitro preparation containing cortical neurons and glia. As a metric of neuronal network activity, spontaneous mean network firing rates (MFR) were measured using microelectorde arrays (MEAs). In addition, the effect of a complex and exposure relevant mixture of the five pyrethroids (containing 52% permethrin, 28.8% cypermethrin, 12.9% ?-cyfluthrin, 3.4% deltamethrin and 2.7% esfenvalerate) was also measured. Data were modeled to determine whether effects of the pyrethroid mixture were predicted by dose-addition. At concentrations up to 10?M, all compounds except permethrin reduced MFR. Deltamethrin and ?-cyfluthrin were the most potent and reduced MFR by as much as 60 and 50%, respectively, while cypermethrin and esfenvalerate were of approximately equal potency and reduced MFR by only ?20% at the highest concentration. Permethrin caused small (?24% maximum), concentration-dependent increases in MFR. Effects of the environmentally relevant mixture did not depart from the prediction of dose-addition. These data demonstrate that an environmentally relevant mixture caused dose-additive effects on spontaneous neuronal network activity in vitro, and is consistent with other in vitro and in vivo assessments of pyrethroid mixtures.
Project description:Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30-46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l-1 and 0.918 ± 0.025 U·µg-1, respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.
Project description:Voltage-gated sodium channels (Na(v)) are essential for initiation and propagation of action potentials. Previous in vitro studies reported that exposure to the Na(v) toxins veratridine and ? scorpion toxin cause persistent downregulation of Na(v) mRNA in vitro. However the mechanism of this downregulation is not well characterized. Here, we report that the type-II pyrethroid deltamethrin, which has a similar mechanism as these toxins, elicited an approximate 25% reduction in Na(v) 1.2 and Na(v) 1.3 mRNA in SK-N-AS cells. Deltamethrin-induced decreases of Na(v) mRNA were blocked with the Na(v) antagonist tetrodotoxin, demonstrating a primary role for interaction with Na(v). Pre-treatment with the intracellular calcium chelator BAPTA-AM and the calpain inhibitor PD-150606 also prevented these decreases, identifying a role for intracellular calcium and calpain activation. Because alterations in Na(v) expression and function can result in neurotoxicity, additional studies are warranted to determine whether or not such effects occur in vivo.
Project description:To investigate a role of mKIAA genes in early stage of neurite outgrowth, in vitro time course experiment was performed using the mouse neuroblastoma Neuro2A cells and all-trans-retinoic acid. Six time points of 8 biological replicates were performed.These data were normalized by median intensities and subtracted background intensities. NOTE: Outlier involved several data were not deposited. Keywords: time-course